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

Antibody Labelling of Resilin in Energy Stores for Jumping in Plant Sucking Insects

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

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

A connectome and analysis of the adult Drosophila central brain.

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

Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors.

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

CG9317 is photoreceptor cell-enriched carcinine transporter.

<p>(A-B) Photoreceptor cells express CG9317 at a high level. (A) qPCR experiments show that <i>CG9317</i> expression is enriched in wild-type (wt: <i>w</i>^<i>1118</i>) heads compared with <i>gl</i>^<i>3</i> heads or wild-type bodies. (B) The ratio of <i>CG9317</i> transcript levels versus <i>gpdh</i> transcript levels was determined using quantitative PCR. The mRNA level was normalized to the wild-type body, relative to which the <i>CG9317</i> transcript levels were increased about 43 fold and 5 fold in the heads of wild-type and <i>gl</i>^<i>3</i> mutant flies respectively. Error bars indicate the SD. (C-F) S2 cells transiently expressed (C) mCherry, (D) Ine-mCherry, (E) CG3790-mCherry or (F) CG9317-mCherry. Carcinine was added to the culture medium at a final concentration of 20μm. Cells were labeled with rabbit anti-carcinine (green) and DAPI (blue). The mCherry (red) signal was observed directly. Scale bar, 25μm.</p

Location and functions of Inebriated in the Drosophila eye

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

Loss of CarT affects the histamine, β-alanine, and carcinine contents <i>in vivo</i>.

<p>(A-C) Head histamine, β-alanine, and carcinine contents in the three genotypes indicated. (A-B) The <i>tan</i>^<i>1</i> and <i>cart</i>^<i>1</i> mutants had significantly less histamine and β-alanine than wild-type flies (<i>w</i>^<i>1118</i>). (C) The <i>tan</i>^<i>1</i> mutants had nearly three times as much carcinine as wild-type flies, and <i>cart</i>^<i>1</i> flies only showed a 35% increase in carcinine content. Error bars indicate SD; significant differences between mutant and wt flies were determined using unpaired t-tests (*p < 0.05; ***p < 0.001). (D) Model of the pathway for histamine recycling. After a light stimulus, the photoreceptor cells (PR) release histamine, synthesized by histidine decarboxylase (Hdc), into the synaptic cleft to activate histamine-gated chloride channels (HisClA) on postsynaptic neurons (LMC). The released histamine is quickly removed by an unknown histamine transporter in epithelial glial cells that express Ebony, and is then deactivated by conjugation to β-alanine. The histamine metabolite carcinine is then transported out of epithelial glial cells (Glia) by a second unknown transporter, and back to photoreceptors by means of the CarT transporter at the photoreceptor cell terminals, where carcinine is then hydrolyzed back into histamine by Tan ready to be pumped into synaptic vesicles in preparation for further release.</p

Mutations in <i>cart</i> eliminate “on” and “off” transients in ERG recordings.

<p>(A) Schemes for <i>cart</i> deletion by sgRNA targeting. The organization of the <i>cart</i> locus and the expected structure of the deletion alleles (<i>cart</i>^<i>1</i> and <i>cart</i>^<i>2</i>) are shown. Boxes represent exons, and deep gray boxes represent the coding region. The sgRNA1 and sgRNA2 primer pair was used to generate the <i>cart</i>^<i>1</i> mutation. The sgRNA1 and sgRNA3 primer pair was used to generate the <i>cart</i>^<i>2</i> mutation. The positions of the DNA primers used for PCR (arrows) are indicated. (B) PCR products obtained from successful <i>cart</i> deletion mutants. The agarose gel electrophoresis of PCR products using the primers indicated in (A) (pF and pR) is shown and genomic DNA templates prepared from wt (<i>w</i>^<i>1118</i>), <i>cart</i>^<i>1</i> and <i>cart</i>^<i>2</i> flies. (C) ERG recordings from wt (<i>w</i>^<i>1118</i>), <i>cart</i>^<i>1</i>, <i>cart</i>^<i>2</i> and <i>cart</i>^<i>1</i>; <i>Pcart-cart</i> flies. Flies (~1 d after eclosion) were dark adapted for 1 min and subsequently exposed to a 5s pulse of orange light. (D) Phototaxis assays revealed a difference in behavior between wt and <i>cart</i> mutant flies. Error bars: SD; significant differences between mutant and wt flies were determined using unpaired <i>t</i>-tests (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).</p

Histamine is reduced in <i>cart</i>^<i>1</i> mutant photoreceptor neurons.

<p>Histamine was immunolabeled in horizontal sections of heads from (A-B) wild type (wt: <i>w</i>^<i>1118</i>) and (C-D) <i>cart</i>^<i>1</i> mutant flies. (A) Strong signals in the lamina neuropile (La) and R7/R8 terminals in the distal medulla (Me) were detected in control <i>w</i>^<i>1118</i>. Additional immunolabel also appeared from cells in the lobula (Lo) (arrowhead). (B) The enlarged image of the wt head section in (A). Arrowheads in the lamina and medulla identified histamine positive photoreceptor terminals. (C) Loss of photoreceptor histamine signals and strong signals in lamina marginal glia were detected in <i>cart</i>^<i>1</i> mutant. (D) The enlarged image of the <i>cart</i>^<i>1</i> head section in (C) showing labeled marginal glia (arrow) but no photoreceptor signals. Scale bars: 50μm</p