Dopaminergic neurons (DANs) drive learning across the animal kingdom, but the upstream circuits that regulate their activity and thereby learning remain poorly understood. We provide the first synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mush-room body (MB) of Drosophila larva. We discover afferent sensory pathways and a large population of neurons that provide feedback from MB output neurons and link distinct memory systems (aversive and appetitive). We combine this with functional studies of DANs and their presynaptic partners and with comprehensive circuit modelling. We find that DANs compare convergent feedback from aversive and appetitive systems which enables the computation of integrated predictions that may improve future learning. Computational modelling reveals that the discovered feedback motifs increase model flexibility and performance on learning tasks. Our study provides the most detailed view to date of biological circuit motifs that support associative learning.

In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides ‘minus-end-out’ microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein.DOI: http://dx.doi.org/10.7554/eLife.10140.001

Cytoplasmic streaming in Drosophila oocytes is a microtubulebased bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.kinesin-1 | microtubules | cytoplasmic streaming | Drosophila | axis determination

The plus-end microtubule (MT) motor kinesin-1 is essential for normal development, with key roles in the nervous system. Kinesin-1 drives axonal transport of membrane cargoes to fulfill the metabolic needs of neurons and maintain synapses. We have previously demonstrated that kinesin-1, in addition to its well-established role in organelle transport, can drive MT-MT sliding by transporting "cargo" MTs along "track" MTs, resulting in dramatic cell shape changes. The mechanism and physiological relevance of this MT sliding are unclear. In addition to its motor domain, kinesin-1 contains a second MT-binding site, located at the C terminus of the heavy chain. Here, we mutated this C-terminal MT-binding site such that the ability of kinesin-1 to slide MTs is significantly compromised, whereas cargo transport is unaffected. We introduced this mutation into the genomic locus of kinesin-1 heavy chain (KHC), generating the Khc mutA allele. Khc mutA neurons displayed significant MT sliding defects while maintaining normal transport of many cargoes. Using this mutant, we demonstrated that MT sliding is required for axon and dendrite outgrowth in vivo. Consistent with these results, Khc mutA flies displayed severe locomotion and viability defects. To test the role of MT sliding further, we engineered a chimeric motor that actively slides MTs but cannot transport organelles. Activation of MT sliding in Khc mutA neurons using this chimeric motor rescued axon outgrowth in cultured neurons and in vivo, firmly establishing the role of sliding in axon outgrowth. These results demonstrate that MT sliding by kinesin-1 is an essential biological phenomenon required for neuronal morphogenesis and normal nervous system development.kinesin-1 | microtubules | Drosophila | axon outgrowth | dendrite outgrowth N eurons are the basic unit of the nervous system, forming vast networks throughout the body that communicate using receptorligand machinery located in long cellular projections called axons and dendrites. Learning how these processes form is key to understanding the early development and pathology of the nervous system. Microtubules (MTs) and actin microfilaments have been implicated in neurite outgrowth, with many studies focusing on the growth cone at the tip of the axon. Previous models suggest that the driving forces for neurite outgrowth are MT polymerization and the treadmilling of F-actin (1, 2). However, other studies demonstrate that F-actin is dispensable to outgrowth and neurites extend even in the absence of F-actin (3-5).Our group has found that the motor protein kinesin-1 can rearrange the MT network by sliding MTs against each other (6). We have shown that kinesin-1 is required for MT sliding in cultured neurons and kinesin-1 depletion inhibits both neurite outgrowth and regeneration (7,8). Additionally, we have observed MT sliding in axons as well as MTs pushing on the axon tip (9). Recent studies from other groups have also implicated MT translocation in axon extension and dendritic organization (10-12). Based on th...