Zhiyuan Lu

A visual motion detection circuit suggested by Drosophila connectomics

Animal behaviour arises from computations in neuronal circuits, but our understanding of these computations has been frustrated by the lack of detailed synaptic connection maps, or connectomes. For example, despite intensive investigations over half a century, the neuronal implementation of local motion detection in the insect visual system remains elusive. Here we develop a semi-automated pipeline using electron microscopy to reconstruct a connectome, containing 379 neurons and 8,637 chemical synaptic contacts, within the Drosophila optic medulla. By matching reconstructed neurons to examples from light microscopy, we assigned neurons to cell types and assembled a connectome of the repeating module of the medulla. Within this module, we identified cell types constituting a motion detection circuit, and showed that the connections onto individual motion-sensitive neurons in this circuit were consistent with their direction selectivity. Our results identify cellular targets for future functional investigations, and demonstrate that connectomes can provide key insights into neuronal computations.

The Neural Substrate of Spectral Preference in Drosophila

Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. Here, we identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, we determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers.

Synaptic circuits of the Drosophila optic lobe: the input terminals to the medulla.

Understanding the visual pathways of the flys compound eye has been blocked for decades at the second optic neuropil, the medulla, a two‐part relay comprising 10 strata (M1–M10), and the largest neuropil in the flys brain. Based on the modularity of its composition, and two previous reports, on Golgi‐impregnated cell types (Fischbach and Dittrich, Cell Tissue Res., 1989 ; 258:441–475) and their synaptic circuits in the first neuropil, the lamina, we used serial‐section electron microscopy to examine inputs to the distal strata M1–M6. We report the morphology of the reconstructed medulla terminals of five lamina cells, L1–L5, two photoreceptors, R7 and R8, and three neurons, medulla cell T1 and centrifugal cells C2 and C3. The morphology of these conforms closely to previous reports from Golgi impregnation. This fidelity provides assurance that our reconstructions are complete and accurate. Synapses of these terminals broadly localize to the terminal and provide contacts to unidentified targets, mostly medulla cells, as well as sites of connection between the terminals themselves. These reveal that R8 forms contacts upon R7 and thus between these two spectral inputs; that L3 provides input upon both pathways, adding an achromatic input; that the terminal of L5 reciprocally connects to that of L1, thus being synaptic in the medulla despite lacking synapses in the lamina; that the motion‐sensing input cells L1 and L2 lack direct interconnection but both receive input from C2 and C3, resembling lamina connections of these cells; and that, as in the lamina, T1 provides no output chemical synapses. J. Comp. Neurol. 509:493–513, 2008.

Candidate neural substrates for off-edge motion detection in Drosophila.

BACKGROUND In the flys visual motion pathways, two cell types-T4 and T5-are the first known relay neurons to signal small-field direction-selective motion responses [1]. These cells then feed into large tangential cells that signal wide-field motion. Recent studies have identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD) [2]. Interneurons relaying the OFF channel from L1s partner, L2, to T5 are so far not known, however. RESULTS Here we report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells. Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron, L3, providing a candidate substrate for L3s combinatorial action with L2 [3]. Most numerous, Tm2 and Tm9s input synapses are spatially segregated on T5s dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express choline acetyltransferase (ChAT). CONCLUSIONS We hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.

Synaptic circuits and their variations within different columns in the visual system of Drosophila

Significance Circuit diagrams of brains are generally reported only as absolute or consensus networks; these diagrams fail to identify the accuracy of connections, however, for which multiple circuits of the same neurons must be documented. For this reason, the modular composition of the Drosophila visual system, with many identified neuron classes, is ideal. Using EM, we identified synaptic connections in the fly’s second visual relay neuropil, or medulla, in the 20 neuron classes in a so-called “core connectome,” those neurons present in seven neighboring columns. These connections identify circuits for motion. Their error rates for wiring reveal that <1% of contacts overall are not part of a consensus circuit but incorporate errors of either omission or commission. Autapses are occasionally seen. We reconstructed the synaptic circuits of seven columns in the second neuropil or medulla behind the fly’s compound eye. These neurons embody some of the most stereotyped circuits in one of the most miniaturized of animal brains. The reconstructions allow us, for the first time to our knowledge, to study variations between circuits in the medulla’s neighboring columns. This variation in the number of synapses and the types of their synaptic partners has previously been little addressed because methods that visualize multiple circuits have not resolved detailed connections, and existing connectomic studies, which can see such connections, have not so far examined multiple reconstructions of the same circuit. Here, we address the omission by comparing the circuits common to all seven columns to assess variation in their connection strengths and the resultant rates of several different and distinct types of connection error. Error rates reveal that, overall, <1% of contacts are not part of a consensus circuit, and we classify those contacts that supplement (E+) or are missing from it (E−). Autapses, in which the same cell is both presynaptic and postsynaptic at the same synapse, are occasionally seen; two cells in particular, Dm9 and Mi1, form ≥20-fold more autapses than do other neurons. These results delimit the accuracy of developmental events that establish and normally maintain synaptic circuits with such precision, and thereby address the operation of such circuits. They also establish a precedent for error rates that will be required in the new science of connectomics.

Age‐related plasticity in the synaptic ultrastructure of neurons in the mushroom body calyx of the adult honeybee Apis mellifera

The mushroom bodies are high‐order sensory integration centers in the insect brain. In the honeybee, their main sensory input regions are large, doubled calyces with modality‐specific, distinct sensory neuropil regions. We investigated adult structural plasticity of input synapses in the microglomeruli of the olfactory lip and visual collar. Synapsin‐immunolabeled whole‐mount brains reveal that during the natural transition from nursing to foraging, a significant volume increase in the calycal subdivisions is accompanied by a decreased packing density of boutons from input projection neurons. To investigate the associated ultrastructural changes at pre‐ and postsynaptic sites of individual microglomeruli, we employed serial‐section electron microscopy. In general, the membrane surface area of olfactory and visual projection neuron boutons increased significantly between 1‐day‐old bees and foragers. Both types of boutons formed ribbon and non‐ribbon synapses. The percentage of ribbon synapses per bouton was significantly increased in the forager. At each presynaptic site the numbers of postsynaptic partners—mostly Kenyon cell dendrites—likewise increased. Ribbon as well as non‐ribbon synapses formed mainly dyads in the 1‐day‐old bee, and triads in the forager. In the visual collar, outgrowing Kenyon cell dendrites form about 140 contacts upon a projection neuron bouton in the forager compared with only about 95 in the 1‐day‐old bee, resulting in an increased divergence ratio between the two stages. This difference suggests that synaptic changes in calycal microcircuits of the mushroom body during periods of altered sensory activity and experience promote behavioral plasticity underlying polyethism and social organization in honeybee colonies. J. Comp. Neurol. 520:3509–3527, 2012.

Different classes of input and output neurons reveal new features in microglomeruli of the adult Drosophila mushroom body calyx

To investigate how sensory information is processed, transformed, and stored within an olfactory system, we examined the anatomy of the input region, the calyx, of the mushroom bodies of Drosophila melanogaster. These paired structures are important for various behaviors, including olfactory learning and memory. Cells in the input neuropil, the calyx, are organized into an array of microglomeruli each comprising the large synaptic bouton of a projection neuron (PN) from the antennal lobe surrounded by tiny postsynaptic neurites from intrinsic Kenyon cells. Extrinsic neurons of the mushroom body also contribute to the organization of microglomeruli. We employed a combination of genetic reporters to identify single cells in the Drosophila calyx by light microscopy and compared these with cell shapes, synapses, and circuits derived from serial‐section electron microscopy. We identified three morphological types of PN boutons, unilobed, clustered, and elongated; defined three ultrastructural types, with clear‐ or dense‐core vesicles and those with a dark cytoplasm having both; reconstructed diverse dendritic specializations of Kenyon cells; and identified Kenyon cell presynaptic sites upon extrinsic neurons. We also report new features of calyx synaptic organization, in particular extensive serial synapses that link calycal extrinsic neurons into a local network, and the numerical proportions of synaptic contacts between calycal neurons. All PN bouton types had more ribbon than nonribbon synapses, dark boutons particularly so, and ribbon synapses were larger and with more postsynaptic elements (2–14) than nonribbon (1–10). The numbers of elements were in direct proportion to presynaptic membrane area. Extrinsic neurons exclusively had ribbon synapses. J. Comp. Neurol. 520:2185–2201, 2012.

A connectome of a learning and memory center in the adult Drosophila brain

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB’s α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall. DOI: http://dx.doi.org/10.7554/eLife.26975.001

Charcot-Marie-Tooth 2B mutations in rab7 cause dosage-dependent neurodegeneration due to partial loss of function

The small GTPase Rab7 is a key regulator of endosomal maturation in eukaryotic cells. Mutations in rab7 are thought to cause the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a gain-of-function mechanism. Here we show that loss of rab7, but not overexpression of rab7 CMT2B mutants, causes adult-onset neurodegeneration in a Drosophila model. All CMT2B mutant proteins retain 10–50% function based on quantitative imaging, electrophysiology, and rescue experiments in sensory and motor neurons in vivo. Consequently, expression of CMT2B mutants at levels between 0.5 and 10-fold their endogenous levels fully rescues the neuropathy-like phenotypes of the rab7 mutant. Live imaging reveals that CMT2B proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. These findings are not consistent with a gain-of-function mechanism. Instead, they indicate a dosage-dependent sensitivity of neurons to rab7-dependent degradation. Our results suggest a therapeutic approach opposite to the currently proposed reduction of mutant protein function. DOI: http://dx.doi.org/10.7554/eLife.01064.001

The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling

Left-right asymmetries in brains are usually minor or cryptic. We report brain asymmetries in the tiny, dorsal tubular nervous system of the ascidian tadpole larva, Ciona intestinalis. Chordate in body plan and development, the larva provides an outstanding example of brain asymmetry. Although early neural development is well studied, detailed cellular organization of the swimming larva’s CNS remains unreported. Using serial-section EM we document the synaptic connectome of the larva’s 177 CNS neurons. These formed 6618 synapses including 1772 neuromuscular junctions, augmented by 1206 gap junctions. Neurons are unipolar with at most a single dendrite, and few synapses. Some synapses are unpolarised, others form reciprocal or serial motifs; 922 were polyadic. Axo-axonal synapses predominate. Most neurons have ciliary organelles, and many features lack structural specialization. Despite equal cell numbers on both sides, neuron identities and pathways differ left/right. Brain vesicle asymmetries include a right ocellus and left coronet cells. DOI: http://dx.doi.org/10.7554/eLife.16962.001