Genetic and Optogenetic Models to Dissect the Role of the Serotonin 1B Receptor

描述（由申请人提供）：攻击性和冲动性是行为的两个未被充分研究的方面，它们是许多精神疾病的重要症状。5-羟色胺1B受体（5-HT1B R）与攻击性和冲动行为有关，包括药理学、组成型敲除小鼠模型和人类基因多态性相关研究。鉴于5-羟色胺能调节大脑发育的证据，5-HT1B R可能在建立潜在的攻击性和冲动行为的电路中发挥发育作用。5-HT1B R是一种抑制5-羟色胺能和非5-羟色胺能神经元轴突末端神经递质释放的GPCR，广泛分布于基底节区、海马、丘脑、小脑和中脑。因此，需要一种可诱导的组织特异性策略来描述5-HT1B Rs调节攻击性和冲动性的敏感期和神经回路。因此，我们生成了一个允许5-HT1B R的时间和空间调控的小鼠系。一个四环素操作符（tetO）被插入到5-HT1B R的启动子和编码区（tetO1B）之间。受体结合和原位杂交显示，与129只WT小鼠相比，这些tetO1B小鼠在大脑中具有正常的基础5-HT1B R表达。将tetO1B小鼠与表达四环素依赖性转录沉默子（tTS）的转基因小鼠杂交，在¿-Actin（普遍存在）、CaMKII（前脑特异性）或Pet （rape特异性）启动子的控制下，产生具有组织特异性和可诱导敲低5-HT1B R的小鼠系。通过放射自显影和原位杂交的初步表征揭示了启动子特异性的5-HT1B R表达模式，可以通过强力霉素调节。tetO1B:：¿-Actin-tTS+小鼠的初步行为分析显示，其行为表型与构成型敲除小鼠相似。，冲动性攻击显著增加。因此，我们有工具来操纵5-HT1B R在空间和时间上的表达，并检查其对攻击性和冲动行为的影响。利用光遗传学的后续研究将通过确定在关键时期干预可能改变了成年期攻击和冲动回路中的哪些大脑区域来解决回路水平的机制。假设是在发育的关键时期缺乏5HT1B Rs会导致过度活跃的“攻击回路”，因此通过抑制这些过度活跃的大脑区域，可以挽救正常的表型。这可以使用光敏抑制Cl-泵，盐视紫红质，通过病毒载体注入目标大脑区域，随后通过光纤植入的黄光激活。总的来说，了解5-HT1B R如何介导潜在的攻击和冲动的神经回路将对治疗产生影响

DESCRIPTION (provided by applicant): Aggression and impulsivity are two understudied aspects of behavior that are significant symptoms in a number of psychiatric disorders. The serotonin 1B receptor (5-HT1B R) has been implicated in aggressive and impulsive behavior using pharmacology, constitutive knock-out mouse models, and human gene polymorphism association studies. Given evidence of serotonergic modulation of brain development, it is likely that 5-HT1B R plays a developmental role in establishing circuits underlying aggressive and impulsive behavior. 5-HT1B R is a GPCR that inhibits neurotransmitter release in axon terminals of serotonergic and non-serotonergic neurons localized broadly in the basal ganglia, hippocampus, thalamus, cerebellum and raphe. Thus, an inducible and tissue specific strategy is necessary to delineate the sensitive period and neural circuits through which 5-HT1B Rs regulate aggression and impulsivity. We have therefore generated a mouse line that permits temporal and spatial regulation of 5-HT1B R. A tetracycline operator (tetO) has been inserted between the promoter and coding region of the 5-HT1B R (tetO1B). Receptor binding and in situ hybridization reveals that these tetO1B mice have normal basal 5-HT1B R expression in the brain compared to 129 WT mice. Crossing tetO1B mice to transgenic mice expressing the tetracycline-dependent transcriptional silencer (tTS) under the control of ¿-Actin (ubiquitous), CaMKII (forebrain-specific) or Pet (raphe-specific) promoters yields mouse lines with tissue specific and inducible knock-down of 5-HT1B R. Initial characterization with autoradiography and in situ hybridization reveals promoter-specific 5-HT1B R expression patterns that can be modulated with doxycycline administration. Preliminary behavioral assays using tetO1B:: ¿-Actin-tTS+ mice reveal a similar behavioral phenotype to the constitutive knock-out mouse-i.e., significantly increased impulsive aggression. Therefore, we have the tools to manipulate the expression of 5-HT1B R spatially and temporally and examine the resulting impact on aggressive and impulsive behaviors. Follow-up studies using optogenetics will address the circuit level mechanisms by determining which brain regions in the aggression and impulsivity circuitry in adulthood may have been altered by the intervention during the critical period. The hypothesis is that a lack of 5HT1B Rs during a critical period of development results in an overactive 'aggression circuit', and so by inhibiting these overactive brain regions, a normal phenotype can be rescued. This is possible using the light-sensitive inhibitory Cl- pump, halorhodopsin, introduced through viral vector injection into target brain regions, with subsequent activation by yellow light administered via fiber optic implant. Overall, understanding how 5-HT1B R mediates the neural circuits underlying aggression and impulsivity will have implications for the treatments of several neuropsychiatric disorders, including addiction, attention-deficit hyperactivity disorde and bipolar disorder, and suicidal depression.