Olfactory receptor and circuit evolution promote host specialization.

The evolution of animal behaviour is poorly understood1,2. Despite numerous correlations between interspecific divergence in behaviour and nervous system structure and function, demonstrations of the genetic basis of these behavioural differences remain rare3-5. Here we develop a neurogenetic model, Drosophila sechellia, a species that displays marked differences in behaviour compared to its close cousin Drosophila melanogaster6,7, which are linked to its extreme specialization on noni fruit (Morinda citrifolia)8-16. Using calcium imaging, we identify olfactory pathways in D. sechellia that detect volatiles emitted by the noni host. Our mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni, and our cross-species allele-transfer experiments demonstrate that the tuning of one of these receptors is important for species-specific host-seeking. We identify the molecular determinants of this functional change, and characterize their evolutionary origin and behavioural importance. We perform circuit tracing in the D. sechellia brain, and find that receptor adaptations are accompanied by increased sensory pooling onto interneurons as well as species-specific central projection patterns. This work reveals an accumulation of molecular, physiological and anatomical traits that are linked to behavioural divergence between species, and defines a model for investigating speciation and the evolution of the nervous system.

Functional integration of "undead" neurons in the olfactory system.

Programmed cell death (PCD) is widespread during neurodevelopment, eliminating the surpluses of neuronal production. Using the Drosophila olfactory system, we examined the potential of cells fated to die to contribute to circuit evolution. Inhibition of PCD is sufficient to generate new cells that express neural markers and exhibit odor-evoked activity. These "undead" neurons express a subset of olfactory receptors that is enriched for relatively recent receptor duplicates and includes some normally found in different chemosensory organs and life stages. Moreover, undead neuron axons integrate into the olfactory circuitry in the brain, forming novel receptor/glomerular couplings. Comparison of homologous olfactory lineages across drosophilids reveals natural examples of fate change from death to a functional neuron. Last, we provide evidence that PCD contributes to evolutionary differences in carbon dioxide-sensing circuit formation in Drosophila and mosquitoes. These results reveal the remarkable potential of alterations in PCD patterning to evolve new neural pathways.

Ionotropic Receptors Specify the Morphogenesis of Phasic Sensors Controlling Rapid Thermal Preference in Drosophila.

Thermosensation is critical for avoiding thermal extremes and regulating body temperature. While thermosensors activated by noxious temperatures respond to hot or cold, many innocuous thermosensors exhibit robust baseline activity and lack discrete temperature thresholds, suggesting they are not simply warm and cool detectors. Here, we investigate how the aristal Cold Cells encode innocuous temperatures in Drosophila. We find they are not cold sensors but cooling-activated and warming-inhibited phasic thermosensors that operate similarly at warm and cool temperatures; we propose renaming them "Cooling Cells." Unexpectedly, Cooling Cell thermosensing does not require the previously reported Brivido Transient Receptor Potential (TRP) channels. Instead, three Ionotropic Receptors (IRs), IR21a, IR25a, and IR93a, specify both the unique structure of Cooling Cell cilia endings and their thermosensitivity. Behaviorally, Cooling Cells promote both warm and cool avoidance. These findings reveal a morphogenetic role for IRs and demonstrate the central role of phasic thermosensing in innocuous thermosensation. VIDEO ABSTRACT.

Ionotropic Receptor-dependent moist and dry cells control hygrosensation in Drosophila.

Insects use hygrosensation (humidity sensing) to avoid desiccation and, in vectors such as mosquitoes, to locate vertebrate hosts. Sensory neurons activated by either dry or moist air ('dry cells' and 'moist cells') have been described in many insects, but their behavioral roles and the molecular basis of their hygrosensitivity remain unclear. We recently reported that Drosophila hygrosensation relies on three Ionotropic Receptors (IRs) required for dry cell function: IR25a, IR93a and IR40a (Knecht et al., 2016). Here, we discover Drosophila moist cells and show that they require IR25a and IR93a together with IR68a, a conserved, but orphan IR. Both IR68a- and IR40a-dependent pathways drive hygrosensory behavior: each is important for dry-seeking by hydrated flies and together they underlie moist-seeking by dehydrated flies. These studies reveal that humidity sensing in Drosophila, and likely other insects, involves the combined activity of two molecularly related but neuronally distinct hygrosensing systems.

Evolution of Acid-Sensing Olfactory Circuits in Drosophilids.

Animals adapt their behaviors to specific ecological niches, but the genetic and cellular basis of nervous system evolution is poorly understood. We have compared the olfactory circuits of the specialist Drosophila sechellia-which feeds exclusively on Morinda citrifolia fruit-with its generalist cousins D. melanogaster and D. simulans. We show that D. sechellia exhibits derived odor-evoked attraction and physiological sensitivity to the abundant Morinda volatile hexanoic acid and characterize how the responsible sensory receptor (the variant ionotropic glutamate receptor IR75b) and attraction-mediating circuit have evolved. A single amino acid change in IR75b is sufficient to recode it as a hexanoic acid detector. Expanded representation of this sensory pathway in the brain relies on additional changes in the IR75b promoter and trans-acting loci. By contrast, higher-order circuit adaptations are not apparent, suggesting conserved central processing. Our work links olfactory ecology to structural and regulatory genetic changes influencing nervous system anatomy and function.

Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila.

Ionotropic Receptors (IRs) are a large subfamily of variant ionotropic glutamate receptors present across Protostomia. While these receptors are most extensively studied for their roles in chemosensory detection, recent work has implicated two family members, IR21a and IR25a, in thermosensation in Drosophila. Here we characterize one of the most evolutionarily deeply conserved receptors, IR93a, and show that it is co-expressed and functions with IR21a and IR25a to mediate physiological and behavioral responses to cool temperatures. IR93a is also co-expressed with IR25a and a distinct receptor, IR40a, in a discrete population of sensory neurons in the sacculus, a multi-chambered pocket within the antenna. We demonstrate that this combination of receptors is required for neuronal responses to dry air and behavioral discrimination of humidity differences. Our results identify IR93a as a common component of molecularly and cellularly distinct IR pathways important for thermosensation and hygrosensation in insects.

Weaker ligands can dominate an odor blend due to syntopic interactions.

Most odors in natural environments are mixtures of several compounds. Perceptually, these can blend into a new "perfume," or some components may dominate as elements of the mixture. In order to understand such mixture interactions, it is necessary to study the events at the olfactory periphery, down to the level of single-odorant receptor cells. Does a strong ligand present at a low concentration outweigh the effect of weak ligands present at high concentrations? We used the fruit fly receptor dOr22a and a banana-like odor mixture as a model system. We show that an intermediate ligand at an intermediate concentration alone elicits the neuron's blend response, despite the presence of both weaker ligands at higher concentration, and of better ligands at lower concentration in the mixture. Because all of these components, when given alone, elicited significant responses, this reveals specific mixture processing already at the periphery. By measuring complete dose-response curves we show that these mixture effects can be fully explained by a model of syntopic interaction at a single-receptor binding site. Our data have important implications for how odor mixtures are processed in general, and what preprocessing occurs before the information reaches the brain.

Calcium imaging of odor-evoked responses in the Drosophila antennal lobe.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions. Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP and Cameleon, the bioluminescent calcium indicator GFP-aequorin, or a reporter of synaptic transmission, synapto-pHluorin has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS, LexA/LexAop, Q system) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed. Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems.

To sense myriad environmental odors, animals have evolved multiple, large families of divergent olfactory receptors. How and why distinct receptor repertoires and their associated circuits are functionally and anatomically integrated is essentially unknown. We have addressed these questions through comprehensive comparative analysis of the Drosophila olfactory subsystems that express the ionotropic receptors (IRs) and odorant receptors (ORs). We identify ligands for most IR neuron classes, revealing their specificity for select amines and acids, which complements the broader tuning of ORs for esters and alcohols. IR and OR sensory neurons exhibit glomerular convergence in segregated, although interconnected, zones of the primary olfactory center, but these circuits are extensively interdigitated in higher brain regions. Consistently, behavioral responses to odors arise from an interplay between IR- and OR-dependent pathways. We integrate knowledge on the different phylogenetic and developmental properties of these receptors and circuits to propose models for the functional contributions and evolution of these distinct olfactory subsystems.

Mind the gap: olfactory trace conditioning in honeybees.

Trace conditioning is a form of classical conditioning, where a neutral stimulus (conditioned stimulus, CS) is associated with a following appetitive or aversive stimulus (unconditioned stimulus, US). Unlike classical delay conditioning, in trace conditioning there is a stimulus-free gap between CS and US, and thus a poststimulus neural representation (trace) of the CS is required to bridge the gap until its association with the US. The properties of such stimulus traces are not well understood, nor are their underlying physiological mechanisms. Using behavioral and physiological approaches, we studied appetitive olfactory trace conditioning in honeybees. We found that single-odor presentation created a trace containing information about odor identity. This trace conveyed odor information about the initial stimulus and was robust against interference by other odors. Memory acquisition decreased with increasing CS-US gap length. The maximum learnable CS-US gap length could be extended by previous trace-conditioning experience. Furthermore, acquisition improved when an additional odor was presented during the CS-US gap. Using calcium imaging, we tested whether projection neurons in the primary olfactory brain area, the antennal lobe, contain a CS trace. We found odor-specific persistent responses after stimulus offset. These post-odor responses, however, did not encode the CS trace, and perceived odor quality could be predicted by the initial but not by the post-odor response. Our data suggest that olfactory trace conditioning is a less reflexive form of learning than classical delay conditioning, indicating that odor traces might involve higher-level cognitive processes.

Olfactory information processing in the Drosophila antennal lobe: anything goes?

When an animal smells an odor, olfactory sensory neurons generate an activity pattern across olfactory glomeruli of the first sensory neuropil, the insect antennal lobe or the vertebrate olfactory bulb. Here, several networks of local neurons interact with sensory neurons and with output neurons--insect projection neurons, or vertebrate mitral/tufted cells. The extent and form of information processing taking place in these local networks has been subject of controversy. To investigate the role of local neurons in odor information processing we have used the calcium sensor G-CaMP to perform in vivo recordings of odor-evoked spatiotemporal activity patterns in five genetically defined neuron populations of the antennal lobe of Drosophila melanogaster: three distinct populations of local neurons (two GABAergic and one cholinergic), as well as sensory neurons and projection neurons. Odor-specific and concentration dependent spatiotemporal response patterns varied among neuron populations. Activity transfer differed along the olfactory pathway for different glomerulus-odor combinations: we found cases of profile broadening and of linear and complex transfer. Moreover, the discriminability between the odors also varied across neuron populations and was maximal in projection neurons. Discriminatory power increased with higher odor concentrations over a wide dynamic range, but decreased at the highest concentration. These results show the complexity and diversity of odor information processing mechanisms across olfactory glomeruli in the fly antennal lobe.

Processing of odor mixtures in the Drosophila antennal lobe reveals both global inhibition and glomerulus-specific interactions.

To understand how odor information is represented and processed in the antennal lobe (AL) of Drosophila melanogaster, we have optically recorded glomerular calcium responses to single odors and odor mixtures from olfactory sensory neurons (OSNs) and projection neurons (PNs). Odor mixtures offer a good tool to analyze odor processing because experimental results can be tested against clear predictions. At the level of the OSNs, the representation of odor mixtures could be predicted from the response patterns of the components in most cases. PN responses to mixtures, however, provide evidences of interglomerular inhibition. Application of picrotoxin (PTX), an antagonist of GABA(A)-like receptors, enhanced odor responses, modified their temporal course, and eliminated mixture suppression at the PN level. Our results can be best explained by postulating the existence of at least two local networks in the fly AL: a glomerulus specific network, which includes excitatory and inhibitory connections and a PTX sensitive inhibitory global network that acts on all glomeruli with proportional strength to the global AL input.

Role of histamine as a putative inhibitory transmitter in the honeybee antennal lobe.

BACKGROUND: Odors are represented by specific spatio-temporal activity patterns in the olfactory bulb of vertebrates and its insect analogue, the antennal lobe. In honeybees inhibitory circuits in the AL are involved in the processing of odors to shape afferent odor responses. GABA is known as an inhibitory transmitter in the antennal lobe, but not all interneurons are GABAergic. Therefore we sought to analyze the functional role of the inhibitory transmitter histamine for the processing of odors in the honeybee AL. RESULTS: We optically recorded the representation of odors before, during and after histamine application at the input level (estimated from a compound signal), and at the output level (by selectively measuring the projection neurons). For both, histamine led to a strong and reversible reduction of odor-evoked responses. CONCLUSION: We propose that histamine, in addition to GABA, acts as an inhibitory transmitter in the honeybee AL and is therefore likely to play a role in odor processing.

Direct interaction of serotonin type 3 receptor ligands with recombinant and native alpha 9 alpha 10-containing nicotinic cholinergic receptors.

In the present work, we characterized the effects of serotonin type 3 receptor ligands on recombinant and native alpha 9 alpha 10-containing nicotinic acetylcholine receptors (nAChRs). Our results indicate that the recombinant alpha 9 alpha 10 nAChR shares striking pharmacological properties with 5-HT(3) ligand-gated ion channels. Thus, 5-HT(3) receptor antagonists block ACh-evoked currents in alpha 9 alpha 10-injected Xenopus laevis oocytes with a rank order of potency of tropisetron (IC(50), 70.1 +/- 0.9 nM) > ondansetron (IC(50), 0.6 +/- 0.1 microM) = MDL 72222 (IC(50), 0.7 +/- 0.1 microM). Although serotonin does not elicit responses in alpha 9 alpha 10-injected oocytes, it blocks recombinant alpha 9 alpha 10 receptors in a noncompetitive and voltage-dependent manner (IC(50), 5.4 +/- 0.6 microM). On the other hand, we demonstrate an in vivo correlate of these properties of the recombinant receptor, with those of the alpha 9 alpha 10-containing nAChR of frog saccular hair cells. The possibility that the biogenic amine serotonin might act as a neuromodulator of the cholinergic efferent transmission in the vestibular apparatus and in the organ of Corti is discussed.