Imaging neuromodulation in the brain

上瘾涉及大脑系统调节动机、唤醒和奖励的内部状态，以及 感情。这样的内部状态会影响目标导向的行为和决策。的一个共同特征 这种内部状态是它们的价态及其持久性：它们可以有正价，也可以有负价，以及 可以比他们触发的刺激持续很多分钟。然而，其背后的神经生物学机制 关于内态的持久性，以及它们与价的编码之间的关系，人们知之甚少。 果蝇为研究神经调节剂如何作用于神经提供了一种易于处理的遗传模式生物 控制持续内部状态的电路，这些内部状态管理目标导向的行为和决策。我们有 研究发现，控制雄性求爱行为的P1中间神经元在激活时可以促进 持久的社会唤醒或动力的内部状态，可持续几分钟。在支持的出版物中 通过基本拨款，我们已经获得了P1中间神经元和对 章鱼胺(OA)，一种去甲肾上腺素(NE)的昆虫同系物，已知有助于精神刺激性自我 对啮齿类动物的管理。我们还确定了P1神经元的一个下游靶点，称为PCD细胞，它 似乎在决定社交唤醒的内部状态的持续性方面起着关键的必要作用。 在延伸期内，我们将继续研究P1神经元如何促进奖赏的内部 状态，以及这些机制与P1刺激的正价或奖赏性质的关系。 在头两年，我们将专注于实现基本赠款的目标3和4。这些目标是：目标3)测试 假设P1神经元的激活是积极的和有益的；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 发现激活P1神经元可以产生实时位置偏爱(RTPP)，而且它也可以 作为条件性嗅觉偏好的无条件刺激(US)。这两个发现 表明P1的激活是正价的，而且它可能是有益的。我们计划调查是否 在COP期间，可塑性发生在P1神经元或其下游，以及P1神经元是否对 缔约方会议的表述(目标3)。初步实验表明，多巴胺(DA)可能在 调节P1刺激的效应效应。我们将证实和扩大这些发现，并进行调查 其他神经调节剂的作用(S)，包括生物胺，如章鱼胺(OA)，我们有 被证明可以调节刺激P1的效果，激活控制社会行为的aSP2神经元9。 此外，我们将调查参与奖赏学习的蘑菇体(MB)神经元是否也 参与P1介导的气味条件作用(目标4)。根据之前的数据，我们预计将为MB找到一个角色， 但目前还不清楚具体涉及到MB神经元的哪一个子集。 在优点扩展目标5中，我们将研究其他神经调节剂在P1诱导的持续性中的作用 社会唤醒和奖励学习。P1神经元的候选神经调节靶点包括5-羟色胺能 (TRH)神经元，其子集被激活以响应P1刺激(初步结果)，以及 神经肽F(NPF)，它在其他情况下与奖赏有关。我们将处理这个问题 使用功能连接学，其中P1神经元的光发生激活与钙成像相结合 在包含这些细胞的假定神经调节靶点的人群中。靶神经元可以用来填充 光可激活绿色荧光蛋白(PA-GFP)及其形态作为识别特定基因的“搜索图像” 标记该单元子集的驱动程序。使用这些驱动程序，这些神经元的激活和沉默可以 在P1介导的奖励(RTPP和COP分析)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，这些神经元是持续性的 由P1神经元激活，是由P1激活触发的持续性社会行为所必需的。作为一名 补充方法，我们将利用我们在技术方面取得的最新进展 成人脑的整装荧光原位杂交(FISH)，可以识别候选基因 使用hr38的FISH探针通过光遗传刺激P1神经元激活跟随细胞 果蝇的早期基因(类似于c-fos)。可以使用hr38和探针进行双标记FISH 神经递质生物合成酶或神经肽，以确定在P1靶标中表达的神经调节剂。 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，在优点扩展目标6中，我们将研究PCD神经元在P1中的作用。 中介奖赏学习，使用这些细胞的功能操作。初步数据显示， 多巴胺调节P1细胞对PCD神经元的持续激活，我们将研究这一过程是如何实现的 施加了调制影响。我们预计，这些实验将产生关于如何 持久的内部奖赏状态是由大脑编码的，在系统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.