Ian A. Meinertzhagen

Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein.

A protein required to localize mitochondria to Drosophila nerve terminals has been identified genetically. Photoreceptors mutant for milton show aberrant synaptic transmission despite normal phototransduction. Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. Milton protein is associated with mitochondria and is present primarily in axons and synapses. A likely explanation of the apparent trafficking defect is offered by the coimmunoprecipitation of Milton and kinesin heavy chain. Transfected into HEK293T cells, Milton induces a redistribution of mitochondria within the cell. We propose that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals.

Digital in-line holography for biological applications

Digital in-line holography with numerical reconstruction has been developed into a new tool, specifically for biological applications, that routinely achieves both lateral and depth resolution, at least at the micron level, in three-dimensional imaging. The experimental and numerical procedures have been incorporated into a program package with a very fast reconstruction algorithm that is now capable of real-time reconstruction. This capability is demonstrated for diverse objects, such as suspension of microspheres and biological samples (diatom, the head of Drosophila melanogaster), and the advantages are discussed by comparing holographic reconstructions with images taken by using conventional compound light microscopy.

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.

Endophilin Mutations Block Clathrin-Mediated Endocytosis but Not Neurotransmitter Release

We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic boutons, indicating an inability to retrieve synaptic membrane. This defect is accompanied by an expansion of the presynaptic membrane, and a depletion of vesicles from the bouton lumen. Interestingly, mutant larvae are still able to sustain release at 15%-20% of the normal rate during high-frequency stimulation. We propose that kiss-and-run maintains neurotransmission at active zones of the larval NMJ in endophilin animals.

Synaptic organization of the mushroom body calyx in Drosophila melanogaster

The calyx neuropil of the mushroom body in adult Drosophila melanogaster contains three major neuronal elements: extrinsic projection neurons, presumed cholinergic, immunoreactive to choline acetyltransferase (ChAT‐ir) and vesicular acetylcholine transporter (VAChT‐ir) antisera; presumed γ‐aminobutyric acid (GABA)ergic extrinsic neurons with GABA‐like immunoreactivity; and local intrinsic Kenyon cells. The projection neurons connecting the calyx with the antennal lobe via the antennocerebral tract are the only source of cholinergic elements in the calyces. Their terminals establish an array of large boutons 2–7 μm in diameter throughout all calycal subdivisions. The GABA‐ir extrinsic neurons, different in origin, form a network of fine fibers and boutons codistributed in all calycal regions with the cholinergic terminals and with tiny profiles, mainly Kenyon cell dendrites. We have investigated the synaptic circuits of these three neuron types using preembedding immuno‐electron microscopy. All ChAT/VAChT‐ir boutons form divergent synapses upon multitudinous surrounding Kenyon cell dendrites. GABA‐ir elements also regularly contribute divergent synaptic input onto these dendrites, as well as occasional inputs to boutons of projection neurons. The same synaptic microcircuits involving these three neuron types are repeatedly established in glomeruli in all calycal regions. Each glomerulus comprises a large cholinergic bouton at its core, encircled by tiny vesicle‐free Kenyon cell dendrites as well as by a number of GABAergic terminals. A single dendritic profile may thereby receive synaptic input from both cholinergic and GABAergic elements in close vicinity at presynaptic sites with T‐bars typical of fly synapses. ChAT‐ir boutons regularly have large extensions of the active zones. Thus, Kenyon cells may receive major excitatory input from cholinergic boutons and considerable postsynaptic inhibition from GABAergic terminals, as well as, more rarely, presynaptic inhibitory signaling. The calycal glomeruli of Drosophila are compared with the cerebellar glomeruli of vertebrates. The cholinergic boutons are the largest identified cholinergic synapses in the Drosophila brain and an eligible prospect for studying the genetic regulation of excitatory presynaptic function. J. Comp. Neurol. 445:211–226, 2002.

Tracking particles in four dimensions with in-line holographic microscopy.

We describe a simple holographic method that has enabled us to capture as a single data set the trajectories of micrometer-sized objects suspended in water. By subtracting consecutive holograms of a particle suspension and then adding these difference holograms, we constructed a final data set that contains the time evolution of the particle trajectories free from spurious background interference effects. The method is illustrated by a recording of the motion of 5-10-microm diameter algae in water.

Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner.

Aplysia VAP-33 (VAMP-associated protein) has been previously proposed to be involved in the control of neurotransmitter release. Here, we show that a Drosophila homolog of VAP-33, DVAP-33A, is localized to neuromuscular junctions. Loss of DVAP-33A causes a severe decrease in the number of boutons and a corresponding increase in bouton size. Conversely, presynaptic overexpression of DVAP-33A induces an increase in the number of boutons and a decrease in their size. Gain-of-function experiments show that the presynaptic dose of DVAP-33A tightly modulates the number of synaptic boutons. Our data also indicate that the presynaptic microtubule architecture is severely compromised in DVAP-33A mutants. We propose that a DVAP-33A-mediated interaction between microtubules and presynaptic membrane plays a pivotal role during bouton budding.

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.

The protocadherin Flamingo is required for axon target selection in the Drosophila visual system.

Photoreceptor neurons (R cells) in the Drosophila visual system elaborate a precise map of visual space in the brain. The eye contains some 750 identical modules called ommatidia, each containing eight photoreceptor cells (R1–R8). Cells R1–R6 synapse in the lamina; R7 and R8 extend through the lamina and terminate in the underlying medulla. In a screen for visual behavior mutants, we identified alleles of flamingo (fmi) that disrupt the precise maps elaborated by these neurons. These mutant R1–R6 neurons select spatially inappropriate targets in the lamina. During target selection, Flamingo protein is dynamically expressed in R1–R6 growth cones. Loss of fmi function in R cells also disrupts the local pattern of synaptic terminals in the medulla, and Flamingo is transiently expressed in R8 axons as they enter the target region. We propose that Flamingo-mediated interactions between R-cell growth cones within the target field regulate target selection.

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.