Search

Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. The bioenergetic machinery of glycolysis and oxidative phosphorylation is highly responsive to such demands, but it must occupy a minimum volume if it is to accommodate these demands. We examined the trade-off between presynaptic power demands and the volume available to the bioenergetic machinery. We quantified the energy demands of six Drosophila motor nerve terminals through direct measurements of neurotransmitter release and Ca2+ entry, and via theoretical estimates of Na+ entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio (∼66 nmol·min-1·μL-1) harbor the largest mitochondria packed at the greatest density. If we assume sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation, then these mitochondria are required to produce ATP at a rate of 52 nmol·min-1·μL-1 at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol·min-1·μL-1 of cytosol at rest, rising to 4.36. These data provide a quantitative framework for presynaptic bioenergetics in situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand.Significance StatementThe remarkable energy demands of the brain are supported by the complete oxidation of its fuel but debate continues regarding a division of labor between glycolysis and oxidative phosphorylation across different cell types. Here we leverage the neuromuscular synapse, a model for studying neurophysiology, to elucidate fundamental aspects of neuronal energy metabolism that ultimately constrain rates of neural processing. We quantified energy production rates required to sustain activity at individual nerve terminals and compared these data with the volume capable of oxidative phosphorylation (mitochondria) and glycolysis (cytosol). We find strong support for oxidative phosphorylation playing a primary role in presynaptic terminals and provide the first in vivo estimates of energy production rates per unit volume of presynaptic mitochondria and cytosol.

Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. Neurons have presumably optimized the volume of their bioenergetic machinery to ensure these power demands are met, but the relationship between presynaptic power demands and the volume available to the bioenergetic machinery has never been quantified. Here, we estimated the power demands of six motor nerve terminals in femaleDrosophilalarvae through direct measurements of neurotransmitter release and Ca2+entry, and via theoretical estimates of Na+entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio (∼66 nmol·min−1·µl−1) harbor the largest mitochondria packed at the greatest density. If we assume sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation, then these mitochondria are required to produce ATP at a rate of 52 nmol·min−1·µl−1at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol·min−1·µl−1of cytosol at rest, rising to 4.36 during activity. These data provide a quantitative framework for presynaptic bioenergeticsin situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand.SIGNIFICANCE STATEMENTThe remarkable energy demands of the brain are supported by the complete oxidation of its fuel but debate continues regarding a division of labor between glycolysis and oxidative phosphorylation across different cell types. Here, we exploit the neuromuscular synapse, a model for studying neurophysiology, to elucidate fundamental aspects of neuronal energy metabolism that ultimately constrain rates of neural processing. We quantified energy production rates required to sustain activity at individual nerve terminals and compared these with the volume capable of oxidative phosphorylation (mitochondria) and glycolysis (cytosol). We find strong support for oxidative phosphorylation playing a primary role in presynaptic terminals and provide the firstin vivoestimates of energy production rates per unit volume of presynaptic mitochondria and cytosol.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly’s brain., eLife digest Animal brains of all sizes, from the smallest to the largest, work in broadly similar ways. Studying the brain of any one animal in depth can thus reveal the general principles behind the workings of all brains. The fruit fly Drosophila is a popular choice for such research. With about 100,000 neurons – compared to some 86 billion in humans – the fly brain is small enough to study at the level of individual cells. But it nevertheless supports a range of complex behaviors, including navigation, courtship and learning. Thanks to decades of research, scientists now have a good understanding of which parts of the fruit fly brain support particular behaviors. But exactly how they do this is often unclear. This is because previous studies showing the connections between cells only covered small areas of the brain. This is like trying to understand a novel when all you can see is a few isolated paragraphs. To solve this problem, Scheffer, Xu, Januszewski, Lu, Takemura, Hayworth, Huang, Shinomiya et al. prepared the first complete map of the entire central region of the fruit fly brain. The central brain consists of approximately 25,000 neurons and around 20 million connections. To prepare the map – or connectome – the brain was cut into very thin 8nm slices and photographed with an electron microscope. A three-dimensional map of the neurons and connections in the brain was then reconstructed from these images using machine learning algorithms. Finally, Scheffer et al. used the new connectome to obtain further insights into the circuits that support specific fruit fly behaviors. The central brain connectome is freely available online for anyone to access. When used in combination with existing methods, the map will make it easier to understand how the fly brain works, and how and why it can fail to work correctly. Many of these findings will likely apply to larger brains, including our own. In the long run, studying the fly connectome may therefore lead to a better understanding of the human brain and its disorders. Performing a similar analysis on the brain of a small mammal, by scaling up the methods here, will be a likely next step along this path.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly’s brain.

The neural circuits responsible for behavior remain largely unknown. Previous efforts have reconstructed the complete circuits of small animals, with hundreds of neurons, and selected circuits for larger animals. Here we (the FlyEM project at Janelia and collaborators at Google) summarize new methods and present the complete circuitry of a large fraction of the brain of a much more complex animal, the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses, and proofread such large data sets; new methods that define cell types based on connectivity in addition to morphology; and new methods to simplify access to a large and evolving data set. From the resulting data we derive a better definition of computational compartments and their connections; an exhaustive atlas of cell examples and types, many of them novel; detailed circuits for most of the central brain; and exploration of the statistics and structure of different brain compartments, and the brain as a whole. We make the data public, with a web site and resources specifically designed to make it easy to explore, for all levels of expertise from the expert to the merely curious. The public availability of these data, and the simplified means to access it, dramatically reduces the effort needed to answer typical circuit questions, such as the identity of upstream and downstream neural partners, the circuitry of brain regions, and to link the neurons defined by our analysis with genetic reagents that can be used to study their functions.Note: In the next few weeks, we will release a series of papers with more involved discussions. One paper will detail the hemibrain reconstruction with more extensive analysis and interpretation made possible by this dense connectome. Another paper will explore the central complex, a brain region involved in navigation, motor control, and sleep. A final paper will present insights from the mushroom body, a center of multimodal associative learning in the fly brain.

The dogma that the synaptic cleft acidifies during neurotransmission is based on the corelease of neurotransmitters and protons from synaptic vesicles, and is supported by direct data from sensory ribbon-type synapses. However, it is unclear whether acidification occurs at non–ribbon-type synapses. Here we used genetically encoded fluorescent pH indicators to examine cleft pH at conventional neuronal synapses. At the neuromuscular junction of femaleDrosophilalarvae, we observed alkaline spikes of over 1 log unit during fictive locomotionin vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only ∼0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca2+movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca2+/H+antiporting activity of the plasma membrane Ca2+-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage gated Ca2+channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extrasynaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non–ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses.SIGNIFICANCE STATEMENTNeurotransmission is highly sensitive to the pH of the extracellular milieu. This is readily evident in the neurological symptoms that accompany systemic acid/base imbalances. Imaging data from sensory ribbon-type synapses show that neurotransmission itself can acidify the synaptic cleft, likely due to the corelease of protons and glutamate. It is not clear whether the same phenomenon occurs at conventional neuronal synapses due to the difficulties in collecting such data. If it does occur, it would provide for an additional layer of activity-dependent modulation of neurotransmission. Our findings of alkalinization, rather than acidification, within the cleft of two different neuronal synapses encourages a reassessment of the scope of activity-dependent pH influences on neurotransmission and short-term synaptic plasticity.

Nerve terminals contain multiple sites specialized for the release of neurotransmitters. Release usually occurs with low probability, a design thought to confer many advantages. High-probability release sites are not uncommon, but their advantages are not well understood. Here, we test the hypothesis that high-probability release sites represent an energy-efficient design. We examined release site probabilities and energy efficiency at the terminals of two glutamatergic motor neurons synapsing on the same muscle fiber in Drosophila larvae. Through electrophysiological and ultrastructural measurements, we calculated release site probabilities to differ considerably between terminals (0.33 versus 0.11). We estimated the energy required to release and recycle glutamate from the same measurements. The energy required to remove calcium and sodium ions subsequent to nerve excitation was estimated through microfluorimetric and morphological measurements. We calculated energy efficiency as the number of glutamate molecules released per ATP molecule hydrolyzed, and high-probability release site terminals were found to be more efficient (0.13 versus 0.06). Our analytical model indicates that energy efficiency is optimal (∼0.15) at high release site probabilities (∼0.76). As limitations in energy supply constrain neural function, high-probability release sites might ameliorate such constraints by demanding less energy. Energy efficiency can be viewed as one aspect of nerve terminal function, in balance with others, because high-efficiency terminals depress significantly during episodic bursts of activity.

SUMMARYThe ability of neurons to distinguish appropriate from inappropriate synaptic partners in their local environment is fundamental to the proper assembly and function of neural circuits. How synaptic partner selection is regulated is a longstanding question in Neurobiology. A prevailing hypothesis is that appropriate partners express complementary molecules that match them together and promote synaptogenesis. Dpr and DIP IgSF proteins bind heterophilically and are expressed in a complementary manner between synaptic partners in the Drosophila visual system. Here, we show that in the lamina, DIP mis-expression is sufficient to promote synapse formation with Dpr-expressing neurons, and that DIP proteins are not necessary for synaptogenesis but rather function to prevent ectopic synapse formation. These findings indicate that Dpr-DIP interactions regulate synaptic specificity by biasing synapse formation towards specific cell-types. We propose that synaptogenesis occurs independent of synaptic partner choice, and that precise synaptic connectivity is established by limiting promiscuous synapse formation.

Histamine (HA) is a neurotransmitter in arthropod photoreceptors. It is recycled via conjugation to β-alanine to form β-alanylhistamine (carcinine). Conjugation occurs in epithelial glia that surround photoreceptor terminals in the first optic neuropil, and carcinine (CA) is then transported back to photoreceptors and cleaved to liberate HA and β-alanine. The gene Inebriated (Ine) encodes an Na+/Cl−-dependent SLC6 family transporter translated as two protein isoforms, long (P1) and short (P2). Photoreceptors specifically express Ine-P2 whereas Ine-P1 is expressed in non-neuronal cells. Both ine1 and ine3 have significantly reduced head HA contents compared with wild type, and a smaller increase in head HA after drinking 1% CA. Similarly, uptake of 0.1% CA was reduced in ine1 and ine3 mutant synaptosomes, but increased by 90% and 84% respectively for fractions incubated in 0.05% β-Ala, compared with wild type. Screening potential substrates in Ine expressing Xenopus oocytes revealed very little response to carcinine and β-Ala but increased conductance with glycine. Both ine1 and ine3 mutant responses in light-dark phototaxis did not differ from wild-type. Collectively our results suggest that Inebriated functions in an adjunct role as a transporter to the previously reported carcinine transporter CarT., Summary: Inebriated isoforms express in alternating glial layers of the Drosophila visual system, consistent with a role in histamine recycling in the eye, and its mutants exhibit blue light phototaxis defects.

Summary Background Neuronal circuits in worms, flies, and mammals are organized so as to minimize wiring length for a functional number of synaptic connections, a phenomenon called wiring optimization. However, the molecular mechanisms that establish optimal wiring during development are unknown. We addressed this question by studying the role of N-cadherin in the development of optimally wired neurite fascicles in the peripheral visual system of Drosophila . Results Photoreceptor axons surround the dendrites of their postsynaptic targets, called lamina cells, within a concentric fascicle called a cartridge. N-cadherin is expressed at higher levels in lamina cells than in photoreceptors, and all genetic manipulations that invert these relative differences displace lamina cells to the periphery and relocate photoreceptor axon terminals into the center. Conclusions Differential expression of a single cadherin is both necessary and sufficient to determine cartridge structure because it positions the most-adhesive elements that make the most synapses at the core and the less-adhesive elements that make fewer synapses at the periphery. These results suggest a general model by which differential adhesion can be utilized to determine the relative positions of axons and dendrites to establish optimal wiring.

SUMMARYFlies recycle the photoreceptor neurotransmitter histamine by conjugating it to β-alanine to form β-alanyl-histamine (carcinine). The conjugation is regulated by Ebony, while Tan hydrolyses carcinine, releasing histamine and β-alanine. In Drosophila, β-alanine synthesis occurs either from uracil or from the decarboxylation of aspartate but detailed roles for the enzymes responsible remain unclear. Immunohistochemically detected β-alanine is present throughout the fly’s entire brain, and is enhanced in the retina especially in the pseudocone, pigment and photoreceptor cells of the ommatidia. HPLC determinations reveal 10.7 ng of β-alanine in the wild-type head, roughly five times more than histamine. When wild-type flies drink uracil their head β-alanine increases more than after drinking l-aspartic acid, indicating the effectiveness of the uracil pathway. Mutants of black, which lack aspartate decarboxylase, cannot synthesize β-alanine from l-aspartate but can still synthesize it efficiently from uracil. Our findings demonstrate a novel function for pigment cells, which not only screen ommatidia from stray light but also store and transport β-alanine and carcinine. This role is consistent with a β-alanine-dependent histamine recycling pathway occurring not only in the photoreceptor terminals in the lamina neuropile, where carcinine occurs in marginal glia, but vertically via a long pathway that involves the retina. The lamina’s marginal glia are also a hub involved in the storage and/or disposal of carcinine and β-alanine.

Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to β-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals., Author Summary Neurotransmitter transporters that remove neurotransmitters and recycle them after their release have particular importance at visual synapses, which must signal at high frequencies and therefore required rapid clearance of neurotransmitters from the synaptic cleft. In this study, we identified a SLC22 family transporter, CarT, in the visual system of Drosophila, which is exclusively located to photoreceptor terminals in the lamina neuropil and is responsible for taking up carcinine, an inactive histamine metabolite, from surrounding glia. Loss of CarT disrupts the regeneration of histamine and blocks neurotransmission at photoreceptor cell synapses. Our work provides direct evidence for a local histamine recycling pathway between glial cells and photoreceptor terminals, and shows that a CarT-dependent histamine/carcinine shuttle pathway is critical for maintaining the normal histamine content of neurons.

In the fly's visual system, two classes of lamina interneuron, L1 and L2, cyclically change both their size and shape in a rhythm that is circadian. Several neurotransmitters and the lamina's glial cells are known to be involved in regulating these rhythms. Moreover, vacuolar-type H + —ATPase (V-ATPase) in the optic lobe is thought also to participate in such regulation. We have detected V-ATPase-like immunoreactivity in the heads of both Drosophilla melanogaster and Musca domestica using antibodies raised against either the B- or H-subunits of V-ATPase from D. melanogaster or against the B-subunit from two other insect species Culex quinquefasciatus and Manduca sexta . In the visual systems of both fly species V-ATPase was localized immunocytochemically to the compound eye photoreceptors. In D. melanogaster immunoreactivity oscillated during the day and night and under constant darkness the signal was stronger during the subjective night than the subjective day. In turn, blocking V-ATPase by injecting a V-ATPase blocker, bafilomycin, in M. domestica increased the axon sizes of L1 and L2, but only when bafilomycin was applied during the night. As a result bafilomycin abolished the day/night difference in axon size in L1 and L2, their sizes being similar during the day and night.

Photoreceptors are richly supplied with mitochondria, where they are required to meet the energetic demands, in the soma, of phototransduction and, in the terminal, of neurotransmitter release. Compromising the latter, we have made photoreceptors R1-R6 in Drosophila ommatidia homozygous for either of two alleles, milt(186) and milt(92), of milton in whole-eye mosaics. Such mutant photoreceptors fail to target mitochondria to their terminals. We show from quantitative electron microscopy (EM) that mitochondria are totally lacking at the terminal but nevertheless abundant and present throughout the soma, where their distribution differs from that of control ommatidia, however, being more heavily concentrated in the nuclear region. Mitochondria are sparse at the basalmost level of mutant ommatidia, and are lacking beneath the basement membrane, in the axons and terminals of these cells. The absence of mitochondria from R1-R6 terminals and concommitant reductions in synaptic vesicle packing density, previously reported, we show here are accompanied by reduced immunoreactivity to the photoreceptor transmitter histamine but not by any change in total head histamine content, as determined by high-performance liquid chromatography. Mutant terminals also contain vesicle profiles with a wider range of sizes. These two phenotypes suggest that the reduced availability of ATP when mutant terminals lack a mitochondrial supply compromises their ability to pump histamine into synaptic vesicles and perturbs membrane distribution within the terminal. In addition, a band of somata in the lamina cortex, at least some of which are postsynaptic neurons not homozygous for milton, also shows altered mitochondrial targeting, with abnormal clusters of mitochondria, as visualized by immunolabeling with anti-hsp and by serial EM. Within the lamina, terminals of mutant photoreceptors are penetrated by neighboring cells with invaginations that frequently contain mitochondria, suggesting that a mechanism exists for intercellular metabolic support. Our findings indicate the direct and compensatory responses in a population of neurons when mitochondria are not correctly targeted to their synaptic terminals.

In Drosophila melanogaster, ebony and tan, two cuticle melanizing mutants, regulate the conjugation (ebony) of beta-alanine to dopamine or hydrolysis (tan) of the beta-alanyl conjugate to liberate dopamine. beta-alanine biosynthesis is regulated by black. ebony and tan also exert unexplained reciprocal defects in the electroretinogram, at ON and OFF transients attributable to impaired transmission at photoreceptor synapses, which liberate histamine. Compatible with this impairment, we show that both mutants have reduced histamine contents in the head, as measured by HPLC, and have correspondingly reduced numbers of synaptic vesicles in their photoreceptor terminals. Thus, the histamine phenotype is associated with sites of synaptic transmission at photoreceptors. We demonstrate that when they receive microinjections into the head, wild-type Sarcophaga bullata (in whose larger head such injections are routinely possible) rapidly (5 sec) convert exogenous [3H]histamine into its beta-alanine conjugate, carcinine, a novel metabolite. Drosophila tan has an increased quantity of [3H]carcinine, the hydrolysis of which is blocked; ebony lacks [3H]carcinine, which it cannot synthesize. Confirming these actions, carcinine rescues the histamine phenotype of ebony, whereas beta-alanine rescues the carcinine phenotype of black;tan double mutants. The equilibrium ratio between [3H]carcinine and [3H]histamine after microinjecting wild-type Sarcophaga favors carcinine hydrolysis, increasing to only 0.5 after 30 min. Our findings help resolve a longstanding conundrum of the involvement of tan and ebony in photoreceptor function. We suggest that reversible synthesis of carcinine occurs in surrounding glia, serving to trap histamine after its release at photoreceptor synapses; subsequent hydrolysis liberates histamine for reuptake.

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., Author Summary Neurons, the cells in the brain responsible for carrying information, communicate with each other using a class of chemicals known as neurotransmitters. One family of neurotransmitters, the monoamines, includes dopamine, serotonin, and histamine, all of which play major physiological roles. However, unlike dopamine and serotonin, the regulation of the brain's histamine content is poorly understood. We are using the fruitfly Drosophila melanogaster to study the storage and release of histamine from brain cells. Both mammals and insects use a class of proteins called transporters to store amines, but, to date, amine transporters have been thought to be restricted to neurons. We have found that the support cells, or glia, that facilitate the function of neurons in the fly's visual system contain a new form of monoamine transporter. Despite its circumscribed distribution, this protein is required to maintain normal levels of histamine throughout the visual system. We speculate that other animals may use a similar strategy to regulate the function of this important neurotransmitter.

SUMMARYMonoamines such as dopamine, histamine and serotonin (5-HT) are widely distributed throughout the brain of the fruit fly Drosophila melanogaster, where many of their actions have been investigated. For example, histamine is released from photoreceptor synapses in the lamina neuropile of the visual system. Mutations of the genes white, an important eye pigmentation marker in fly genetics that encodes an ABC transporter, and its binding partner brown, cause neural phenotypes not readily reconciled solely with actions in eye pigmentation. We find that flies mutant for these genes, and another binding partner, scarlet, have about half the wild-type amount of histamine in the head, as well as reduced 5-HT and dopamine. These differences parallel reductions in immunoreactivity to the corresponding biogenic amines. They also correlate with the amine content of fractions after differential centrifugation of head homogenates. Thus, most of the amine is found in the vesicle-rich fraction of wild-type head homogenates, whereas it is found in the supernatant fractions from white, brown and scarlet flies. White co-expresses in lamina epithelial glia with Ebony, which conjugates histamine to β-alanine. Histamine is then released when the conjugate is hydrolyzed in photoreceptors, by Tan. Mutant white ameliorates the effects of tan on head histamine whereas it exacerbates the effects of ebony. Our results are consistent with the proposal that histamine uptake by the epithelial glia may be white dependent. Behavioral abnormalities in white, brown and scarlet mutants could arise because aminergic neurons in the Drosophila brain have reduced amine for release.

Histamine, a ubiquitous aminergic messenger throughout the body, also serves as a neurotransmitter in both vertebrates and invertebrates. In particular, the photoreceptors of adult arthropods use histamine, modulating its release to signal increases and decreases in light intensity. Strong evidence from various arthropod species indicates that histamine is synthesized and stored in photoreceptors, undergoes Ca-dependent release, inhibits postsynaptic interneurons by gating Cl channels, and is then recycled. In Drosophila, the synthetic enzyme, histidine decarboxylase, and the subunits of the histamine-gated chloride channel have been cloned. Possible histamine transporters at synaptic vesicles and for reuptake remain elusive. Indeed, the mechanisms that remove histamine from the synaptic cleft, and that help terminate histamine's action, are unexpectedly complex, their details remaining unresolved. A major pathway in Drosophila, and possibly other arthropod species, is by conjugation of histamine to beta-alanine to form carcinine in adjacent glia. This conjugate then returns to the photoreceptors where it is hydrolysed to liberate histamine, which is then loaded into synaptic vesicles. Evidence from other species suggests that direct reuptake of histamine into the photoreceptors may also occur. Light depolarizes the photoreceptors, causing histamine release and postsynaptic inhibition; dimming hyperpolarizes the photoreceptors, causing a decrease in histamine release and an "off" response in the postsynaptic cell. Further pursuit of histamine's action at these highly specialized synapses should lead to an understanding of how they signal minute changes in presynaptic membrane potential, how they reliably extract signals from noise, and how they adapt to a wide range of presynaptic membrane potentials.

Reliable estimates of the quantum size in histaminergic neurons are not available. We have exploited two unusual opportunities in the fly's (Drosophila melanogaster) visual system to make such determinations for histaminergic photoreceptor synapses: 1) the possibility to microdissect successively from whole fly heads freeze-dried in acetone: the compound eyes; the first optic neuropils, or lamina; and the rest of the brain; and 2) the uniform sheaves of lamina synaptic terminals of photoreceptors R1-R6. We used this organization to count scrupulously the numbers of 30-nm synaptic vesicles from electron micrographs of R1-R6 profiles, and from microdissections we determined the regional contents of histamine in the compound eye, lamina, and central brain. Total head histamine averages 1.98 ng of which 9% was lost after freeze-drying in acetone and a further 28% after the brain was microdissected. Of the remainder, 71% was in the eye and lamina. Assuming that histamine loss from the tissue occurred mostly by diffusion evenly distributed among all regions, the overall lamina content of the head would be 0.1935 ng before dissection. From published values for the volumes of the brain's compartments, the computed regional concentrations of histamine are highest in the lamina (4.35 mM) because of the terminals of R1-R6. The concentration in the retina is approximately 13% that in the lamina, suggesting that most histamine is vesicular. There are approximately 43,500 +/- 7,400 (SD) synaptic vesicles per terminal and, if all histamine is allocated equally and exclusively among these, the vesicle contents would be 858 +/- 304 x 10(-21) moles or approximately 5,000 +/- 1,800 (SD) molecules at an approximate concentration of 670 mM. These values are compared with the vesicle contents at synapses using acetylcholine and catecholamines.

The rubbery protein resilin appears to form an integral part of the energy storage structures that enable many insects to jump by using a catapult mechanism. In plant sucking bugs that jump (Hemiptera, Auchenorrhyncha), the energy generated by the slow contractions of huge thoracic jumping muscles is stored by bending composite bow-shaped parts of the internal thoracic skeleton. Sudden recoil of these bows powers the rapid and simultaneous movements of both hind legs that in turn propel a jump. Until now, identification of resilin at these storage sites has depended exclusively upon characteristics that may not be specific: its fluorescence when illuminated with specific wavelengths of ultraviolet (UV) light and extinction of that fluorescence at low pH. To consolidate identification we have labelled the cuticular structures involved with an antibody raised against a product of the Drosophila CG15920 gene. This encodes pro-resilin, the first exon of which was expressed in E. coli and used to raise the antibody. We show that in frozen sections from two species, the antibody labels precisely those parts of the metathoracic energy stores that fluoresce under UV illumination. The presence of resilin in these insects is thus now further supported by a molecular criterion that is immunohistochemically specific.