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充分研究的方面，是许多精神疾病的重要症状。5-羟色胺1B受体（5-HT 1B R）与攻击性和冲动性行为有关，使用药理学，组成性敲除小鼠模型和人类基因多态性关联研究。鉴于大脑发育的血清素能调节证据，5-HT 1B R很可能在建立攻击性和冲动行为背后的回路中发挥发育作用。5-HT 1B R是一种GPCR，其抑制广泛位于基底神经节、海马、丘脑、小脑和中缝中的多巴胺能和非多巴胺能神经元的轴突末端中的神经递质释放。因此，诱导和组织特异性的策略是必要的，以描绘敏感期和神经回路，通过5-HT 1B R调节侵略性和冲动性。因此，我们已经产生了一个小鼠线，允许5-HT 1B R的时间和空间调节。四环素操纵基因（tetO）被插入到5-HT 1B R（tetO 1B）的启动子和编码区之间。受体结合和原位杂交显示，这些tetO 1B小鼠有正常的基础5-HT 1B R表达在大脑中相比，129 WT小鼠。将tetO 1B小鼠与表达四环素依赖性转录沉默物（tTS）的转基因小鼠杂交，所述四环素依赖性转录沉默物（tTS）在<$-肌动蛋白（普遍存在的）、CaMKII（前脑特异性）或Pet（中缝特异性）启动子的控制下产生具有组织特异性和可诱导的5-HT 1B R敲低的小鼠品系。放射自显影和原位杂交的初步表征揭示了启动子特异性5-HT 1B R的表达模式，可以与多西环素管理调制。使用tetO 1B：：<$-Actin-tTS+小鼠的初步行为测定揭示了与组成型敲除小鼠相似的行为表型-即，冲动攻击性显著增加因此，我们有工具来操纵5-HT 1B R的表达空间和时间，并检查由此产生的影响，攻击和冲动行为。使用光遗传学的后续研究将通过确定成年期攻击性和冲动性回路中的哪些大脑区域可能在关键时期被干预改变来解决回路水平机制。该假说认为，在发育的关键时期缺乏5 HT 1B R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。这是可能的，使用光敏抑制性Cl-泵，盐视紫红质，通过病毒载体注射到目标脑区域，随后激活黄光通过光纤植入管理。总的来说，了解5-HT 1B R如何调节攻击性和冲动性背后的神经回路将对治疗产生影响 几种神经精神疾病，包括成瘾，注意力缺陷多动障碍和双相情感障碍，以及自杀性抑郁症。

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.