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Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. Drosophila central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, we mapped 18 Drosophila central brain lineages. While we found large aggregate differences between lineages, we also discovered shared patterns of morphological diversification. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.

The efficiency of gene targeting can vary drastically. Even utilizing CRISPR/Cas9 does not ensure rapid, successful gene targeting. InDrosophila, we previously established Golic+ to augment gene-targeting productivity simply through fly pushing. This transgene-based system elicits gene targeting in germ cells. It further implements lethality selection to enrich for progeny with accurate gene targeting. However, limitations still remain. Here we deliver an improved Golic+ technique that we name Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to eliminate false positives while simultaneously boosting efficiency. Strikingly, male germ cells are exceptionally susceptible to gene targeting using E-Golic+. With male founders, we easily achieve 100% recovery of correct gene targeting. Given the superior efficiency/specificity and relatively effortless scalability, E-Golic+ promises to triumph in any challenging and otherwise unattainable gene targeting projects inDrosophila.

SummaryWiring a complex brain requires enormous cell specificity. This specificity is laid out via a developmental process where neural stem cells produce countless diverse neurons. To help elucidate this process and resolve the considerable dynamic specificity, we need to observe the development of multiple neuronal lineages.Drosophilacentral brain lineages are predetermined, comprised of a fixed set of neurons born in pairs in a specific order. To reveal specific roles of lineage identity, Notch-dependent sister fate specification, and temporal patterning in morphological diversification, we mapped approximately one quarter of theDrosophilacentral brain lineages. While we found large aggregate differences, we also discovered similar patterns of morphological specification and diversification. Lineage identity plus Notch state govern primary neuronal trajectories, whereas temporal fates diversify terminal elaborations in target-specific manners. In addition, we identified ‘related’ lineages of analogous neuron types produced in similar temporal patterns. Two stem cells even yield identical series of dopaminergic neuron types, but with completely disparate sister neurons. These phenomena suggest that large changes in morphological diversity can be the consequence of relatively small differences in lineage fating. Taken together, this large-scale lineage mapping study reveals that relatively simple rules drive incredible neuronal complexity.

In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Our interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted us to develop a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies' preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. We show that optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments and that the preference for NPF neuron activation is dependent on NPF signaling. Finally, we identify a small subset of NPF-expressing neurons located in the dorsomedial posterior brain that are sufficient to elicit preference in our assay. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.

Memory-retrieval processing of cross-modal sensory preconditioning is vital for understanding the plasticity underlying the interactions between modalities. As part of the sensory preconditioning paradigm, it has been hypothesized that the conditioned response to an unreinforced cue depends on the memory of the reinforced cue via a sensory link between the two cues. To test this hypothesis, we studied cross-modal memory-retrieval processing in a genetically tractable model organism,Drosophila melanogaster. By expressing the dominant temperature-sensitiveshibirets1(shits1) transgene, which blocks synaptic vesicle recycling of specific neural subsets with theGal4/UASsystem at the restrictive temperature, we specifically blocked visual and olfactory memory retrieval, either alone or in combination; memory acquisition remained intact for these modalities. Blocking the memory retrieval of the reinforced olfactory cues did not impair the conditioned response to the unreinforced visual cues or vice versa, in contrast to the canonical memory-retrieval processing of sensory preconditioning. In addition, these conditioned responses can be abolished by blocking the memory retrieval of the two modalities simultaneously. In sum, our results indicated that a conditioned response to an unreinforced cue in cross-modal sensory preconditioning can be recalled through parallel pathways.

Reversal learning has been widely used to probe the implementation of cognitive flexibility in the brain. Previous studies in monkeys identified an essential role of the orbitofrontal cortex (OFC) in reversal learning. However, the underlying circuits and molecular mechanisms are poorly understood. Here, we use the T-maze to investigate the neural mechanism of olfactory reversal learning in Drosophila. By adding a reversal training cycle to the classical learning protocol, we show that wild-type flies are able to reverse their choice according to the alteration of conditioned stimulus (CS)-unconditioned stimulus (US) contingency. The reversal protocol induced a specific suppression of the initial memory, an effect distinct from memory decay or extinction. GABA down-regulation in the anterior paired lateral (APL) neurons, which innervate the mushroom bodies (MBs), eliminates this suppression effect and impairs normal reversal. These findings reveal that inhibitory regulation from the GABAergic APL neurons facilitates olfactory reversal learning by suppressing initial memory in Drosophila.

Drosophila, with a tiny brain, can accomplish many goal-directed behaviors, which might shed the light of intelligence. We reviewed our exploratory trip in the recent years concerning the neural circuitry mechanism of decision-making in Drosophila, and proposed a general Gating and Gain Rule in central nervous processing of cognitive function, based on our neurogenetics studies and the functional connectivity mapping of mushroom bodies (MB) which has been believed to be a center of ‘free will’ in fly’s brain. We provide a Tai-ji model to reflect the balance of regulation in MB, and assigned dopaminergic and GABAergic systems as the two antagonistical parts.