Methods for acute and long-term imaging of the ventral nerve cord in behaving adult Drosophila

Understanding how neural circuits remodel and adapt to animal behavior is a central theme in the field of Neuroscience. One strategy to reach this goal is to repeatedly record the same animal's neural circuits and observe how they adapt when facing different challenges. Research has primarily focused on identifying neural circuits and connectivity maps within the brains of model organisms. Nevertheless, it has become evident that functional recordings are essential to have a complete understanding of how neural circuits generate behaviors. Numerous techniques have been introduced to record activity from brain circuits in model animals spanning from monkeys to insects. However, imaging the spinal cord in mammals or the ventral nerve cord in insects remains an open challenge due to the complexity of obtaining optical access without impacting the animal's behavior and damaging neural tissues. As a result, we have very limited information on how neural circuits in the spinal cord generate behaviors and how they adapt to different experiences such as injury or aging. Drosophila is a perfect model organism for carrying out this line of research thanks to its small size, genetically tractable nervous system, and behavioral diversity. The results are relevant to human physiology and behavior as many mechanisms have homologs in mammals. What is missing is a technological advancement that allows recording of behavior, structural changes in the circuits, and spatiotemporal dynamics of neural activity on the same animal.This thesis addresses this gap and introduces two novel methods for in vivo functional recordings of the fly's ventral nerve cord (VNC). The first one gains short-term access to the fly's VNC using a novel dissection preparation. The second one enables long-term functional recordings of the fly's VNC thanks to a toolkit combining four microengineered devices: a surgical arm, a V-shape implant, an optically transparent and numbered window and a remounting stage. The toolkit is suitable for both male and female flies and provides optical access to the VNC for at least one month. Additionally, the long-term toolkit does not impact flies' behaviors compared to intact flies and can be used to image both sparse and large populations of neural activity in flies' cervical connective and VNC. The toolkit was tested in two proof-of-concept (POC) applications. In the first POC, the toolkit monitored the degradation of sensory neurons in the VNC over weeks following limb amputation. In the second POC, the toolkit monitored global changes in neural dynamics following ingestion of a high-concentration caffeine solution. In conclusion, this thesis introduces new techniques for functional recordings of the fly's cervical connective and VNC over short and long time scales. They enable optical access to the fly's cervical connective that can be leveraged to study the content of information transmitted between the brain and the VNC while the fly is behaving. The two POCs demonstrate that the novel technology is ideally suited for long-term experiments and could be applied to a wide range of studies. For instance, the toolkit could be used to investigate how behaviors are encoded in the fly's VNC and how they adapt over time to experiences such as disease, aging, drug intake and learning.

Uncovering the neural encoding of behavior in the adult Drosophila motor system

Chin-Lin Chen

One of the most important goals in neuroscience research has always been to understand how animals control their behavior. However, the long focus on the role of brain neurons in behavioral control might be missing the full story. In fact, brain-wide fluctuations in neural activity are highly correlated to the behavioral state, suggesting a closed-loop system where the behavioral command and the body state interact to interpret sensory inputs and dynamically guide action selection. However, to date, little is known about how and where information about the behavioral state is generated. In principle, information about the behavioral state is a relatively high-level encoding of the body state derived from the integration of signals from primary sensors and the local network. Local motor circuits residing in the spinal cord of mammals or the ventral nerve cord (VNC) of insects may give rise to the behavioral state and pass to the brain via brain-projecting interneurons---termed ascending neurons (ANs). However, what information ANs carry and where this information originates is still a mystery due to the technical challenges in measuring these signals in behaving animals. Therefore, it is critical to develop tools to track neuron identity and record neuronal activity during behavior to crack the functional network. In this work, a method is introduced for imaging the motor circuit in the VNC of Drosophila melanogaster. This preparation demonstrates how to retain fly limb function after dissection and allows simultaneous recording of a tethered fly's behavior and its neural activity in the VNC. Incorporating this methodology with other approaches can verify the conclusions from the experiments such as those manipulating neuronal excitability, and motivate further investigation into neuronal function in specific types of behavior. Since it has long been theorized that ANs are the cellular basis of the behavioral state information sent in the brain, the technique introduced in this work was designed to resolve questions relating to what type of information ANs convey. Here, a large-scale functional screen was performed to relate neural activity, behavior, and neuronal morphology for 247 genetically identified ANs in the adult fly. This is the first large-scale screen of neural activity in a behaving animal, and this work uncovered three fundamental features of AN populations. First, rather than low-level limb movements, ANs encode high-level behaviors like walking, grooming, and resting. Second, ANs project nearly exclusively to an integrative sensory center (AVLP) and an action selection hub (GNG). Remarkably, the encodings projected to these distinct areas reflect their potential roles in contextualizing sensory cues (AVLP) and guiding future actions (GNG). Third, the behavioral encodings of ANs are closely linked to their motor system patterning. In conclusion, this work provides an indispensable tool to study the motor system beyond the brain network by overcoming a major technical barrier in in-vivo recording of the VNC of a behaving fly. This tool was used to help resolve what is encoded by ANs as a higher-level behavioral state, suggesting that the primary cellular origin of the behavioral information that is used to modulate brain function comes from the VNC. Overall, the technique and the findings on AN encoding should inspire follow-up studies to uncover additional neural mechanisms used in adaptive behavior generation.

EPFL2022

Uncovering the neural substrates of communication between the brain and ventral nerve cord in Drosophila melanogaster (italic)

Florian Aymanns

Despite a long history of research in motor control, the exact mechanism of how the brain communicates with the the invertebrate ventral nerve cord (VNC) and the vertebrate spinal cord on a single neuron basis remains largely elusive.Drosophila melanogaster is an ideal model organism to address the role of individual neurons, thanks to its stereotyped nervous system.Descending neurons (DNs), interneurons in the brain that project to the invertebrate ventral nerve cord or vertebrate spinal cord, have been shown to elicit specific behaviors upon optogenetic activation in Drosophila.These results suggest that behavior is controlled by sparse sets of command neurons, each eliciting a particular behavior.However, it remains to be seen how many DNs are involved in the control of naturalistic behaviors and what other information they might encode.In order to avoid commands leading to physically impossible or destabilizing actions, the brain has to be aware of the current behavior state.Ascending neurons (ANs), interneurons in the invertebrate ventral nerve cord or vertebrate spinal cord projecting to the brain, are likely conveying this information to the brain.To which degree of fidelity and in what way ANs encode behavior state remains unclear.To address these questions, we developed a dissection approach that allows functional imaging of ANs and DNs in the cervical connective.This dissection gives us optical access to the cervical connective and parts of the VNC in behaving adult Drosophila such that a two-photon fluorescence microscope can be used to image neural activity via calcium indicators.We began by imaging a new library of sparse split GAL4 driver lines targeting ANs.Our recordings from 247 genetically identifiable ANs revealed neurons encoding walking, resting, turning, eye grooming, and foreleg movements.Anatomical characterization of these ANs showed that they predominantly project to two brain regions: the anterior ventrolateral protocerebrum (AVLP) and the gnathal ganglion (GNG).Our results suggest that ANs encoding the motion of the animal with respect to the surrounding environment (self-motion) predominantly project to the AVLP, an integrative sensory hub. ANs projecting to the GNG mostly encode discrete actions.We then proceeded to image populations of DNs.The majority of DNs we recorded encoded walking behavior, with smaller fractions encoding resting and head grooming.Some of these neurons are likely to drive walking, but we also identified neurons encoding walking speed and turning.This suggests that a core set of DNs drives a behavior whose features can be modulated by additional DNs.Besides encoding behaviors, some DNs encode sensory information, including the presence of odors and deflection of the antennae.

EPFL2022

A conditional glutamatergic synaptic vesicle marker for Drosophila

Brian Donal McCabe, Evelyne Ruchti

Glutamate is a principal neurotransmitter used extensively by the nervous systems of all vertebrate and invertebrate animals. It is primarily an excitatory neurotransmitter that has been implicated in nervous system development, as well as a myriad of brain functions from the simple transmission of information between neurons to more complex aspects of nervous system function including synaptic plasticity, learning, and memory. Identification of glutamatergic neurons and their sites of glutamate release are thus essential for understanding the mechanisms of neural circuit function and how information is processed to generate behavior. Here, we describe and characterize smFLAG-vGlut, a conditional marker of glutamatergic synaptic vesicles for the Drosophila model system. smFLAG-vGlut is validated for functionality, conditional expression, and specificity for glutamatergic neurons and synaptic vesicles. The utility of smFLAG-vGlut is demonstrated by glutamatergic neurotransmitter phenotyping of 26 different central complex neuron types of which nine were established to be glutamatergic. This illumination of glutamate neurotransmitter usage will enhance the modeling of central complex neural circuitry and thereby our understanding of information processing by this region of the fly brain. The use of smFLAG for glutamatergic neurotransmitter phenotyping and identification of glutamate release sites can be extended to any Drosophila neuron(s) represented by a binary transcription system driver.

OXFORD UNIV PRESS INC2022

Uncovering the neural encoding of behavior in the adult Drosophila motor system

Chin-Lin Chen

EPFL2022

Uncovering the neural substrates of communication between the brain and ventral nerve cord in Drosophila melanogaster (italic)

Florian Aymanns

EPFL2022