Hybrid Neurons in a MicroRNA Mutant Are Putative Evolutionary Intermediates in Insect CO_2 Sensory Systems

Carbon dioxide (CO_2) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO_2 are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. We found in Drosophila that loss of a microRNA, miR-279, leads to formation of CO_2 neurons in the MPs. miR-279 acts through down-regulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO_2 receptors and form connections characteristic of CO_2 neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). We propose that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO_2

A Neural Circuit Arbitrates between Persistence and Withdrawal in Hungry Drosophila.

In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, we show that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. We further show that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, our data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive

Internal State Dependent Odor Processing and Perception—The Role of Neuromodulation in the Fly Olfactory System

Animals rely heavily on their sense of olfaction to perform various vital interactions with an ever-in-flux environment. The turbulent and combinatorial nature of air-borne odorant cues demands the employment of various coding strategies, which allow the animal to attune to its internal needs and past or present experiences. Furthermore, these internal needs can be dependent on internal states such as hunger, reproductive state and sickness. Neuromodulation is a key component providing flexibility under such conditions. Understanding the contributions of neuromodulation, such as sensory neuron sensitization and choice bias requires manipulation of neuronal activity on a local and global scale. With Drosophila's genetic toolset, these manipulations are feasible and even allow a detailed look on the functional role of classical neuromodulators such as dopamine, octopamine and neuropeptides. The past years unraveled various mechanisms adapting chemosensory processing and perception to internal states such as hunger and reproductive state. However, future research should also investigate the mechanisms underlying other internal states including the modulatory influence of endogenous microbiota on Drosophila behavior. Furthermore, sickness induced by pathogenic infection could lead to novel insights as to the neuromodulators of circuits that integrate such a negative postingestive signal within the circuits governing olfactory behavior and learning. The enriched emporium of tools Drosophila provides will help to build a concrete picture of the influence of neuromodulation on olfaction and metabolism, adaptive behavior and our overall understanding of how a brain works

Gogo Receptor Contributes to Retinotopic Map Formation and Prevents R1-6 Photoreceptor Axon Bundling

<div><p>Background</p><p>Topographic maps form the basis of neural processing in sensory systems of both vertebrate and invertebrate species. In the Drosophila visual system, neighboring R1–R6 photoreceptor axons innervate adjacent positions in the first optic ganglion, the lamina, and thereby represent visual space as a continuous map in the brain. The mechanisms responsible for the establishment of retinotopic maps remain incompletely understood.</p><p>Results</p><p>Here, we show that the receptor Golden goal (Gogo) is required for R axon lamina targeting and cartridge elongation in a partially redundant fashion with local guidance cues provided by neighboring axons. Loss of function of Gogo in large clones of R axons results in aberrant R1–R6 fascicle spacing. Gogo affects target cartridge selection only indirectly as a consequence of the disordered lamina map. Interestingly, small clones of <i>gogo</i> deficient R axons perfectly integrate into a proper retinotopic map suggesting that surrounding R axons of the same or neighboring fascicles provide complementary spatial guidance. Using single photoreceptor type rescue, we show that Gogo expression exclusively in R8 cells is sufficient to mediate targeting of all photoreceptor types in the lamina. Upon lamina targeting and cartridge selection, R axons elongate within their individual cartridges. Interestingly, here Gogo prevents bundling of extending R1-6 axons.</p><p>Conclusion</p><p>Taken together, we propose that Gogo contributes to retinotopic map formation in the Drosophila lamina by controlling the distribution of R1–R6 axon fascicles. In a later developmental step, the regular position of R1–R6 axons along the lamina plexus is crucial for target cartridge selection. During cartridge elongation, Gogo allows R1–R6 axons to extend centrally in the lamina cartridge.</p></div

Overexpression in single R cell types disrupts cartridge formation.

<p>(A–F) Confocal stacks of wild-type and Gogo overexpression in R3/R4 using the <i>mδ</i>–Gal4 driver. (A–D) Horizontal (A,B) and lateral (C, D) patterns in adult brains. Wild-type control in (A, C) and overexpression of Gogo in (B, D). Termini of R1–R6 axons are visualized using the directly fused construct Rh1-lacZ. (A, B) Compared to wild-type (A), the pattern of R1–R6 axons in gain of function flies is disrupted (B). Arrowheads mark the start points and arrows the end of R4 extensions. In wild-type (C) cartridges are formed by six axon termini (rings). The number of termini per cartridge is altered when Gogo levels are increased in R3/R4 (D). (E, F’) Wild-type control (E, E’) and overexpression (F, F’) in pupal laminae. Asterisks mark the start points and arrowheads the end of R4 extensions. R1–R6 cells are labeled with mAB24B10 antibody staining and R4 axons are visualized by mCD8-GFP expression. Increasing Gogo levels in R3/R4 axons does not influence R4 target selection or the overall pattern of cartridge assembly. Scale bars: A–B’, E–F = 5 µm, C, D: 10 µm.</p

In<i>gogo</i> mosaic eyes R axons bundle with R axons from neighboring cartridges.

<p>(A–B’’’) Confocal sections showing the developing lamina in wild-type and <i>gogo</i>- mosaic eyes and corresponding schematics at 51 hr APF. R1–R6 axons are labeled with Gmr-KO (magenta) and R4 axons are labeled with mCD8-GFP (green). Arrowheads indicate the start and arrows the end of axon elongation within the cartridge. (A–A’’’) In wild-type controls R axons elongate parallel in separate columns. (B–B’’’) When Gogo is absent in the majority of ommatidia R axons fail to project in a parallel fashion. Moreover, single R axons (R4) leave their original target cartridge and bundle with axons of adjacent cartridges. Note that axon termini are not yet fully extended at this developmental stage. (C) In the wild-type control all R4 axons follow the original tracts of their target cartridge. In the <i>ey3.5flp:gogo</i> background almost half of R4 axons project away from their target cartridge to join a neighboring column. (D–E) Model of R1–R6 axon extensions during cartridge formation in wild-type (D) and mutant (E) backgrounds. When axons arrive at the lamina plexus they extend lateral to the target cartridges, turn and elongate along lamina neurons (grey). Scale bars: 5 µm.</p

Mutant R4 axons target correctly to areas innervated by wild-type R axons.

<p>(A–C’’’) Cartridge pattern and behavior of R4 axons in small <i>ey3.5flp:gogo</i> clones and controls. All R1–R6 cells are labeled with mAb24B10 (magenta). Gmr-KO is located in <i>trans</i> to the <i>gogo</i> mutant allele. Thus, wild-type areas express Gmr-KO (blue) and mutant areas appear black (indicated by dashed lines). mCD8-GFP is expressed under the control of the <i>mδ-Gal4 driver.</i> The Gal4 repressor Gal80 is located in <i>trans</i> to the control (A–A’’) or the <i>gogo</i> mutant allele. Therefore only R4 axons that are homozygous for the control or the <i>gogo</i> mutant allele express mCD8-GFP. (A–A’’’) Laminae of control animals display a uniform R4 and a well ordered overall projection pattern. (B–B’’’) Mutant R4 axons express mCD8-GFP, whereas wild-type R4 axons are not visible. The overall pattern of lamina cartridges displays a regular distribution indistinguishable from wild-type. Mutant R4 axons project parallel to each other when R axons of the target cartridge area are wild-type. (D–E) Orientation vectors of control (D) and mutant R4 axons that target to wild-type areas (E). Scale bar: 5 µm.</p

Gogo function in R8 is sufficient to maintain retinotopic map formation.

<p>(A–D) Pupal lamina in <i>eyflp;gogo</i> eyes (control) and specific rescue experiments stained with mAb24B10 and plot profiles. (A, C) Absence of Gogo in the majority of R axons and the target area strongly disrupts the overall pattern of the pupal lamina (42 hr APF). (B) When FL Gogo is specifically restored in R8 axons the orderly organized pattern of the topographic map is fully rescued. (C) However, FL Gogo expression in R4 axons in the mutant background cannot restore the orderly arrangement of cartridges. (E–H) Pooled plot profiles of 4 cartridges in each experiment provide an example of the pattern regularity. The R8 rescue displays a periodic profile while in mutant controls and R4 rescue a periodicity is not detectable. (I-J’’’) Larval optic lobes in control and <i>gogo</i> mosaic animals and corresponding magnifications. mAb24B10 staining visualizes R1–R8 axons (magenta) and GFP is expressed in the R4 subtype (green). While <i>gogo</i> mutant axons strongly bundle within the medulla (asterisk), mutant R cell targeting to the lamina plexus (defined by chevrons) shows only mild irregularities compared to wild-type. Single wild-type (I’’) and <i>gogo</i> mutant (J’’) R4 axons strictly remain within their fascicle. (K, L) In <i>eyflp;gogo</i> eyes R fascicle (white arrows) distribute along the anterior-posterior axis indistinguishable from wild-type. a: anterior; p: posterior; d: dorsal; v: ventral. Scale bars: 20 µm.</p

Absence of Gogo in single R cells does not influence cartridge selection.

<p>(A) Schematic of cartridge target selection. R1–R6 projection pattern is specific and uniform for each R1–R6 fascicle. Axon extensions occur in the control 180° rotated with respect to the position of their cell bodies. (B–E) Single cells were made homozygous for <i>gogo</i> null allele via mitotic recombination. Asterisks mark start and arrowheads end of axon extensions. (B, C) In the complementary MARCM (cMARCM) approach mutant R axons are labeled by mCD8-GFP (green) and wild-type cells are labeled with Gmr-KO (magenta). <i>gogo</i> mutant R axons surrounded by wild-type cells choose correct targets in the lamina (C) with respect to the position of their ommatidial cell bodies (B). (D, E) In the reverse MARCM approach (rMARCM) single wild-type axons are labeled with mCD8-GFP in a wild-type Gmr-KO labeled background (magenta) and unlabeled <i>gogo</i> mutant R cells that appear in black (dashed lines). Wild-type axons adjacent to mutant axons target correctly (E) with respect to their cell-bodies (D). Scale bars: 5 µm.</p

Retinotopic map formation and target cartridge selection is disrupted in the absence of Gogo.

<p>(A–B) Schematics of cartridge assembly. (A) Within each fascicle, the R8 axon extends first to the lamina plexus during larval development, followed by a sequential outgrowth of R1–R7. R1-6 axon fascicles reach the lamina plexus in a precise spatial pattern, forming the initial topographic map (30 hr APF), whereas R8 projects through the lamina to innervate the medulla. (B) Subsequently, R1-6 fascicles separate and project to different specific cartridges. Six R1–R6 axons from six different ommatidia in turn converge with one set of lamina neurons (not shown) to a single target cartridge. (C–D’’’) Confocal images and schematics of retinotopic mapping of R1–R6 fascicles in wild-type and <i>eyflp;gogo</i> background at 30 hr APF. (C–C’’’) In wild-type FRT80 controls R1–R6 fascicles terminate at the lamina plexus maintaining their equal spacing and the spatial order of their ommatidia. (D–D’’’) In <i>gogo</i> mutant background, R1–R6 fascicles fail to arrange in the correct order to neighboring axons. (E–F’’’) Sections of midpupal lamina at the onset of target cartridge selection at 42 hr APF in control and in <i>gogo</i> mutant background. Arrowheads (E–E’’’, F–F’’’) and dots (E’’’, F’’’) mark the start points and asterisks (E–E’’’, F–F’’’) the end of R4 extensions. (E–E’’’) In control animals, R4 projection pattern (green) is uniform in direction and length and the overall pattern (magenta) displays orderly distributed and uniformly sized cartridges. (F–F’’’) When Gogo is removed from the majority of R cells R4 extensions (green) are not parallel and the overall pattern of the lamina (magenta) is highly disrupted. R4 cells sometimes form long growth cones (orange arrow) or two axons converge to a single target (red arrow). (G, J) Polar plots visualize orientation vectors of R4 axons in wild-type control and mutant. (H, I) DeLaunay (green strokes) and Voronoi diagrams (grey strokes) display the uniform and irregular retinotopic mapping in wild-type (E–E’’’) and mutant (F–F’’’), respectively. Scale bars: 10 µm.</p