Non-canonical odor coding in the mosquito

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor cod-ing. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demon-strates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes

An analysis of candidate ionotropic receptors mutations that block synaptic homeostasis: PPK11 and PPK16 drive homeostatic synaptic plasticity.

Homeostatic plasticity restricts changes in neural activity to allow for a stable yet plastic nervous system. At the synapse, homeostatic regulation maintains synaptic efficacy when there is a disruption of postsynaptic neurotransmitter sensitivity or excitability (Turrigiano, 2012; Davis, 2006). At the Drosophila neuromuscular junction (NMJ), when glutamate receptor sensitivity is decreased, either genetically or pharmacologically, the motoneuron potentiates neurotransmitter release to precisely offset changes in receptor sensitivity. This restores muscle excitation within minutes. The molecular mechanisms that underlie this form of synaptic homeostasis are beginning to be discovered, in large part due to an ongoing forward genetic screen for mutations that block synaptic homeostasis (Dickman and Davis, 2009; Müller et al., 2011). This thesis work focuses on the examination of two candidate mutations identified by this genetic screen. These were chosen for study because they completely blocked synaptic homeostasis and were likely to disrupt genes encoding ion channel subunits. In chapter 2 we provide evidence that two Degenerin/Epithelial Sodium Channel (DEG/ENaC) subunits, pickpocket11 (ppk11) and pickpocket16 (ppk16), are necessary for synaptic homeostasis. These genes are required for both the rapid induction and sustained expression of synaptic homeostasis. Mutations in ppk11 and ppk16 do not disrupt baseline synaptic transmission or NMJ development. ppk11 and ppk16 reside in a single locus and are coregulated as an operon-like genetic unit, with increased transcription accompanying prolonged expression of synaptic homeostasis. We use pharmacology to show that DEG/ENaC channel conductance is continuously required for the potentiation of release during homeostasis. Lastly we show that DEG/ENaC channel function is necessary to enhance action-potential evoked presynaptic calcium influx specifically during synaptic homeostasis, but not baseline release. Taken together this identifies PPK11 and PPK16 as components of the molecular pathway that potentiates release during synaptic homeostasis and we present a model for how PPK11 and PPK16 enhance calcium influx during synaptic homeostasis. In chapter 3 we examine a third gene encoding a DEG/ENaC channel subunit, pickpocket 18 (ppk18), which is in the same genetic locus as ppk11 and ppk16. We found that despite the proximity to ppk11 and ppk16, ppk18 is transcribed independently from the other two genes, and the quantity of ppk18 transcript is unchanged during the sustained expression of synaptic homeostasis. Furthermore, mutations in ppk18 do not block the rapid induction of synaptic homeostasis, indicating that it does not act in concert with ppk11 and ppk16 to potentiate release during synaptic homeostasis. In chapter 4 we examine a separate mutation that was identified in the same forward genetic screen for mutations that block synaptic homeostasis. This mutation resides in a complicated locus containing four genes: Ionotropic Receptor 75a (IR75a), Ionotropic Receptor 75b (IR75b), Ionotropic Receptor 75c (IR75c), and GABA and glycine-like receptor of Drosophila (GRD). We use a combination of electrophysiology, quantitative PCR, immunostaining and genetics to examine the requirement of different genes in this locus for the homeostatic potentiation of release. We show that multiple mutations within this genomic region block synaptic homeostasis, and that the distribution of postsynaptic glutamate receptors is altered by a mutation within this locus, which may be an underlying cause. We are unable to identify the responsible gene, although our data points to GRD as the most promising candidate. This work provides a basis for future investigation into this locus

A Presynaptic ENaC Channel Drives Homeostatic Plasticity

An electrophysiology-based forward genetic screen has identified two genes, pickpocket11 (ppk11) and pickpocket16 (ppk16), as being necessary for the homeostatic modulation of presynaptic neurotransmitter release at the Drosophila neuromuscular junction (NMJ). Pickpocket genes encode Degenerin/Epithelial Sodium channel subunits (DEG/ENaC). We demonstrate that ppk11 and ppk16 are necessary in presynaptic motoneurons for both the acute induction and long-term maintenance of synaptic homeostasis. We show that ppk11 and ppk16 are cotranscribed as a single mRNA that is upregulated during homeostatic plasticity. Acute pharmacological inhibition of a PPK11- and PPK16-containing channel abolishes the expression of short- and long-term homeostatic plasticity without altering baseline presynaptic neurotransmitter release, indicating remarkable specificity for homeostatic plasticity rather than NMJ development. Finally, presynaptic calcium imaging experiments support a model in which a PPK11- and PPK16-containing DEG/ENaC channel modulates presynaptic membrane voltage and, thereby, controls calcium channel activity to homeostatically regulate neurotransmitter release

Composition and Control of a Deg/ENaC Channel during Presynaptic Homeostatic Plasticity.

The homeostatic control of presynaptic neurotransmitter release stabilizes information transfer at synaptic connections in the nervous system of organisms ranging from insect to human. Presynaptic homeostatic signaling centers upon the regulated membrane insertion of an amiloride-sensitive degenerin/epithelial sodium (Deg/ENaC) channel. Elucidating the subunit composition of this channel is an essential step toward defining the underlying mechanisms of presynaptic homeostatic plasticity (PHP). Here, we demonstrate that the ppk1 gene encodes an essential subunit of this Deg/ENaC channel, functioning in motoneurons for the rapid induction and maintenance of PHP. We provide genetic and biochemical evidence that PPK1 functions together with PPK11 and PPK16 as a presynaptic, hetero-trimeric Deg/ENaC channel. Finally, we highlight tight control of Deg/ENaC channel expression and activity, showing increased PPK1 protein expression during PHP and evidence for signaling mechanisms that fine tune the level of Deg/ENaC activity during PHP

Distinct visual pathways mediate drosophila larval light avoidance and circadian clock entrainment

Visual organs perceive environmental stimuli required for rapid initiation of behaviors and can also entrain the circadian clock. The larval eye of Drosophila is capable of both functions. Each eye contains only 12 photoreceptors (PRs), which can be subdivided into two subtypes. Four PRs express blue-sensitive rhodopsin5 (rh5) and eight express green-sensitive rhodopsin6 (rh6). We found that either PR-subtype is sufficient to entrain the molecular clock by light, while only the Rh5-PR subtype is essential for light avoidance. Acetylcholine released from PRs confers both functions. Both subtypes of larval PRs innervate the main circadian pacemaker neurons of the larva, the neuropeptide PDF (pigment-dispersing factor)-expressing lateral neurons (LNs), providing sensory input to control circadian rhythms. However, we show that PDF-expressing LNs are dispensable for light avoidance, and a distinct set of three clock neurons is required. Thus we have identified distinct sensory and central circuitry regulating light avoidance behavior and clock entrainment. Our findings provide insights into the coding of sensory information for distinct behavioral functions and the underlying molecular and neuronal circuitry

Mosquito brains encode unique features of human odour to drive host seeking

A globally invasive form of the mosquito Aedes aegypti specializes in biting humans, making it an efficient disease vector(1). Host-seeking female mosquitoes strongly prefer human odour over the odour of animals(2,3), but exactly how they distinguish between the two is not known. Vertebrate odours are complex blends of volatile chemicals with many shared components(4-7), making discrimination an interesting sensory coding challenge. Here we show that human and animal odours evoke activity in distinct combinations of olfactory glomeruli within the Ae. aegypti antennal lobe. One glomerulus in particular is strongly activated by human odour but responds weakly, or not at all, to animal odour. This human-sensitive glomerulus is selectively tuned to the long-chain aldehydes decanal and undecanal, which we show are consistently enriched in human odour and which probably originate from unique human skin lipids. Using synthetic blends, we further demonstrate that signalling in the human-sensitive glomerulus significantly enhances long-range host-seeking behaviour in a wind tunnel, recapitulating preference for human over animal odours. Our research suggests that animal brains may distil complex odour stimuli of innate biological relevance into simple neural codes and reveals targets for the design of next-generation mosquito-control strategies