The first and second neural projections of the insect eye

1. The patterns of projection of some of the perpendicular neurones between the retina and medulla of the optic lobes of various insects have been studied. Axon paths have been studied from consecutive semi-thin plastic sections cut transversely and stained with toluidine blue. The termination positions and the paths of axons are both highly ordered and predictable. 2. In all insects with fused-rhabdomere eyes the axons of one ommatidium project to one cartridge of the lamina and the array of cartridges duplicates the array of ommatidia. In insects with open-rhabdomere eyes visual information is distributed amongst a number of lamina cartridges so that each cartridge receives information originating from one visual axis. 3. In both open- and fused-rhabdomere types the cartridge, array of the lamina is exactly duplicated in the medulla but by the intervention of the chiasma is reversed about a dorso-ventral plane. The axons of one lamina cartridge contribute to one medulla cartridge. Thus, in all insects studied, the visual field is projected exactly from retina to medulla. 4. Most of the retinula axons from one ommatidium terminate in the lamina but usually one pair passes directly to the medulla. These are from the central retinula cells (open-rhabdomere eyes) or from the small retinula cells (apposition type fused-rhabdomere eyes). Retinal responses are known mainly only for the short retinula axons so that visual information delivered to the medulla cartridge is still largely unresolved. 5. The lamina neuropile probably contains the elements responsible for the lateral correlation between parallel receptor inputs which is necessary for movement perception, but units with long lasting responses which could act as the delay circuit of movement perception are unknown. 6. The occurrence of errors in termination of the first projection of the optic lobe of the fly, which are reported for the first time in this work, provide no direct clues to the developmental processes by which such a morphologically complex system arises. Nevertheless errors may arise within a sequence of growth processes which are fundamentally quite simple and not obvious from knowledge of the generalized perfect pattern of connections

Cholinergic Circuits Integrate Neighboring Visual Signals in a Drosophila Motion Detection Pathway

SummaryDetecting motion is a feature of all advanced visual systems [1], nowhere more so than in flying animals, like insects [2, 3]. In flies, an influential autocorrelation model for motion detection, the elementary motion detector circuit (EMD; [4, 5]), compares visual signals from neighboring photoreceptors to derive information on motion direction and velocity. This information is fed by two types of interneuron, L1 and L2, in the first optic neuropile, or lamina, to downstream local motion detectors in columns of the second neuropile, the medulla. Despite receiving carefully matched photoreceptor inputs, L1 and L2 drive distinct, separable pathways responding preferentially to moving “on” and “off” edges, respectively [6, 7]. Our serial electron microscopy (EM) identifies two types of transmedulla (Tm) target neurons, Tm1 and Tm2, that receive apparently matched synaptic inputs from L2. Tm2 neurons also receive inputs from two retinotopically posterior neighboring columns via L4, a third type of lamina neuron. Light microscopy reveals that the connections in these L2/L4/Tm2 circuits are highly determinate. Single-cell transcript profiling suggests that nicotinic acetylcholine receptors mediate transmission within the L2/L4/Tm2 circuits, whereas L1 is apparently glutamatergic. We propose that Tm2 integrates sign-conserving inputs from neighboring columns to mediate the detection of front-to-back motion generated during forward motion

The Synaptic Vesicle SNARE Neuronal Synaptobrevin Promotes Endolysosomal Degradations and Prevents Neurodegeneration

Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-syb (neuronal Synaptobrevin) plays a key role during synaptic vesicle exocytosis. In this paper, we report that loss of n-syb caused slow neurodegeneration independent of its role in neurotransmitter release in adult Drosophila melanogaster photoreceptor neurons. In addition to synaptic vesicles, n-Syb localized to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. Our evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescued n-syb–dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. Our findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity

Drosophila tan Encodes a Novel Hydrolase Required in Pigmentation and Vision

Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-β-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5′ untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function

En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics

Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly’s delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging

Activity-Independent Prespecification of Synaptic Partners in the Visual Map of Drosophila

SummarySpecifying synaptic partners and regulating synaptic numbers are at least partly activity-dependent processes during visual map formation in all systems investigated to date [1–5]. In Drosophila, six photoreceptors that view the same point in visual space have to be sorted into synaptic modules called cartridges in order to form a visuotopically correct map [6, 7]. Synapse numbers per photoreceptor terminal and cartridge are both precisely regulated [8–10]. However, it is unknown whether an activity-dependent mechanism or a genetically encoded developmental program regulates synapse numbers. We performed a large-scale quantitative ultrastructural analysis of photoreceptor synapses in mutants affecting the generation of electrical potentials (norpA, trp;trpl), neurotransmitter release (hdc, syt), vesicle endocytosis (synj), the trafficking of specific guidance molecules during photoreceptor targeting (sec15), a specific guidance receptor required for visual map formation (Dlar), and 57 other novel synaptic mutants affecting 43 genes. Remarkably, in all these mutants, individual photoreceptors form the correct number of synapses per presynaptic terminal independently of cartridge composition. Hence, our data show that each photoreceptor forms a precise and constant number of afferent synapses independently of neuronal activity and partner accuracy. Our data suggest cell-autonomous control of synapse numbers as part of a developmental program of activity-independent steps that lead to a “hard-wired” visual map in the fly brain

High probability neurotransmitter release sites represent an energy efficient design

At most synapses, the probability of neurotransmitter release from an active zone (PAZ) is low, a design thought to confer many advantages. Yet, high PAZ can also be found at synapses. Speculating that high PAZ confers high energy efficiency, we examined energy efficiency at terminals of two Drosophila motor neurons (MNs) synapsing on the same muscle fiber, but with contrasting average PAZ. Through electrophysiological and ultrastructural measurements we calculated average PAZ for MNSNb/d-Is and MN6-Ib terminals (0.33±0.10 and 0.11±0.02 respectively). Using a miles-per-gallon analogy, we calculated efficiency as the number of glutamate molecules released for each ATP molecule that powers the release and recycling of glutamate and the removal of calcium (Ca2+) and sodium (Na+). Ca2+ and Na+ entry were calculated by microfluorimetry and morphological measurements respectively. Terminals with the highest PAZ release more glutamate but admit less Ca2+ and Na+, supporting the hypothesis that high PAZ confers greater energy efficiency than low PAZ (0.13±0.02 and 0.06±0.01 respectively). In an analytical treatment of parameters that influence efficiency we found that efficiency could be optimized in either terminal by increasing PAZ. Terminals with highest PAZ operate closest to this optimum but are less active and less able to sustain high release rates. Adopting an evolutionary biological perspective, we interpret the persistence of low PAZ release sites at more active terminals to be the result of selection pressures for sustainable neurotransmitter release dominating selection pressures for high energy efficiency

Glutamate, GABA and Acetylcholine Signaling Components in the Lamina of the Drosophila Visual System

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to γ-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABAB type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABAB receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABAA receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in α-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types

A Glial Variant of the Vesicular Monoamine Transporter Is Required To Store Histamine in the Drosophila Visual System

Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems