Heterogeneity of synaptic connectivity in the fly visual system.

Visual systems are homogeneous structures, where repeating columnar units retinotopically cover the visual field. Each of these columns contain many of the same neuron types that are distinguished by anatomic, genetic and - generally - by functional properties. However, there are exceptions to this rule. In the 800 columns of the Drosophila eye, there is an anatomically and genetically identifiable cell type with variable functional properties, Tm9. Since anatomical connectivity shapes functional neuronal properties, we identified the presynaptic inputs of several hundred Tm9s across both optic lobes using the full adult female fly brain (FAFB) electron microscopic dataset and FlyWire connectome. Our work shows that Tm9 has three major and many sparsely distributed inputs. This differs from the presynaptic connectivity of other Tm neurons, which have only one major, and more stereotypic inputs than Tm9. Genetic synapse labeling showed that the heterogeneous wiring exists across individuals. Together, our data argue that the visual system uses heterogeneous, distributed circuit properties to achieve robust visual processing.

NECAB2 is an endosomal protein important for striatal function.

Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.

Multifaceted luminance gain control beyond photoreceptors in Drosophila.

Animals navigating in natural environments must handle vast changes in their sensory input. Visual systems, for example, handle changes in luminance at many timescales, from slow changes across the day to rapid changes during active behavior. To maintain luminance-invariant perception, visual systems must adapt their sensitivity to changing luminance at different timescales. We demonstrate that luminance gain control in photoreceptors alone is insufficient to explain luminance invariance at both fast and slow timescales and reveal the algorithms that adjust gain past photoreceptors in the fly eye. We combined imaging and behavioral experiments with computational modeling to show that downstream of photoreceptors, circuitry taking input from the single luminance-sensitive neuron type L3 implements gain control at fast and slow timescales. This computation is bidirectional in that it prevents the underestimation of contrasts in low luminance and overestimation in high luminance. An algorithmic model disentangles these multifaceted contributions and shows that the bidirectional gain control occurs at both timescales. The model implements a nonlinear interaction of luminance and contrast to achieve gain correction at fast timescales and a dark-sensitive channel to improve the detection of dim stimuli at slow timescales. Together, our work demonstrates how a single neuronal channel performs diverse computations to implement gain control at multiple timescales that are together important for navigation in natural environments.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

First-order visual interneurons distribute distinct contrast and luminance information across ON and OFF pathways to achieve stable behavior.

The accurate processing of contrast is the basis for all visually guided behaviors. Visual scenes with rapidly changing illumination challenge contrast computation because photoreceptor adaptation is not fast enough to compensate for such changes. Yet, human perception of contrast is stable even when the visual environment is quickly changing, suggesting rapid post receptor luminance gain control. Similarly, in the fruit fly Drosophila, such gain control leads to luminance invariant behavior for moving OFF stimuli. Here, we show that behavioral responses to moving ON stimuli also utilize a luminance gain, and that ON-motion guided behavior depends on inputs from three first-order interneurons L1, L2, and L3. Each of these neurons encodes contrast and luminance differently and distributes information asymmetrically across both ON and OFF contrast-selective pathways. Behavioral responses to both ON and OFF stimuli rely on a luminance-based correction provided by L1 and L3, wherein L1 supports contrast computation linearly, and L3 non-linearly amplifies dim stimuli. Therefore, L1, L2, and L3 are not specific inputs to ON and OFF pathways but the lamina serves as a separate processing layer that distributes distinct luminance and contrast information across ON and OFF pathways to support behavior in varying conditions.

Populations of local direction-selective cells encode global motion patterns generated by self-motion.

Self-motion generates visual patterns on the eye that are important for navigation. These optic flow patterns are encoded by the population of local direction­selective cells in the mouse retina, whereas in flies, local direction­selective T4/T5 cells are thought to be uniformly tuned. How complex global motion patterns can be computed downstream is unclear. We show that the population of T4/T5 cells in Drosophila encodes global motion patterns. Whereas the mouse retina encodes four types of optic flow, the fly visual system encodes six. This matches the larger number of degrees of freedom and the increased complexity of translational and rotational motion patterns during flight. The four uniformly tuned T4/T5 subtypes described previously represent a local subset of the population. Thus, a population code for global motion patterns appears to be a general coding principle of visual systems that matches local motion responses to modes of the animal's movement.

The physiological basis for contrast opponency in motion computation in Drosophila.

In Drosophila, direction-selective neurons implement a mechanism of motion computation similar to cortical neurons, using contrast-opponent receptive fields with ON and OFF subfields. It is not clear how the presynaptic circuitry of direction-selective neurons in the OFF pathway supports this computation if all major inputs are OFF-rectified neurons. Here, we reveal the biological substrate for motion computation in the OFF pathway. Three interneurons, Tm2, Tm9 and CT1, provide information about ON stimuli to the OFF direction-selective neuron T5 across its receptive field, supporting a contrast-opponent receptive field organization. Consistent with its prominent role in motion detection, variability in Tm9 receptive field properties transfers to T5, and calcium decrements in Tm9 in response to ON stimuli persist across behavioral states, while spatial tuning is sharpened by active behavior. Together, our work shows how a key neuronal computation is implemented by its constituent neuronal circuit elements to ensure direction selectivity.

m6A RNA methylation regulates promoter- proximal pausing of RNA polymerase II.

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.

Adaptive temporal processing of odor stimuli.

The olfactory system translates chemical signals into neuronal signals that inform behavioral decisions of the animal. Odors are cues for source identity, but if monitored long enough, they can also be used to localize the source. Odor representations should therefore be robust to changing conditions and flexible in order to drive an appropriate behavior. In this review, we aim at discussing the main computations that allow robust and flexible encoding of odor information in the olfactory neural pathway.

Luminance Information Is Required for the Accurate Estimation of Contrast in Rapidly Changing Visual Contexts.

Visual perception scales with changes in the visual stimulus, or contrast, irrespective of background illumination. However, visual perception is challenged when adaptation is not fast enough to deal with sudden declines in overall illumination, for example, when gaze follows a moving object from bright sunlight into a shaded area. Here, we show that the visual system of the fly employs a solution by propagating a corrective luminance-sensitive signal. We use in vivo 2-photon imaging and behavioral analyses to demonstrate that distinct OFF-pathway inputs encode contrast and luminance. Predictions of contrast-sensitive neuronal responses show that contrast information alone cannot explain behavioral responses in sudden dim light. The luminance-sensitive pathway via the L3 neuron is required for visual processing in such rapidly changing light conditions, ensuring contrast constancy when pure contrast sensitivity underestimates a stimulus. Thus, retaining a peripheral feature, luminance, in visual processing is required for robust behavioral responses.

Distinct expression of potassium channels regulates visual response properties of lamina neurons in Drosophila melanogaster.

The computational organization of sensory systems depends on the diversification of individual cell types with distinct signal-processing capabilities. The Drosophila visual system, for instance, splits information into channels with different temporal properties directly downstream of photoreceptors in the first-order interneurons of the OFF pathway, L2 and L3. However, the biophysical mechanisms that determine this specialization are largely unknown. Here, we show that the voltage-gated Ka channels Shaker and Shal contribute to the response properties of the major OFF pathway input L2. L3 calcium response kinetics postsynaptic to photoreceptors resemble the sustained calcium signals of photoreceptors, whereas L2 neurons decay transiently. Based on a cell-type-specific RNA-seq data set and endogenous protein tagging, we identified Shaker and Shal as the primary candidates to shape L2 responses. Using in vivo two-photon imaging of L2 calcium signals in combination with pharmacological and genetic perturbations of these Ka channels, we show that the wild-type Shaker and Shal function is to enhance L2 responses and cell-autonomously sharpen L2 kinetics. Our results reveal a role for Ka channels in determining the signal-processing characteristics of a specific cell type in the visual system.

The Drosophila NCAM homolog Fas2 signals independently of adhesion.

The development of tissues and organs requires close interaction of cells. To achieve this, cells express adhesion proteins such as the neural cell adhesion molecule (NCAM) or its Drosophila ortholog Fasciclin 2 (Fas2). Both are members of the Ig-domain superfamily of proteins that mediate homophilic adhesion. These proteins are expressed as isoforms differing in their membrane anchorage and their cytoplasmic domains. To study the function of single isoforms, we have conducted a comprehensive genetic analysis of Fas2 We reveal the expression pattern of all major Fas2 isoforms, two of which are GPI anchored. The remaining five isoforms carry transmembrane domains with variable cytoplasmic tails. We generated Fas2 mutants expressing only single isoforms. In contrast to the null mutation, which causes embryonic lethality, these mutants are viable, indicating redundancy among the different isoforms. Cell type-specific rescue experiments showed that glial-secreted Fas2 can rescue the Fas2 mutant phenotype to viability. This demonstrates that cytoplasmic Fas2 domains have no apparent essential functions and indicate that Fas2 has function(s) other than homophilic adhesion. In conclusion, our data suggest novel mechanistic aspects of a long-studied adhesion protein.

ON selectivity in the Drosophila visual system is a multisynaptic process involving both glutamatergic and GABAergic inhibition.

Sensory systems sequentially extract increasingly complex features. ON and OFF pathways, for example, encode increases or decreases of a stimulus from a common input. This ON/OFF pathway split is thought to occur at individual synaptic connections through a sign-inverting synapse in one of the pathways. Here, we show that ON selectivity is a multisynaptic process in the Drosophila visual system. A pharmacogenetics approach demonstrates that both glutamatergic inhibition through GluClα and GABAergic inhibition through Rdl mediate ON responses. Although neurons postsynaptic to the glutamatergic ON pathway input L1 lose all responses in GluClα mutants, they are resistant to a cell-type-specific loss of GluClα. This shows that ON selectivity is distributed across multiple synapses, and raises the possibility that cell-type-specific manipulations might reveal similar strategies in other sensory systems. Thus, sensory coding is more distributed than predicted by simple circuit motifs, allowing for robust neural processing.

TAF-ChIP: an ultra-low input approach for genome-wide chromatin immunoprecipitation assay.

Chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) is a powerful technique to study transcriptional regulation. However, the requirement of millions of cells to generate results with high signal-to-noise ratio precludes it in the study of small cell populations. Here, we present a tagmentation-assisted fragmentation ChIP (TAF-ChIP) and sequencing method to generate high-quality histone profiles from low cell numbers. The data obtained from the TAF-ChIP approach are amenable to standard tools for ChIP-Seq analysis, owing to its high signal-to-noise ratio. The epigenetic profiles from TAF-ChIP approach showed high agreement with conventional ChIP-Seq datasets, thereby underlining the utility of this approach.

Publisher Correction: Diverse populations of local interneurons integrate into the Drosophila adult olfactory circuit.

The original version of this Article contained errors in Figs. 4 and 6. In Fig. 4, panel a, text labels UAS-FLP and LexAop2>stop>myr::smGdP-HA were shifted upwards during typesetting of the figure, and in Fig. 6, panel h, the number 15 was incorrectly placed on the heat map scale. These have now been corrected in both the PDF and HTML versions of the Article.

Diverse populations of local interneurons integrate into the Drosophila adult olfactory circuit.

Drosophila olfactory local interneurons (LNs) in the antennal lobe are highly diverse and variable. How and when distinct types of LNs emerge, differentiate, and integrate into the olfactory circuit is unknown. Through systematic developmental analyses, we found that LNs are recruited to the adult olfactory circuit in three groups. Group 1 LNs are residual larval LNs. Group 2 are adult-specific LNs that emerge before cognate sensory and projection neurons establish synaptic specificity, and Group 3 LNs emerge after synaptic specificity is established. Group 1 larval LNs are selectively reintegrated into the adult circuit through pruning and re-extension of processes to distinct regions of the antennal lobe, while others die during metamorphosis. Precise temporal control of this pruning and cell death shapes the global organization of the adult antennal lobe. Our findings provide a road map to understand how LNs develop and contribute to constructing the olfactory circuit.

Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila.

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.

Drosophila Fezf coordinates laminar-specific connectivity through cell-intrinsic and cell-extrinsic mechanisms.

Laminar arrangement of neural connections is a fundamental feature of neural circuit organization. Identifying mechanisms that coordinate neural connections within correct layers is thus vital for understanding how neural circuits are assembled. In the medulla of the Drosophila visual system neurons form connections within ten parallel layers. The M3 layer receives input from two neuron types that sequentially innervate M3 during development. Here we show that M3-specific innervation by both neurons is coordinated by Drosophila Fezf (dFezf), a conserved transcription factor that is selectively expressed by the earlier targeting input neuron. In this cell, dFezf instructs layer specificity and activates the expression of a secreted molecule (Netrin) that regulates the layer specificity of the other input neuron. We propose that employment of transcriptional modules that cell-intrinsically target neurons to specific layers, and cell-extrinsically recruit other neurons is a general mechanism for building layered networks of neural connections.

Different modes of APC/C activation control growth and neuron-glia interaction in the developing Drosophila eye.

The development of the nervous system requires tight control of cell division, fate specification and migration. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that affects different steps of cell cycle progression, as well as having postmitotic functions in nervous system development. It can therefore link different developmental stages in one tissue. The two adaptor proteins, Fizzy/Cdc20 and Fizzy-related/Cdh1, confer APC/C substrate specificity. Here, we show that two distinct modes of APC/C function act during Drosophila eye development. Fizzy/Cdc20 controls the early growth of the eye disc anlage and the concomitant entry of glial cells onto the disc. In contrast, fzr/cdh1 acts during neuronal patterning and photoreceptor axon growth, and subsequently affects neuron-glia interaction. To further address the postmitotic role of Fzr/Cdh1 in controlling neuron-glia interaction, we identified a series of novel APC/C candidate substrates. Four of our candidate genes are required for fzr/cdh1-dependent neuron-glia interaction, including the dynein light chain Dlc90F Taken together, our data show how different modes of APC/C activation can couple early growth and neuron-glia interaction during eye disc development.

A Class of Visual Neurons with Wide-Field Properties Is Required for Local Motion Detection.

Visual motion cues are used by many animals to guide navigation across a wide range of environments. Long-standing theoretical models have made predictions about the computations that compare light signals across space and time to detect motion. Using connectomic and physiological approaches, candidate circuits that can implement various algorithmic steps have been proposed in the Drosophila visual system. These pathways connect photoreceptors, via interneurons in the lamina and the medulla, to direction-selective cells in the lobula and lobula plate. However, the functional architecture of these circuits remains incompletely understood. Here, we use a forward genetic approach to identify the medulla neuron Tm9 as critical for motion-evoked behavioral responses. Using in vivo calcium imaging combined with genetic silencing, we place Tm9 within motion-detecting circuitry. Tm9 receives functional inputs from the lamina neurons L3 and, unexpectedly, L1 and passes information onto the direction-selective T5 neuron. Whereas the morphology of Tm9 suggested that this cell would inform circuits about local points in space, we found that the Tm9 spatial receptive field is large. Thus, this circuit informs elementary motion detectors about a wide region of the visual scene. In addition, Tm9 exhibits sustained responses that provide a tonic signal about incoming light patterns. Silencing Tm9 dramatically reduces the response amplitude of T5 neurons under a broad range of different motion conditions. Thus, our data demonstrate that sustained and wide-field signals are essential for elementary motion processing.