Identifying Neural Substrates of Behavior in Drosophila Melanogaster

这项研究计划的目标是开发和进一步开发操纵神经活动的技术，以识别特定行为背后的大脑回路。利用果蝇Gal4-UAS基因打靶系统来驱动其产物抑制神经元兴奋性的基因的表达，我们选择性地抑制神经元亚群的活动，并分析这种操纵对行为的影响。我们特别感兴趣的是成蝇在从蛹中羽化后不久执行的一套激素协调和发育编程的行为，立即关注的是那些对翅膀扩展所必需的行为。 我们用这种方法识别了两组功能不同的神经元，它们是翅膀扩张所必需的。两组都表达一种被称为甲壳类心脏活性多肽(CCAP)的共同神经肽，但其中一组(输出组)也表达并分泌激素法氏囊到血淋巴(血液)中，而另一组(调节组)调节输出组的活动。这两个组都由多个神经元组成，其中只有一些可能是翅膀扩张所必需的。此外，每组内的单个神经元(或神经元的子集)可能具有不同的功能角色。为了进一步确定我们已经识别的两个广泛组中神经元的功能同一性，我们开发了Gal4-UAS技术的修改版本，允许我们选择性地操作表达CCAP的神经元的小亚集。 我们的Split Gal4系统结合了酵母双杂交系统的技术，将Gal4分子分为DNA结合(DBD)和转录激活(TA)两个区域。我们已经将每个结构域融合到两个互补的异源二聚亮氨酸拉链中的一个，以便DBD和TA结构域在表达这两个结构域的细胞中关联，以重建Gal4的转录活性。通过在体内单独靶向这两个区域，我们可以在表达这两个区域的细胞子集中选择性地激活UAS转基因基因。我们利用了这一系统，将DBD结构域定位于CCAP表达的神经元，并建立了TA增强子陷阱系，以包括CCAP表达神经元的不同子集的任意模式表达Gal4 TA(或HSV-1 VP16转录因子更有效的TA)。在初步筛选中，我们已经产生了允许在大约38个表达CCAP的神经元的独特亚群中表达UAS转基因的品系。我们目前正在分析通过靶向表达细胞死亡基因收割者来消融这些神经元亚群的后果。初步分析已经使我们能够确定调节组中16个神经元的关键子集。我们的分析进一步表明，输出组的14个神经元可能在功能上是冗余的。 为了补充现有的靶向抑制神经元活动的方法，我们还一直在开发允许选择性增强或诱导神经元活动的工具。正如抑制活动的技术可以用来证明哪些神经元是特定行为所必需的一样，增强活动的技术也可以用来证明哪些神经元足以驱动这种行为。此前，我们已经利用编码细菌钠通道的基因NaChBac(融合到绿色荧光蛋白)来结构性地增强细胞的兴奋性，并表明表达CCAP的神经元调节组的兴奋性增强扰乱了翅膀的扩张和激素法氏囊的分泌。最近，我们成功地开发了大鼠寒冷和薄荷醇受体(TRPM8)，作为一种尖锐激活靶向神经元的工具。我们已经使用这个工具将表达CCAP的神经元的兴奋性增强的关键期映射到从蛹中出现之前不久的时间窗口。我们现在开始使用TRPM8敏锐地激活CCAP和滑囊素表达神经元的子集，试图确定一个可能足以诱导翅膀扩张计划的最小子集。 使用我们正在开发的广泛工具来研究果蝇退缩后行为的神经元底物，应该可以作为电路映射方法概念的证明，这种方法以后可以扩展到哺乳动物行为的研究。

The goal of this research program is to exploit, and further develop, techniques for manipulating neural activity to identify the brain circuits underlying specific behaviors. Using the Gal4-UAS gene targeting system of Drosophila melanogaster to drive the expression of genes whose products inhibit neuronal excitability, we are selectively suppressing the activity of subsets of neurons and analyzing the effects of this manipulation on behavior. We are particularly interested in the suite of hormonally coordinated and developmentally programmed behaviors executed by the adult fly shortly after emergence from the pupal case, with an immediate focus on those necessary for wing expansion. We have used this approach to identify two functionally distinct groups of neurons which are necessary for wing expansion. Both groups express a common neuropeptide known as Crustacean Cardioactive Peptide (CCAP), but one group (the output group) also expresses and secretes the hormone bursicon into the hemolymph (blood), while the other (the regulatory group) modulates the activity of the output group. Both of these groups consist of multiple neurons, only some of which may be necessary for wing expansion. In addition, individual neurons (or subsets of neurons) within each group may subserve different functional roles. To further determine the functional identities of neurons within the two broad groups we have identified, we have developed a modified version of the Gal4-UAS technique which allows us to selectively manipulate small subsets of CCAP-expressing neurons. Our Split Gal4 system incorporates technology from the yeast two-hybrid system in that it divides the Gal4 molecule into its component DNA-binding (DBD) and transcription activation (TA) domains. We have fused each domain to one of two complementary, heterodimerizing leucine zippers so that the DBD and TA domains associate in cells that express both domains to reconstitute Gal4 transcriptional activity. By independently targeting the two domains in vivo, we can activate UAS transgenes selectively in the subset of cells that expresses both domains. We have exploited this system by targeting the DBD domain to CCAP-expressing neurons and making TA enhancer trap lines that express the Gal4 TA (or the more potent TA of the HSV-1 VP16 transcription factor) in arbitrary patterns that include different subsets of the CCAP-expressing neurons. In a preliminary screen, we have generated lines that permit expression of UAS-transgenes in approximately 38 unique subsets of CCAP-expressing neurons. We are currently analyzing the consequences of ablating these subsets of neurons by tartgeted expression of the cell death gene reaper. Preliminary analysis has already allowed us to identify a critical subset of 16 neurons within the regulatory group. Our analysis further indicates that the 14 neurons of the output group are likely to be functionally redundant. To supplement the available methods for targeted suppression of neuronal activity, we have also been developing tools that permit the selective enhancement or induction of neuronal activity. Just as techniques for suppressing activity can be used to demonstrate which neurons are necessary for a specific behavior, techniques for enhancing activity can be used to demonstrate which neurons are sufficient to drive that behavior. Previously, we have exploited the gene encoding the bacterial sodium channel, NaChBac (fused to Green Fluorescent Protein), to constitutively enhance cellular excitability and have shown that enhanced excitability in the regulatory group of CCAP-expressing neurons disrupts wing expansion and secretion of the hormone bursicon. More recently, we have succeeded in developing the rat cold and menthol receptor (TRPM8) as a tool for acutely activating targeted neurons. We have used this tool to map the critical period for enhancement of excitability in CCAP-expressing neurons to a time window shortly preceding emergence from the pupal case. We are now beginning to acutely activate subsets of CCAP- and bursicon-expressing neurons using TRPM8 in an attempt to identify a minimal subset that might be sufficient to induce the wing expansion program. Investigation of the neuronal substrates of posteclosion behavior in Drosophila using the broad palette of tools we are developing should serve as a proof of concept of a circuit mapping approach that can later be extended to studies of mammalian behavior.