Nonsynaptic Transmission Mediates Light Context-Dependent Odor Responses in Drosophila melanogaster.

Recent connectome analyses of the entire synaptic circuit in the nervous system have provided tremendous insights into how neural processing occurs through the synaptic relay of neural information. Conversely, the extent to which ephaptic transmission which does not depend on the synapses contributes to the relay of neural information, especially beyond a distance between adjacent neurons and to neural processing remains unclear. We show that ephaptic transmission mediated by extracellular potential changes in female Drosophila melanogaster can reach >200 µm, equivalent to the depth of its brain. Furthermore, ephaptic transmission driven by retinal photoreceptor cells mediates light-evoked firing rate increases in olfactory sensory neurons. These results indicate that ephaptic transmission contributes to sensory responses that can change momentarily in a context-dependent manner.SIGNIFICANCE STATEMENT Although extracellular field potential activities are commonly observed in many nervous systems, this activity has been generally considered as a side effect of synchronized spiking of neurons. This study, however, shows that field potential changes in retinae evoked by a sensory stimulus can control the excitability of distant neurons in vivo and mediates multimodal sensory integration in Drosophila melanogaster As such ephaptic transmission is more effective at a short distance, the ephaptic transmission from the retinae may contribute significantly to firing rate changes in downstream neurons of the photoreceptor cells in the optic lobe.

A Sexually Dimorphic Olfactory Neuron Mediates Fixed Action Transition during Courtship Ritual in Drosophila melanogaster.

Animals perform a series of actions in a fixed order during ritualistic innate behaviors. Although command neurons and sensory pathways responding to external stimuli that trigger these behaviors have been identified, how each action is induced in a fixed order in response to multimodal sensory stimuli remains unclear. Here, the sexually dimorphic lateral antennal lobe tract projection neuron 4 (lPN4) in male Drosophila melanogaster mediates the expression of a fixed behavioral action pattern at the beginning of the courtship ritual, in which a male taps a female body and then extends a wing unilaterally to produce a courtship song. We found that blocking the synaptic output of lPN4 caused an increase in the ratio of male flies that extended a wing unilaterally without tapping the female body, whereas excitation of lPN4 suppressed the transition from the tapping phase to the unilateral wing extension phase. Real-time calcium imaging showed that lPN4 is activated by a volatile pheromone, palmitoleic acid, whose responses were inhibited by simultaneous gustatory stimulation with female cuticular hydrocarbons, showing the existence of an "AND-gate" for multimodal sensory inputs during male courtship behaviors. These results suggest that the function of lPN4 is to suppress unilateral wing extension while responding to a female smell, which is released by appropriate contact chemosensory inputs received when tapping a female. As the female smell also promotes male courtship behaviors, the olfactory system is ready to simultaneously promote and suppress the progress of courtship actions while responding to a female smell.SIGNIFICANCE STATEMENT Although it has been 80 years since Konrad Lorenz and Niko Tinbergen introduced how multiple acts comprising separate innate behaviors are released in a fixed order according to external stimuli, the neural circuits responsible for such fixed action patterns remain largely unknown. The male courtship behavior of Drosophila melanogaster is a good model to investigate how such a fixed behavioral sequence is determined in the brain. Here, we show that lateral antennal lobe tract projection neuron 4 (lPN4) in D. melanogaster functions as an "AND-gate" for volatile and contact chemosensory inputs, mediating the expression of tapping behaviors before unilateral wing extension during male courtship rituals.

The color pattern inducing gene wingless is expressed in specific cell types of campaniform sensilla of a polka-dotted fruit fly, Drosophila guttifera.

A polka-dotted fruit fly, Drosophila guttifera, has a unique pigmentation pattern on its wings and is used as a model for evo-devo studies exploring the mechanism of evolutionary gain of novel traits. In this species, a morphogen-encoding gene, wingless, is expressed in species-specific positions and induces a unique pigmentation pattern. To produce some of the pigmentation spots on wing veins, wingless is thought to be expressed in developing campaniform sensillum cells, but it was unknown which of the four cell types there express(es) wingless. Here we show that two of the cell types, dome cells and socket cells, express wingless, as indicated by in situ hybridization together with immunohistochemistry. This is a unique case in which non-neuronal SOP (sensory organ precursor) progeny cells produce Wingless as an inducer of pigmentation pattern formation. Our finding opens a path to clarifying the mechanism of evolutionary gain of a unique wingless expression pattern by analyzing gene regulation in dome cells and socket cells.

Calcium imaging method to visualize the spatial patterns of neural responses in the pygmy squid, Idiosepius paradoxus, central nervous system.

BACKGROUND: Cephalopods exhibit unique behaviors such as camouflage and tactile learning. The brain functions correlated to these behaviors have long been analyzed through behavioral observations of animals subject to surgical manipulation or electrical stimulation of brain lobes. However, physiological methods have rarely been introduced to investigate the functions of each individual lobe, though physiological work on giant axons and slices of the vertical lobe system of the cephalopods have provided deep insights into ion conductance of nerves and long-term synaptic plasticity. The lack of in vivo physiological work is partly due to difficulties in immobilizing the brain which is contained within the soft body and applying calcium indicators to the cephalopod central nervous system. NEW METHOD: We here present a calcium imaging method to visualize neural responses in the central nervous system of the smallest squid, Idiosepius paradoxus. RESULTS: We injected calcium indicator Cal-520 into the brachial lobes and revealed a spatiotemporal pattern of neural responses to the electrical stimulations of the axial nerve cord in the first arm. COMPARISON WITH EXISTING METHODS: We established a method to immobilize the central nervous system which is contained within the soft body and record the calcium responses from the intact central nervous system. CONCLUSIONS: Our method provides a novel approach to investigate the mechanisms of how the characteristic organization of the cephalopod brain functions to induce their unique behaviors.

Cephalopod biology and care, a COST FA1301 (CephsInAction) training school: anaesthesia and scientific procedures.

Cephalopods are the sole invertebrates included in the list of regulated species following the Directive 2010/63/EU. According to the Directive, achieving competence through adequate training is a requisite for people having a role in the different functions (article 23) as such carrying out procedures on animals, designing procedures and projects, taking care of animals, killing animals. Cephalopod Biology and Care Training Program is specifically designed to comply with the requirements of the "working document on the development of a common education and training framework to fulfil the requirements under the Directive 2010/63/EU". The training event occurred at the ICM-CSIC in Barcelona (Spain) where people coming from Europe, America and Asia were instructed on how to cope with regulations for the use of cephalopod molluscs for scientific purposes. The training encompasses discussion on the guidelines for the use and care of animals and their welfare with particular reference to procedures that may be of interest for neuroscience. Intensive discussion has been carried out during the training sessions with focus on behavioural studies and paradigms, welfare assessment, levels of severity of scientific procedures, animal care, handling, transport, individual identification and marking, substance administration, anaesthesia, analgesia and humane killing.

The role of PDF neurons in setting the preferred temperature before dawn in Drosophila.

Animals have sophisticated homeostatic controls. While mammalian body temperature fluctuates throughout the day, small ectotherms, such as Drosophila achieve a body temperature rhythm (BTR) through their preference of environmental temperature. Here, we demonstrate that pigment dispersing factor (PDF) neurons play an important role in setting preferred temperature before dawn. We show that small lateral ventral neurons (sLNvs), a subset of PDF neurons, activate the dorsal neurons 2 (DN2s), the main circadian clock cells that regulate temperature preference rhythm (TPR). The number of temporal contacts between sLNvs and DN2s peak before dawn. Our data suggest that the thermosensory anterior cells (ACs) likely contact sLNvs via serotonin signaling. Together, the ACs-sLNs-DN2s neural circuit regulates the proper setting of temperature preference before dawn. Given that sLNvs are important for sleep and that BTR and sleep have a close temporal relationship, our data highlight a possible neuronal interaction between body temperature and sleep regulation.

Upregulation of Juvenile Hormone Titers in Female Drosophila melanogaster Through Mating Experiences and Host Food Occupied by Eggs and Larvae.

Juvenile hormone (JH) plays a crucial role in the determination of developmental timing in insects. In Drosophila melanogaster, reports indicate that JH titers are the highest immediately following eclosion and that the mating experience increases the titers in females. However, the titers have not been successively measured for an extended period of time after eclosion. This study reveals that JH titers are increased after eclosion in virgin females when supplied with food that is occupied by eggs and larvae as well as in mated females. With the presence of eggs and larvae, food induced the virgin females to lay unfertilized eggs. When combined with previous work indicating that females are attracted to such food where they prefer to lay eggs, these results suggest that flies can prepare themselves to lay eggs by changing the titers of JH under the presence of growing larvae, ensuring that the food is an appropriate place to oviposit.

Convergence of multimodal sensory pathways to the mushroom body calyx in Drosophila melanogaster.

Detailed structural analyses of the mushroom body which plays critical roles in olfactory learning and memory revealed that it is directly connected with multiple primary sensory centers in Drosophila. Connectivity patterns between the mushroom body and primary sensory centers suggest that each mushroom body lobe processes information on different combinations of multiple sensory modalities. This finding provides a novel focus of research by Drosophila genetics for perception of the external world by integrating multisensory signals.

Three-dimensional brain atlas of pygmy squid, Idiosepius paradoxus, revealing the largest relative vertical lobe system volume among the cephalopods.

Cephalopods have the largest and most complex nervous system of all invertebrates, and the brain-to-body weight ratio exceeds those of most fish and reptiles. The brain is composed of lobe units, the functions of which have been studied through surgical manipulation and electrical stimulation. However, how information is processed in each lobe for the animal to make a behavioral decision has rarely been investigated. To perform such functional analyses, it is necessary to precisely describe how brain lobes are spatially organized and mutually interconnected as a whole. We thus made three-dimensional digital brain atlases of both hatchling and juvenile pygmy squid, Idiosepius paradoxus. I. paradoxus is the smallest squid and has a brain small enough to scan as a whole region in the field-of-view of a low-magnification laser scan microscope objective. Precise analyses of the confocal images of the brains revealed one newly identified lobe and also that the relative volume of the vertical lobe system, the higher association center, in the pygmy squid represents the largest portion compared with the cephalopod species reported previously. In addition, principal component analyses of relative volumes of lobe complexes revealed that the organization of I. paradoxus brain is comparable to those of Decapodiformes species commonly used to analyze complex behaviors such as Sepia officinalis and Sepioteuthis sepioidea. These results suggest that the pygmy squid can be a good model to investigate the brain functions of coleoids utilizing physiological methods. J. Comp. Neurol. 524:2142-2157, 2016. © 2016 Wiley Periodicals, Inc.

Organization of antennal lobe-associated neurons in adult Drosophila melanogaster brain.

The primary olfactory centers of both vertebrates and insects are characterized by glomerular structure. Each glomerulus receives sensory input from a specific type of olfactory sensory neurons, creating a topographic map of the odor quality. The primary olfactory center is also innervated by various types of neurons such as local neurons, output projection neurons (PNs), and centrifugal neurons from higher brain regions. Although recent studies have revealed how olfactory sensory input is conveyed to each glomerulus, it still remains unclear how the information is integrated and conveyed to other brain areas. By using the GAL4 enhancer-trap system, we conducted a systematic mapping of the neurons associated with the primary olfactory center of Drosophila, the antennal lobe (AL). We identified in total 29 types of neurons, among which 13 are newly identified in the present study. Analyses of arborizations of these neurons in the AL revealed how glomeruli are linked with each other, how different PNs link these glomeruli with multiple secondary sites, and how these secondary sites are organized by the projections of the AL-associated neurons.

Dye fills reveal additional olfactory tracts in the protocerebrum of wild-type Drosophila.

The antennal lobe (AL) is the primary olfactory center in insect brains. It receives sensory input from the olfactory sensory neurons (OSNs) and sends, through its projection neurons (PNs), reformatted output to secondary olfactory centers, including the mushroom body (MB) calyx and the lateral horn (LH) in the protocerebrum. By injecting dye into the AL of wild-type Drosophila, we identified previously unknown direct pathways between the AL and the ventrolateral, superior medial, and posterior lateral protocerebra. We found that most of these areas in the protocerebrum are connected with the AL through multiple tracts, suggesting that these areas are sites of convergence for olfactory information. Furthermore, areas such as the superior medial protocerebrum now appear to receive olfactory output both directly from the AL and indirectly from lobes of the MB and the LH, suggesting a degree of functional interaction among these areas. We also analyzed the length and number of fibers in each tract. We compare our results obtained from wild-type flies with recent results from transgenic strains and discuss how information about odorants is distributed to multiple protocerebral areas.

Dual-labeling method for electron microscopy to characterize synaptic connectivity using genetically encoded fluorescent reporters in Drosophila.

Light and electron microscopy (LM and EM) both offer important advantages for characterizing neuronal circuitry in intact brains: LM can reveal the general patterns neurons trace between brain areas, and EM can confirm synaptic connections between identified neurons within a small area. In a few species, genetic labeling with fluorescent proteins has been used with LM to visualize many kinds of neurons and to analyze their morphologies and projection patterns. However, combining these large-scale patterns with the fine detail available in EM analysis has been a technical challenge. To analyze the synaptic connectivity of neurons expressing fluorescent markers with EM, we developed a dual-labeling method for use with pre-embedded brains. In Drosophila expressing genetic labels and also injected with markers we visualized synaptic connections among two populations of neurons in the AL, one of which has been shown to mediate a specific function, odor evoked neural oscillation.

Odor-evoked neural oscillations in Drosophila are mediated by widely branching interneurons.

Stimulus-evoked oscillatory synchronization of neurons has been observed in a wide range of species. Here, we combined genetic strategies with paired intracellular and local field potential (LFP) recordings from the intact brain of Drosophila to study mechanisms of odor-evoked neural oscillations. We found common food odors at natural concentrations elicited oscillations in LFP recordings made from the mushroom body (MB), a site of sensory integration and analogous to the vertebrate piriform cortex. The oscillations were reversibly abolished by application of the GABA(a) blocker picrotoxin. Intracellular recordings from local and projection neurons within the antennal lobe (AL) (analogous to the olfactory bulb) revealed odor-elicited spikes and subthreshold membrane potential oscillations that were tightly phase locked to LFP oscillations recorded downstream in the MBs. These results suggested that, as in locusts, odors may elicit the oscillatory synchronization of AL neurons by means of GABAergic inhibition from local neurons (LNs). An analysis of the morphologies of genetically distinguished LNs revealed two populations of GABAergic neurons in the AL. One population of LNs innervated parts of glomeruli lacking terminals of receptor neurons, whereas the other branched more widely, innervating throughout the glomeruli, suggesting that the two populations might participate in different neural circuits. To test the functional roles of these LNs, we used the temperature-sensitive dynamin mutant gene shibire to conditionally and reversibly block chemical transmission from each or both of these populations of LNs. We found only the more widely branching population of LNs is necessary for generating odor-elicited oscillations.

Neuronal assemblies of the Drosophila mushroom body.

The mushroom body (MB) of the insect brain has important roles in odor learning and memory and in diverse other brain functions. To elucidate the anatomical basis underlying its function, we studied how the MB of Drosophila is organized by its intrinsic and extrinsic neurons. We screened for the GAL4 enhancer-trap strains that label specific subsets of these neurons and identified seven subtypes of Kenyon cells and three other intrinsic neuron types. Laminar organization of the Kenyon cell axons divides the pedunculus into at least five concentric strata. The alpha', beta', alpha, and beta lobes are each divided into three strata, whereas the gamma lobe appears more homogeneous. The outermost stratum of the alpha/beta lobes is specifically connected with a small, protruded subregion of the calyx, the accessory calyx, which does not receive direct olfactory input. As for the MB extrinsic neurons (MBENs), we found three types of antennal lobe projection neurons, among which two are novel. In addition, we resolved 17 other types of MBENs that arborize in the calyx, lobes, and pedunculus. Lobe-associated MBENs arborize in only specific areas of the lobes, being restricted along their longitudinal axes, forming two to five segmented zones in each lobe. The laminar arrangement of the Kenyon cell axons and segmented organization of the MBENs together divide the lobes into smaller synaptic units, possibly facilitating characteristic interaction between intrinsic and extrinsic neurons in each unit for different functional activities along the longitudinal lobe axes and between lobes. Structural differences between lobes are also discussed.

Activity-dependent plasticity in an olfactory circuit.

Olfactory sensory neurons (OSNs) form synapses with local interneurons and second-order projection neurons to form stereotyped olfactory glomeruli. This primary olfactory circuit is hard-wired through the action of genetic cues. We asked whether individual glomeruli have the capacity for stimulus-evoked plasticity by focusing on the carbon dioxide (CO2) circuit in Drosophila. Specialized OSNs detect this gas and relay the information to a dedicated circuit in the brain. Prolonged exposure to CO2 induced a reversible volume increase in the CO2-specific glomerulus. OSNs showed neither altered morphology nor function after chronic exposure, but one class of inhibitory local interneurons showed significantly increased responses to CO2. Two-photon imaging of the axon terminals of a single PN innervating the CO2 glomerulus showed significantly decreased functional output following CO2 exposure. Behavioral responses to CO2 were also reduced after such exposure. We suggest that activity-dependent functional plasticity may be a general feature of the Drosophila olfactory system.

Roles for Drosophila mushroom body neurons in olfactory learning and memory.

Olfactory learning assays in Drosophila have revealed that distinct brain structures known as mushroom bodies (MBs) are critical for the associative learning and memory of olfactory stimuli. However, the precise roles of the different neurons comprising the MBs are still under debate. The confusion surrounding the roles of the different neurons may be due, in part, to the use of different odors as conditioned stimuli in previous studies. We investigated the requirements for the different MB neurons, specifically the alpha/beta versus the gamma neurons, and whether olfactory learning is supported by different subsets of MB neurons irrespective of the odors used as conditioned stimuli. We expressed the rutabaga (rut)-encoded adenylyl cyclase in either the gamma or alpha/beta neurons and examined the effects on restoring olfactory associative learning and memory of rut mutant flies. We also expressed a temperature-sensitive shibire (shi) transgene in these neuron sets and examined the effects of disrupting synaptic vesicle recycling on Drosophila olfactory learning. Our results indicate that although we did not detect odor-pair-specific learning using GAL4 drivers that primarily express in gamma neurons, expression of the transgenes in a subset of alpha/beta neurons resulted in both odor-pair-specific rescue of the rut defect as well as odor-pair-specific disruption of learning using shi(ts1).

Stereotypic and random patterns of connectivity in the larval mushroom body calyx of Drosophila.

The larval brain of Drosophila is a useful model to study olfactory processing because of its cellular simplicity. The early stages of central olfactory processing involve the detection of odor features, but the coding mechanisms that transform them into a representation in higher brain centers is not clear. Here we examine the pattern of connectivity of the main neurons that process olfactory information in the calyx (dendritic region) of the mushroom bodies, a higher brain center essential for associative olfactory learning. The larval calyx has a glomerular organization. We generated a map of calyx glomeruli, using both anatomical criteria and the pattern of innervation by subsets of its input neurons (projection neurons), molecularly identified by GAL4 markers. Thus, we show that projection neurons innervate calyx glomeruli in a stereotypic manner. By contrast, subsets of mushroom body neurons (Kenyon cells) that are labeled by GAL4 markers show no clear preference for specific glomeruli. Clonal subsets of Kenyon cells show some preference for subregions of the calyx, implying that they receive distinct input. However, at the level of individual glomeruli, dendritic terminals of larval-born Kenyon cells innervate about six glomeruli, apparently randomly. These results are consistent with a model in which Kenyon cells process olfactory information by integrating different inputs from several calyx glomeruli in a combinatorial manner.

Developmentally programmed remodeling of the Drosophila olfactory circuit.

Neural circuits are often remodeled after initial connections are established. The mechanisms by which remodeling occurs, in particular whether and how synaptically connected neurons coordinate their reorganization, are poorly understood. In Drosophila, olfactory projection neurons (PNs) receive input by synapsing with olfactory receptor neurons in the antennal lobe and relay information to the mushroom body (MB) calyx and lateral horn. Here we show that embryonic-born PNs participate in both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the MB calyx. They persist in the adult olfactory circuit and are prespecified by birth order to innervate a subset of glomeruli distinct from larval-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon terminal branches locally during early metamorphosis. Electron microscopy analysis reveals that these PNs synapse with MB gamma neurons in the larval calyx and that these synaptic profiles are engulfed by glia during early metamorphosis. As with MB gamma neurons, PN pruning requires cell-autonomous reception of the nuclear hormone ecdysone. Thus, these synaptic partners are independently programmed to prune their dendrites and axons.

Integration of chemosensory pathways in the Drosophila second-order olfactory centers.

BACKGROUND: Behavioral responses to odorants require neurons of the higher olfactory centers to integrate signals detected by different chemosensory neurons. Recent studies revealed stereotypic arborizations of second-order olfactory neurons from the primary olfactory center to the secondary centers, but how third-order neurons read this odor map remained unknown. RESULTS: Using the Drosophila brain as a model system, we analyzed the connectivity patterns between second-order and third-order olfactory neurons. We first isolated three common projection zones in the two secondary centers, the mushroom body (MB) and the lateral horn (LH). Each zone receives converged information via second-order neurons from particular subgroups of antennal-lobe glomeruli. In the MB, third-order neurons extend their dendrites across various combinations of these zones, and axons of this heterogeneous population of neurons converge in the output region of the MB. In contrast, arborizations of the third-order neurons in the LH are constrained within a zone. Moreover, different zones of the LH are linked with different brain areas and form preferential associations between distinct subsets of antennal-lobe glomeruli and higher brain regions. CONCLUSIONS: MB is known to be an indispensable site for olfactory learning and memory, whereas LH function is reported to be sufficient for mediating direct nonassociative responses to odors. The structural organization of second-order and third-order neurons suggests that MB is capable of integrating a wide range of odorant information across glomeruli, whereas relatively little integration between different subsets of the olfactory signal repertoire is likely to occur in the LH.

Developmental origin of wiring specificity in the olfactory system of Drosophila.

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.