Biomechanical origins of proprioceptor feature selectivity and topographic maps in the Drosophila leg.

Our ability to sense and move our bodies relies on proprioceptors, sensory neurons that detect mechanical forces within the body. Different subtypes of proprioceptors detect different kinematic features, such as joint position, movement, and vibration, but the mechanisms that underlie proprioceptor feature selectivity remain poorly understood. Using single-nucleus RNA sequencing (RNA-seq), we found that proprioceptor subtypes in the Drosophila leg lack differential expression of mechanosensitive ion channels. However, anatomical reconstruction of the proprioceptors and connected tendons revealed major biomechanical differences between subtypes. We built a model of the proprioceptors and tendons that identified a biomechanical mechanism for joint angle selectivity and predicted the existence of a topographic map of joint angle, which we confirmed using calcium imaging. Our findings suggest that biomechanical specialization is a key determinant of proprioceptor feature selectivity in Drosophila. More broadly, the discovery of proprioceptive maps reveals common organizational principles between proprioception and other topographically organized sensory systems.

TUG1-mediated R-loop resolution at microsatellite loci as a prerequisite for cancer cell proliferation.

Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.

Functional architecture of neural circuits for leg proprioception in Drosophila.

To effectively control their bodies, animals rely on feedback from proprioceptive mechanosensory neurons. In the Drosophila leg, different proprioceptor subtypes monitor joint position, movement direction, and vibration. Here, we investigate how these diverse sensory signals are integrated by central proprioceptive circuits. We find that signals for leg joint position and directional movement converge in second-order neurons, revealing pathways for local feedback control of leg posture. Distinct populations of second-order neurons integrate tibia vibration signals across pairs of legs, suggesting a role in detecting external substrate vibration. In each pathway, the flow of sensory information is dynamically gated and sculpted by inhibition. Overall, our results reveal parallel pathways for processing of internal and external mechanosensory signals, which we propose mediate feedback control of leg movement and vibration sensing, respectively. The existence of a functional connectivity map also provides a resource for interpreting connectomic reconstruction of neural circuits for leg proprioception.

Neural Coding of Leg Proprioception in Drosophila.

Animals rely on an internal sense of body position and movement to effectively control motor behavior. This sense of proprioception is mediated by diverse populations of mechanosensory neurons distributed throughout the body. Here, we investigate neural coding of leg proprioception in Drosophila, using in vivo two-photon calcium imaging of proprioceptive sensory neurons during controlled movements of the fly tibia. We found that the axons of leg proprioceptors are organized into distinct functional projections that contain topographic representations of specific kinematic features. Using subclass-specific genetic driver lines, we show that one group of axons encodes tibia position (flexion/extension), another encodes movement direction, and a third encodes bidirectional movement and vibration frequency. Overall, our findings reveal how proprioceptive stimuli from a single leg joint are encoded by a diverse population of sensory neurons, and provide a framework for understanding how proprioceptive feedback signals are used by motor circuits to coordinate the body.

Antennal mechanosensory neurons mediate wing motor reflexes in flying Drosophila.

Although many behavioral studies have shown the importance of antennal mechanosensation in various aspects of insect flight control, the identities of the mechanosensory neurons responsible for these functions are still unknown. One candidate is the Johnston's organ (JO) neurons that are located in the second antennal segment and detect phasic and tonic rotations of the third antennal segment relative to the second segment. To investigate how different classes of JO neurons respond to different types of antennal movement during flight, we combined 2-photon calcium imaging with a machine vision system to simultaneously record JO neuron activity and the antennal movement from tethered flying fruit flies (Drosophila melanogaster). We found that most classes of JO neurons respond strongly to antennal oscillation at the wing beat frequency, but not to the tonic deflections of the antennae. To study how flies use input from the JO neurons during flight, we genetically ablated specific classes of JO neurons and examined their effect on the wing motion. Tethered flies flying in the dark require JO neurons to generate slow antiphasic oscillation of the left and right wing stroke amplitudes. However, JO neurons are not necessary for this antiphasic oscillation when visual feedback is available, indicating that there are multiple pathways for generating antiphasic movement of the wings. Collectively, our results are consistent with a model in which flying flies use JO neurons to detect increases in the wing-induced airflow and that JO neurons are involved in a response that decreases contralateral wing stoke amplitude.

Octopamine neurons mediate flight-induced modulation of visual processing in Drosophila.

BACKGROUND: Activity-dependent modulation of sensory systems has been documented in many organisms and is likely to be essential for appropriate processing of information during different behavioral states. However, the mechanisms underlying these phenomena remain poorly characterized. RESULTS: We investigated the role of octopamine neurons in the flight-dependent modulation observed in visual interneurons in Drosophila. The vertical system (VS) cells exhibit a boost in their response to visual motion during flight compared to quiescence. Pharmacological application of octopamine evokes responses in quiescent flies that mimic those observed during flight, and octopamine cells that project to the optic lobes increase in activity during flight. Using genetic tools to manipulate the activity of octopamine neurons, we find that they are both necessary and sufficient for the flight-induced visual boost. CONCLUSIONS: This study provides the first evidence that endogenous release of octopamine is involved in state-dependent modulation of visual interneurons in flies.

Active and passive antennal movements during visually guided steering in flying Drosophila.

Insects use feedback from a variety of sensory modalities, including mechanoreceptors on their antennae, to stabilize the direction and speed of flight. Like all arthropod appendages, antennae not only supply sensory information but may also be actively positioned by control muscles. However, how flying insects move their antennae during active turns and how such movements might influence steering responses are currently unknown. Here we examined the antennal movements of flying Drosophila during visually induced turns in a tethered flight arena. In response to both rotational and translational patterns of visual motion, Drosophila actively moved their antennae in a direction opposite to that of the visual motion. We also observed two types of passive antennal movements: small tonic deflections of the antenna and rapid oscillations at wing beat frequency. These passive movements are likely the result of wing-induced airflow and increased in magnitude when the angular distance between the wing and the antenna decreased. In response to rotational visual motion, increases in passive antennal movements appear to trigger a reflex that reduces the stroke amplitude of the contralateral wing, thereby enhancing the visually induced turn. Although the active antennal movements significantly increased antennal oscillation by bringing the arista closer to the wings, it did not significantly affect the turning response in our head-fixed, tethered flies. These results are consistent with the hypothesis that flying Drosophila use mechanosensory feedback to detect changes in the wing induced airflow during visually induced turns and that this feedback plays a role in regulating the magnitude of steering responses.

Neural representations of airflow in Drosophila mushroom body.

The Drosophila mushroom body (MB) is a higher olfactory center where olfactory and other sensory information are thought to be associated. However, how MB neurons of Drosophila respond to sensory stimuli other than odor is not known. Here, we characterized the responses of MB neurons to a change in airflow, a stimulus associated with odor perception. In vivo calcium imaging from MB neurons revealed surprisingly strong and dynamic responses to an airflow stimulus. This response was dependent on the movement of the 3(rd) antennal segment, suggesting that Johnston's organ may be detecting the airflow. The calyx, the input region of the MB, responded homogeneously to airflow on. However, in the output lobes of the MB, different types of MB neurons responded with different patterns of activity to airflow on and off. Furthermore, detailed spatial analysis of the responses revealed that even within a lobe that is composed of a single type of MB neuron, there are subdivisions that respond differently to airflow on and off. These subdivisions within a single lobe were organized in a stereotypic manner across flies. For the first time, we show that changes in airflow affect MB neurons significantly and these effects are spatially organized into divisions smaller than previously defined MB neuron types.

Imaging of an early memory trace in the Drosophila mushroom body.

Extensive molecular, genetic, and anatomical analyses have suggested that olfactory memory is stored in the mushroom body (MB), a higher-order olfactory center in the insect brain. The MB comprises three subtypes of neurons with axons that extend into different lobes. A recent functional imaging study has revealed a long-term memory trace manifested as an increase in the Ca(2+) activity in an axonal branch of a subtype of MB neurons. However, early memory traces in the MB remain elusive. We report here learning-induced changes in Ca(2+) activities during early memory formation in a different subtype of MB neurons. We used three independent in vivo and in vitro preparations, and all of them showed that Ca(2+) activities in the axonal branches of alpha'/beta' neurons in response to a conditioned olfactory stimulus became larger compared with one that was not conditioned. The changes were dependent on proper G-protein signaling in the MB. The importance of these changes in the Ca(2+) activity of alpha'/beta' neurons during early memory formation was further tested behaviorally by disrupting G-protein signaling in these neurons or blocking their synaptic outputs during the learning and memory process. Our results suggest that increased Ca(2+) activity in response to a conditioned olfactory stimulus may be a neural correlate of early memory in the MB.

Target-specific short-term dynamics are important for the function of synapses in an oscillatory neural network.

Short-term dynamics such as facilitation and depression are present in most synapses and are often target-specific even for synapses from the same type of neuron. We examine the dynamics and possible functions of two synapses from the same presynaptic neuron in the rhythmically active pyloric network of the spiny lobster. Using simultaneous recordings, we show that the synapses from the lateral pyloric (LP) neuron to the pyloric dilator (PD; a member of the pyloric pacemaker ensemble) and the pyloric constrictor (PY) neurons both show short-term depression. However, the postsynaptic potentials produced by the LP-to-PD synapse are larger in amplitude, depress less, and recover faster than those produced by the LP-to-PY synapse. The main function of the LP-to-PD synapse is to slow down the pyloric rhythm. However, in some cases, it slows down the rhythm only when it is fast and has no effect or to speeds up when it is slow. In contrast, the LP-to-PY synapse functions to delay the activity of the PY neuron; this delay increases as the cycle period becomes longer. Using a computational model, we show that the short-term dynamics of synaptic depression observed for each of these synapses are tailored to their individual functions and that replacing the dynamics of either synapse with the other would disrupt these functions. Together, the experimental and modeling results suggest that the target-specific features of short-term synaptic depression are functionally important for synapses efferent from the same presynaptic neuron.

Dynamic interaction of oscillatory neurons coupled with reciprocally inhibitory synapses acts to stabilize the rhythm period.

In the rhythmically active pyloric circuit of the spiny lobster, the pyloric dilator (PD) neurons are members of the pacemaker group of neurons that make inhibitory synapses onto the follower lateral pyloric (LP) neuron. The LP neuron, in turn, makes a depressing inhibitory synapse to the PD neurons, providing the sole inhibitory feedback from the pyloric network to its pacemakers. This study investigates the dynamic interaction between the pyloric cycle period, the two types of neurons, and the feedback synapse in biologically realistic conditions. When the rhythm period was changed, the membrane potential waveform of the LP neuron was affected with a consistent pattern. These changes in the LP neuron waveform directly affected the dynamics of the LP to PD synapse and caused the postsynaptic potential (PSP) in the PD neurons to both peak earlier in phase and become larger in amplitude. Using an artificial synapse implemented in dynamic clamp, we show that when the LP to PD PSP occurred early in phase, it acted to speed up the pyloric rhythm, and larger PSPs also strengthened this trend. Together, these results indicate that interactions between these two types of neurons can dynamically change in response to increases in the rhythm period, and this dynamic change provides a negative feedback to the pacemaker group that could work to stabilize the rhythm period.

Short-term dynamics of a mixed chemical and electrical synapse in a rhythmic network.

In the rhythmically active pyloric circuit of the spiny lobster, the synapse between the lateral pyloric (LP) neuron and pyloric constrictor (PY) neuron has an inhibitory depressing chemical and an electrical component. To understand how the dynamics of the LP-->PY synapse affect the relative firing times between these two neurons in an ongoing rhythm, we characterized the dynamics of the LP-->PY synapse after a pharmacological block of ongoing activity. When a train of voltage pulses was applied to the voltage-clamped LP neuron, the inhibitory chemical component of the postsynaptic potential (PSP) in the PY neuron rapidly depressed. Thus, after the first few pulses, the PSP was either hyperpolarizing or depolarizing, depending on the interpulse duration, with shorter interpulse durations producing depolarizing PSPs. To characterize the synaptic response during rhythmic activity, we played back prerecorded realistic waveforms in the voltage-clamped LP neuron. After an initial transient, the resulting PSP in PY was always depolarizing, suggesting that in an ongoing rhythm, the electrical component of the synapse is dominant. However, our results indicate that the chemical component of the synapse acts to delay the peak time of the PSP and to reduce its amplitude, and that these effects become more important at slower cycle periods.