SUMMARY Saltatory conduction requires high-density accumulation of Na+ channels at the nodes of Ranvier. Nodal Na+ channel clustering in the peripheral nervous system is regulated by myelinating Schwann cells through unknown mechanisms. During development, Na+ channels are first clustered at heminodes that border each myelin segment, and later in the mature nodes that are formed by the fusion of two heminodes. Here we show that initial clustering of Na+ channels at heminodes requires glial NrCAM and gliomedin, as well as their axonal receptor neurofascin 186 (NF186). We further demonstrate that heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na+ channels to accumulate at mature nodes by restricting their distribution between two growing myelin internodes. We propose that Schwann cells assemble the nodes of Ranvier by capturing Na+ channels at heminodes and by constraining their distribution to the nodal gap. Together, these two cooperating mechanisms ensure fast and efficient conduction in myelinated nerves.

Animals exhibit innate behaviours to a variety of sensory stimuli including olfactory cues. In Drosophila, one higher olfactory centre, the lateral horn (LH), is implicated in innate behaviour. However, our structural and functional understanding of the LH is scant, in large part due to a lack of sparse neurogenetic tools for this region. We generate a collection of split-GAL4 driver lines providing genetic access to 82 LH cell types. We use these to create an anatomical and neurotransmitter map of the LH and link this to EM connectomics data. We find ~30% of LH projections converge with outputs from the mushroom body, site of olfactory learning and memory. Using optogenetic activation, we identify LH cell types that drive changes in valence behavior or specific locomotor programs. In summary, we have generated a resource for manipulating and mapping LH neurons, providing new insights into the circuit basis of innate and learned olfactory behavior.

SummaryThe Drosophila sex pheromone cVA elicits different behaviors in males and females. First- and second-order olfactory neurons show identical pheromone responses, suggesting that sex genes differentially wire circuits deeper in the brain. Using in vivo whole-cell electrophysiology, we now show that two clusters of third-order olfactory neurons have dimorphic pheromone responses. One cluster responds in females; the other responds in males. These clusters are present in both sexes and share a common input pathway, but sex-specific wiring reroutes pheromone information. Regulating dendritic position, the fruitless transcription factor both connects the male-responsive cluster and disconnects the female-responsive cluster from pheromone input. Selective masculinization of third-order neurons transforms their morphology and pheromone responses, demonstrating that circuits can be functionally rewired by the cell-autonomous action of a switch gene. This bidirectional switch, analogous to an electrical changeover switch, provides a simple circuit logic to activate different behaviors in males and females.

Most sensory systems are organized into parallel neuronal pathways that process distinct aspects of incoming stimuli. In the insect olfactory system, second order projection neurons target both the mushroom body, required for learning, and the lateral horn (LH), proposed to mediate innate olfactory behavior. Mushroom body neurons form a sparse olfactory population code, which is not stereotyped across animals. In contrast, odor coding in the LH remains poorly understood. We combine genetic driver lines, anatomical and functional criteria to show that the Drosophila LH has ~1400 neurons and >165 cell types. Genetically labeled LHNs have stereotyped odor responses across animals and on average respond to three times more odors than single projection neurons. LHNs are better odor categorizers than projection neurons, likely due to stereotyped pooling of related inputs. Our results reveal some of the principles by which a higher processing area can extract innate behavioral significance from sensory stimuli.

Drosophila phototransduction results in the opening of two classes of cation channels, composed of the channel subunits transient receptor potential (TRP), TRP-like (TRPL), and TRPgamma. Here, we report that one of these subunits, TRPL, is translocated back and forth between the signaling membrane and an intracellular compartment by a light-regulated mechanism. A high level of rhabdomeral TRPL, characteristic of dark-raised flies, is functionally manifested in the properties of the light-induced current. These flies are more sensitive than flies with no or reduced TRPL level to dim background lights, and they respond to a wider range of light intensities, which fit them to function better in darkness or dim background illumination. Thus, TRPL translocation represents a novel mechanism to fine tune visual responses.

SummaryThe behavioral response to a sensory stimulus may depend on both learned and innate neuronal representations. How these circuits interact to produce appropriate behavior is unknown. In Drosophila, the lateral horn (LH) and mushroom body (MB) are thought to mediate innate and learned olfactory behavior, respectively, although LH function has not been tested directly. Here we identify two LH cell types (PD2a1 and PD2b1) that receive input from an MB output neuron required for recall of aversive olfactory memories. These neurons are required for aversive memory retrieval and modulated by training. Connectomics data demonstrate that PD2a1 and PD2b1 neurons also receive direct input from food odor-encoding neurons. Consistent with this, PD2a1 and PD2b1 are also necessary for unlearned attraction to some odors, indicating that these neurons have a dual behavioral role. This provides a circuit mechanism by which learned and innate olfactory information can interact in identified neurons to produce appropriate behavior.Video Abstract

Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, highsignal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples.immunohistochemistry | neural circuits | protein labeling | fluorescence microscopy T he revolution in live imaging resulting from the use of genetically encoded fluorescent proteins (FPs) is widely appreciated (1, 2), but FPs also have had a major impact on studies of fixed, whole-mount specimens or thick sections. Processing of large or intact pieces of tissue has obvious advantages over sectioning, such as reduced tissue damage, compatibility with fast imaging modalities (e.g., light sheet microscopy), and easy subsequent 3D reconstruction. The drastic increase in imaging throughput by using whole-mount brains had a major impact on Drosophila neurobiology, in which reconstruction of neural circuits is a key requirement. Recently, several methods have been developed that allow whole-mount imaging of the mouse brain: CLARITY (3), Scale (4), SeeDB (5), and CUBIC (6) all render the brain optically transparent (although to different degrees). In such samples, imaging the native fluorescence of genetically encoded FPs offers the advantages of immediate visualization, low background, and spatially even signal. However, FP signals are easily quenched by fixation or other staining procedures, suffer from limited spectral flexibility, and often emit weak signals. Therefore, antibody detection of marker proteins remains essential in many experimental situations. Immunostaining, however, is notoriously slow, highly nonlinear, and often results in uneven labeling with high background levels. Therefore, there are undesirable tradeoffs in the antibody vs. FP labeling techniques.These tradeoffs are a major practical issue for our research in neural circuit tracing in Drosophila (7-12). We therefore sought staining methods that combine the positive aspects of both FPs and antibody-based staining, notably fast, even, strong, and spectrally diverse signals with low background labeling. We have developed an approach based on four commercially available, orthogonal labeling chemistries (SNAP-, CLIP-, Halo-and TMP-tag) characteriz...

Most sensory systems are organized into parallel neuronal pathways that process distinct aspects of incoming stimuli. For example, second order olfactory neurons make divergent projections onto functionally distinct brain areas relevant to different behaviors. In insects, one area, the mushroom body has been intensively studied for its role in 15 olfactory learning while the lateral horn is proposed to mediate innate olfactory behavior. Some lateral horn neurons (LHNs) show selective responses to sex pheromones but its functional principles remain poorly understood. We have carried out a comprehensive anatomical analysis of the Drosophila lateral horn and identified genetic driver lines targeting many LHNs. We find that the lateral horn contains >1300 neurons and by combining genetic, anatomical and functional criteria, we identify >150 cell types. In particular we show that genetically labeled LHNs show stereotyped 20 odor responses from one animal to the next. Although LHN tuning can be ultra-sparse (1/40 odors tested), as a population they respond to three times more odors than their inputs; this coding change can be rationalized by our observation that LHNs are better odor categorizers. Our results reveal some of the principles by which a higher sensory processing area can extract innate behavioral significance from sensory stimuli.