A conditional glutamatergic synaptic vesicle marker for Drosophila

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.

Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons

Brian Donal McCabe

Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both "classical" and "modulatory" signals that could produce diverse and/or complementary effects in associated circuits. Here, we establish that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. Our findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network. Author summary Neurons communicate with each other via electrical events and the release of chemical signals. An emerging challenge in understanding neuron communication is the realization that many neurons release more than one type of chemical signal or neurotransmitter. Here we ask how does the release of more than one neurotransmitter from a single neuron impact circuits that control behavior? We determined the monoamine octopamine and the classical transmitter glutamate are co-expressed in the Drosophila adult CNS. By manipulating the release of glutamate in OA-glutamate neurons, we demonstrated glutamate has both separable actions and complementary actions with OA on aggression and reproductive behaviors respectively. Aggression is a behavior that is highly conserved between organisms and present in many human disease states, including depression and Alzheimer's disease. Our results show that aggressive behavior requires the release of both neurotransmitters in dual-transmitting neurons and suggests within this set of neurons, glutamate may provide a new therapeutic target to modulate aggression in pathological conditions.

2020

A conditional GABAergic synaptic vesicle marker for Drosophila

Brian Donal McCabe

Background: Throughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others. New method: Since the structure and features of GABA synapses are critical to understanding their function within specific neural circuits of interest, here we developed and characterized a conditional marker of GABAergic synaptic vesicles for Drosophila, 9XV5-vGAT. Results: 9XV5-vGAT is validated for conditionality of expression, specificity for localization to synaptic vesicles, specificity for expression in GABAergic neurons, and functionality. Its utility for GABAergic neurotransmitter phenotyping and identification of GABA release sites was verified for ellipsoid body neurons of the central complex. In combination with previously reported conditional SV markers for acetylcholine and glutamate, 9XV5-vGAT was used to demonstrate fast neurotransmitter phenotyping of subesophageal ganglion neurons. Comparison with existing methods: This method is an alternative to single cell transcriptomics for neurotransmitter phenotyping and can be applied to any neurons of interest represented by a binary transcription system driver. Conclusion: A conditional GABAergic synaptic vesicle marker has been developed and validated for GABA neurotransmitter phenotyping and subcellular localization of GABAergic synaptic vesicles.

2022

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 encoding of behavior in the adult Drosophila motor system

Chin-Lin Chen

EPFL2022

Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons

Brian Donal McCabe

2020