Imaging neuromodulation in the brain

成瘾涉及大脑系统调节动机，唤醒和奖励的内部状态，以及 情绪这种内部状态影响目标导向的行为和决策。的共同特征 这种内部状态是它们的价和它们的持久性：它们可以具有正价或负价， 能比触发刺激持续好几分钟然而，神经生物学机制， 内部状态的持久性以及它们与价编码的关系知之甚少。 果蝇为研究神经调质如何作用于神经元提供了一种易处理的遗传模式生物 控制持久的内部状态的回路，这些状态支配着目标导向的行为和决策。我们有 发现控制雄性求偶行为的P1中间神经元在被激活时可以促进 持续的内部社会唤醒或动机状态，可持续数分钟。在一份出版物中， 通过基础研究，我们已经获得了P1中间神经元和响应神经元之间联系的证据。 章鱼胺（OA），一种昆虫去甲肾上腺素（NE）的同系物，已知其促进精神刺激性自我兴奋。 啮齿动物给药。我们还确定了P1神经元的下游靶点，称为pCd细胞， 似乎在决定社会唤醒的内部状态的持续性方面发挥了关键的必要作用。 在延长期内，我们将继续研究P1神经元如何促进奖励性的内部 状态，以及这些机制的积极效价，或奖励性质，P1刺激的关系。 在头两年，我们将专注于实现基本补助金的目标3和4。这些目标是：目标3）测试 P1神经元激活具有正效价和奖励性的假设;目的4）研究 参与P1奖赏学习的神经调节机制。在未发表的实验中，我们有 发现P1神经元的激活可以产生实时位置偏好（RTPP），它也可以 作为条件性嗅觉偏爱（COP）的无条件刺激（US）。这两个发现 这表明P1激活是积极的，并且它可以是奖励的。我们计划调查 COP过程中的可塑性发生在P1神经元处或下游，以及P1神经元是否是COP所必需的。 COP的表达（目标3）。初步实验表明，多巴胺（DA）可能在 调节P1刺激的效应。我们将确认和扩展这些发现，并调查 其他神经调节剂的作用，包括生物胺，如章鱼胺（OA），我们有 显示调节P1刺激的效果以激活控制社会行为的aSP 2神经元9。 此外，我们将研究是否蘑菇体（MB）神经元参与奖励学习也是如此。 参与P1介导的气味调节（目的4）。根据以前的数据，我们希望找到MB的作用， 但确切地说，涉及MB神经元的哪个子集尚不清楚。 在Merit扩展目标5中，我们将研究其他神经调质在P1诱导的持续性脑缺血中的作用。 社会唤醒和奖励学习。P1神经元的候选神经调节靶点包括多巴胺能 (Trh+）神经元，其子集响应于P1刺激而被激活（初步结果），以及 神经肽F（NPF），在其他情况下与奖励有关。我们将着手解决这个问题 使用功能性连接组学，其中P1神经元的光遗传学激活与钙成像相结合 在含有这些细胞的假定神经调节靶点的群体中。目标神经元可以使用 光活化GFP（PA-GFP），并且它们的形态用作“搜索图像”以鉴定特定的遗传标记。 驱动程序来标记细胞的子集。使用这些驱动程序，可以激活和沉默这些神经元。 在P1介导的奖励（RTPP和COP测定）和持续的社会唤醒的背景下进行。 我们已经成功地建立了这种方法，并用它来识别pCd神经元，这是持续的 由P1神经元激活，并且是由P1激活触发的持续社会行为所必需的。作为 作为补充方法，我们将利用我们在技术方面取得的最新进展， 在成人大脑中进行整体荧光原位杂交（FISH）， 使用FISH探针对P1神经元进行hr 38的光遗传学刺激激活跟随细胞， 早期基因（类似于c-fos）。双标记FISH可以使用hr 38和探针进行， 神经递质生物合成酶或神经肽，以鉴定P1靶中表达的神经调质。 一个基本的问题是，介导P1诱导的持续活动的机制是否也参与了 奖励为了解决这个问题，在Merit Extension Aim 6中，我们将研究pCd神经元在P1- 介导的奖励学习，使用这些细胞的功能操作。初步数据显示， P1细胞对pCd神经元的持续激活受到DA的调节，我们将研究这是如何发生的。 施加调节影响。我们预计这些实验将产生一般原则， 持久的内部奖励状态是由大脑编码的，在整个发育过程中具有潜在的广泛相关性。

Addiction involves brain systems mediating internal states of motivation, arousal and reward, as well as emotions. Such internal states influence goal-directed behaviors and decision-making. A common feature of such internal states is their valence and their persistence: they can have a positive or negative valence, and can outlast their triggering stimulus for many minutes. However the neurobiological mechanisms that underlie the persistence of internal states, and their relationship to the encoding of valence, are poorly understood. Drosophila provides a tractable genetic model organism for studying how neuromodulators act on neural circuits to control persistent internal states that govern goal-directed behavior and decision-making. We have discovered that P1 interneurons, which control male courtship behavior, can when activated promote a persistent internal state of social arousal or motivation, which can last for minutes. In a publication supported by the base grant, we have obtained evidence of a link between P1 interneurons and neurons that respond to octopamine (OA), an insect homolog of norepinephrine (NE), which is known to facilitate psychostimulant self- administration in rodents. We have also identified a downstream target of P1 neurons, called pCd cells, which appear to play a key requisite role in determining the persistence of an internal state of social arousal. During the extension period, we will continue our studies of how P1 neurons promote a rewarding internal state, and the relationship of these mechanisms to the positive valence, or rewarding nature, of P1 stimulation. In the first 2 years, we will focus on pursuing Aims 3 and 4 of the base grant. These aims were: Aim 3) to test the hypothesis that P1 neuron activation is positively valenced and rewarding; Aim 4) to investigate neuromodulatory mechanisms involved in P1 reward learning. In unpublished experiments, we have discovered that activation of P1 neurons can produce a real-time place preference (RTPP), and that it can also serve as an unconditional stimulus (US) for conditioned olfactory preference (COP). Both of these findings indicate that P1 activation is positively valenced, and that it can be rewarding. We plan to investigate whether plasticity during COP occurs at or downstream of P1 neurons, and whether P1 neurons are necessary for expression of the COP (Aim 3). Preliminary experiments suggest that dopamine (DA) may play a role in modulating the effects effects of P1 stimulation. We will confirm and extend these findings, and also investigate the role(s) of other neuromodulators including biogenic amines such as octopamine (OA), which we have shown to modulate the effect of P1 stimulation to activate aSP2 neurons that control social behavior9. Furthermore, we will investigate whether mushroom body (MB) neurons involved in reward learning are also involved in P1-mediated odor conditioning (Aim 4). Given previous data, we expect to find a role for the MB, but precisely which subset of MB neurons are involved is not clear. In Merit Extension Aim 5, we will investigate the role of other neuromodulators in P1-induced persistent social arousal and reward learning. Candidate neuromodulatory targets of P1 neurons include serotonergic (Trh+) neurons, a subset of which is activated in response to P1 stimulation (preliminary results), and neuropeptide F (NPF), which has been implicated in reward in other contexts. We will approach this problem using functional connectomics, in which optogenetic activation of P1 neurons is combined with calcium imaging in populations containing putative neuromodulatory targets of these cells. Target neurons can be “filled” using photo-activatable GFP (PA-GFP), and their morphology used as a “search image” to identify specific genetic drivers that label that subset of cells. Using these drivers, activation and silencing of these neurons can be performed in the context of both P1-mediated reward (RTPP and COP assays), and persistent social arousal. We have successfully established this approach and used it to identify pCd neurons, which are persistently activated by P1 neurons and required for persistent social behaviors triggered by P1 activation. As a complementary approach, we will take advantage of recent advances that we have made in techniques for whole-mount fluorescent in situ hybridization (FISH) in the adult brain, which allow identification of candidate follower cells activated by optogenetic stimulation of P1 neurons using FISH probes for hr38, an immediate early gene (analogous to c-fos) in Drosophila. Double-label FISH can be performed using hr38 and probes for neurotransmitter biosynthetic enzymes or neuropeptides, to identify neuromodulators expressed in P1 targets. A fundamental question is whether the mechanism mediating P1-induced persistent activity is also involved in reward. To address this question, in Merit Extension Aim 6 we will investigate the role of pCd neurons in P1- mediated reward learning, using functional manipulations of these cells. Preliminary data suggest that persistent activation of pCd neurons by P1 cells is modulated by DA, and we will investigate how this modulatory influence is exerted. We anticipate that these experiments will yield general principles of how persistent internal reward states are encoded by brains, with potentially broad relevance across phylogeny.