taci-an-imagej-plugin-for-3d-calcium-imaging-analysis

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging ...

behavioral-assays-for-optogenetic-manipulation-neural-circuits

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in <em>Drosophila melanogaster</em>

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Virginia Polytechnic Institute and State University

Virginia Tech

PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.

PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis.

Temperature is a critical environmental variable that affects the distribution, survival and reproduction of most animals. Although temperature receptors have been identified in many animals, how these receptors respond to temperature is still unclear. Here, we describe an automated tracking method for studying the thermotactic behaviors of Drosophila larvae and adults. We built optimal experimental setups to capture behavioral recordings and analyzed them using free software, Fiji and TrackMate, which do not require programming knowledge. Then, we applied the adult thermotactic two-choice assay to examine the movement and temperature preferences of nine Drosophila species. The ability or inclination to move varied among these species and at different temperatures. Distinct species preferred various ranges of temperatures. Wild-type D. melanogaster flies avoided the warmer temperature in the warm avoidance assay and the cooler temperature in the cool avoidance assay. Conversely, D. bipectinata and D. yakuba did not avoid warm or cool temperatures in the respective assays, and D. biarmipes and D. mojavensis did not avoid the warm temperature in the warm avoidance assay. These results demonstrate that Drosophila species have different mobilities and temperature preferences, which will benefit further research in exploring molecular mechanisms of temperature responsiveness.

Responses of different Drosophila species to temperature changes.

Thermosensation is critical for avoiding thermal extremes and regulating body temperature. While thermosensors activated by noxious temperatures respond to hot or cold, many innocuous thermosensors exhibit robust baseline activity and lack discrete temperature thresholds, suggesting they are not simply warm and cool detectors. Here, we investigate how the aristal Cold Cells encode innocuous temperatures in Drosophila. We find they are not cold sensors but cooling-activated and warming-inhibited phasic thermosensors that operate similarly at warm and cool temperatures; we propose renaming them "Cooling Cells." Unexpectedly, Cooling Cell thermosensing does not require the previously reported Brivido Transient Receptor Potential (TRP) channels. Instead, three Ionotropic Receptors (IRs), IR21a, IR25a, and IR93a, specify both the unique structure of Cooling Cell cilia endings and their thermosensitivity. Behaviorally, Cooling Cells promote both warm and cool avoidance. These findings reveal a morphogenetic role for IRs and demonstrate the central role of phasic thermosensing in innocuous thermosensation. VIDEO ABSTRACT.

Ionotropic Receptors Specify the Morphogenesis of Phasic Sensors Controlling Rapid Thermal Preference in Drosophila.

Ionotropic receptors (IRs) are a highly divergent subfamily of ionotropic glutamate receptors (iGluR) and are conserved across Protostomia, a major branch of the animal kingdom that encompasses both Ecdysozoa and Lophothrochozoa. They are broadly expressed in peripheral sensory systems, concentrated in sensory dendrites, and function in chemosensation, thermosensation, and hygrosensation. As iGluRs, four IR subunits form a functional ion channel to detect environmental stimuli. Most IR receptors comprise individual stimulus-specific tuning receptors and one or two broadly expressed coreceptors. This review summarizes the discoveries of the structure of IR complexes and the expression and function of each IR, as well as discusses the future direction for IR studies.

The Structure and Function of Ionotropic Receptors in .

Temperature sensation guides animals to avoid temperature extremes and to seek their optimal temperatures. The larval stage of Drosophila development has a dramatic effect on temperature preference. While early-stage Drosophila larvae pursue a warm temperature, late-stage larvae seek a significantly lower temperature. Previous studies suggest that this transition depends on multiple rhodopsins at the late larval stage. Here, we show that early-stage larvae, in which dorsal organ cool cells (DOCCs) are functionally blocked, exhibit similar cool preference to that of wild type late-stage larvae. The molecular thermoreceptors in DOCCs are formed by three members of the Ionotropic Receptor (IR) family, IR21a, IR93a, and IR25a. Early-stage larvae of each Ir mutant pursue a cool temperature, similar to that of wild type late-stage larvae. At the late larval stage, DOCCs express decreased IR proteins and exhibit reduced cool responses. Importantly, late-stage larvae that overexpress IR21a, IR93a, and IR25a in DOCCs exhibit similar warm preference to that of wild type early-stage larvae. These data suggest that IR21a, IR93a, and IR25a in DOCCs navigate early-stage larvae to avoid cool temperatures and the reduction of these IR proteins in DOCCs results in animals remaining in cool regions during the late larval stage. Together with previous studies, we conclude that multiple temperature-sensing systems are regulated for the transition of temperature preference in fruit fly larvae.

Ionotropic Receptor-dependent cool cells control the transition of temperature preference in Drosophila larvae.

A neural circuit is composed of a population of neurons that are interconnected by synapses and carry out a specific function when activated. It is the structural framework for all brain functions. Its impairments often cause diseases in the nervous system. To understand computations and functions in a brain circuit, it is of crucial importance to identify how neurons in this circuit are connected. Genetic transsynaptic techniques provide opportunities to efficiently answer this question. These techniques label synapses or across synapses to unbiasedly label synaptic partners. They allow for mapping neural circuits with high reproducibility and throughput, as well as provide genetic access to synaptically connected neurons that enables visualization and manipulation of these neurons simultaneously. This review focuses on three recently developed  genetic transsynaptic tools for detecting chemical synapses, highlights their advantages and potential pitfalls, and discusses the future development needs of these techniques.

Genetic Transsynaptic Techniques for Mapping Neural Circuits in .

Animals are continuously confronted with different rates of temperature variation. The mechanism underlying how temperature-sensing systems detect and respond to fast and slow temperature changes is not fully understood in fly larvae. Here, we applied two-choice behavioral assays to mimic fast temperature variations and a gradient assay to model slow temperature changes. Previous research indicates that Rhodopsin 1 (Rh1) and its phospholipase C (PLC) cascade regulate fast and slow temperature responses. We focused on the ionotropic receptors (IRs) expressed in dorsal organ ganglions (DOG), in which dorsal organ cool-activated cells (DOCCs) and warm-activated cells (DOWCs) rely on IR-formed cool and warm receptors to respond to temperature changes. In two-choice assays, both cool and warm IRs are sufficient for selecting 18°C between 18°C and 25°C but neither function in cool preferences between 25°C and 32°C. The Rh1 pathway, on the other hand, contributes to choosing preferred temperatures in both assays. In a gradient assay, cool and warm IR receptors exert opposite effects to guide animals to ∼25°C. Cool IRs drive animals to avoid cool temperatures, whereas warm IRs guide them to leave warm regions. The Rh1 cascade and warm IRs may function in the same pathway to drive warm avoidance in gradient assays. Moreover, IR92a is not expressed in temperature-responsive neurons but regulates the activation of DOWCs and the deactivation of DOCCs. Together with previous studies, we conclude that multiple thermosensory systems, in various collaborative ways, help larvae to make their optimal choices in response to different rates of temperature change.

Cool and warm ionotropic receptors control multiple thermotaxes in  larvae.

Optogenetics has made a significant impact on neuroscience, allowing activation and inhibition of neural activity with exquisite spatiotemporal precision in response to light. In this lab session, we use fruit flies to help students understand the fundamentals of optogenetics through hands-on activities. The CsChrimson channelrhodopsin, a light-activated cation channel, is expressed in sweet and bitter sensory neurons. Sweet sensory neurons guide animals to identify nutrient-rich food and drive appetitive behaviors, while bitter sensory neurons direct animals to avoid potentially toxic substances and guide aversive behavior. Students use two-choice assays to explore the causality between the stimulation activation of these neurons and the appetitive and avoidance behaviors of the fruit flies. To quantify their observations, students calculate preference indices and use the Student's t-test to analyze their data. After this lab session, students are expected to have a basic understanding of optogenetics, fly genetics, sensory perception, and how these relate to sensory-guided behaviors. They will also learn to conduct, quantify, and analyze two-choice behavioral assays.

Lina Ni

School of Neuroscience

Virginia Polytechnic Institute and State University

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Lina Ni

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Neuroscience

Virginia Polytechnic Institute and State University

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

School of Neuroscience