in-vivo-imaging-neural-activity-unanesthetized-drosophila-adult

<em>In Vivo</em> Imaging of Neural Activity in Unanesthetized <em>Drosophila</em> Adult Flies

In vivo imaging is crucial for studying neurobiology in Drosophila as it allows real-time visualization of neuronal activity, development, and plasticity ...

Georgetown University

Active memory forgetting is a well-regulated biological process thought to be adaptive and to allow proper cognitive functions. Recent efforts have elucidated several molecular players involved in the regulation of olfactory forgetting in , including the small G protein Rac1, the dopamine receptor DAMB, and the scaffold protein Scribble. Similarly, we recently reported that dopaminergic neurons mediate both learning- and forgetting-induced plasticity in the mushroom body output neuron MBON-γ2α'1. Two open questions remain: how does forgetting affect plasticity in other, highly plastic, mushroom body compartments and how do genes that regulate forgetting affect this cellular plasticity? Here, we show that forgetting reverses short-term synaptic depression induced by aversive conditioning in the highly plastic mushroom body output neuron MBON-γ1pedc>α/β. In addition, our results indicate that genetic tampering with normal forgetting by inhibition of small G protein Rac1 impairs restoration of depressed odor responses to learned odor by intrinsic forgetting through time passing and forgetting induced acutely by shock stimulation after conditioning or reversal learning. These data further indicate that some forms of forgetting truly erase physiological changes generated by memory encoding.

Rac1 Impairs Forgetting-Induced Cellular Plasticity in Mushroom Body Output Neurons.

Understanding the nature of the molecular mechanisms underlying memory formation, consolidation, and forgetting are some of the fascinating questions in modern neuroscience. The encoding, stabilization and elimination of memories, rely on the structural reorganization of synapses. These changes will enable the facilitation or depression of neural activity in response to the acquisition of new information. In other words, these changes affect the weight of specific nodes within a neural network. We know that these plastic reorganizations require  protein synthesis in the context of Long-term memory (LTM). This process depends on neural activity triggered by the learned experience. The use of model organisms like  has been proven essential for advancing our knowledge in the field of neuroscience. Flies offer an optimal combination of a more straightforward nervous system, composed of a limited number of cells, and while still displaying complex behaviors. Studies in  neuroscience, which expanded over several decades, have been critical for understanding the cellular and molecular mechanisms leading to the synaptic and behavioral plasticity occurring in the context of learning and memory. This is possible thanks to sophisticated technical approaches that enable precise control of gene expression in the fruit fly as well as neural manipulation, like chemogenetics, thermogenetics, or optogenetics. The search for the identity of genes expressed as a result of memory acquisition has been an active interest since the origins of behavioral genetics. From screenings of more or less specific candidates to broader studies based on transcriptome analysis, our understanding of the genetic control behind LTM has expanded exponentially in the past years. Here we review recent literature regarding how the formation of memories induces a rapid, extensive and, in many cases, transient wave of transcriptional activity. After a consolidation period, transcriptome changes seem more stable and likely represent the synthesis of new proteins. The complexity of the circuitry involved in memory formation and consolidation is such that there are localized changes in neural activity, both regarding temporal dynamics and the nature of neurons and subcellular locations affected, hence inducing specific temporal and localized changes in protein expression. Different types of neurons are recruited at different times into memory traces. In LTM, the synthesis of new proteins is required in specific subsets of cells. This  translation can take place in the somatic cytoplasm and/or locally in distinct zones of compartmentalized synaptic activity, depending on the nature of the proteins and the plasticity-inducing processes that occur. We will also review recent advances in understanding how localized changes are confined to the relevant synapse. These recent studies have led to exciting discoveries regarding proteins that were not previously involved in learning and memory processes. This invaluable information will lead to future functional studies on the roles that hundreds of new molecular actors play in modulating neural activity.

The Making of Long-Lasting Memories: A Fruit Fly Perspective.

Learning and memory storage is a complex process that has proven challenging to tackle. It is likely that, in nature, the instructive value of reinforcing experiences is acquired rather than innate. The association between seemingly neutral stimuli increases the gamut of possibilities to create meaningful associations and the predictive power of moment-by-moment experiences. Here, we report physiological and behavioral evidence of olfactory unimodal sensory preconditioning in fruit flies. We show that the presentation of a pair of odors (S1 and S2) before one of them (S1) is associated with electric shocks elicits a conditional response not only to the trained odor (S1) but to the odor previously paired with it (S2). This occurs even if the S2 odor was never presented in contiguity with the aversive stimulus. In addition, we show that inhibition of the small G protein , a known forgetting regulator, facilitates the association between S1/S2 odors. These results indicate that flies can infer value to olfactory stimuli based on the previous associative structure between odors, and that inhibition of  lengthens the time window of the olfactory 'sensory buffer', allowing the establishment of associations between odors presented in sequence.

Higher-order unimodal olfactory sensory preconditioning in .

Understanding the physiological mechanisms that modulate memory acquisition and consolidation remains among the most ambitious questions in neuroscience. Massive efforts have been dedicated to deciphering how experience affects behavior, and how different physiological and sensory phenomena modulate memory. Our ability to encode, consolidate and retrieve memories depends on internal drives, and sleep stands out among the physiological processes that affect memory: one of the most relatable benefits of sleep is the aiding of memory that occurs in order to both prepare the brain to learn new information, and after a learning task, to consolidate those new memories.  lends itself to the study of the interactions between memory and sleep. The fruit fly provides incomparable genetic resources, a mapped connectome, and an existing framework of knowledge on the molecular, cellular, and circuit mechanisms of memory and sleep, making the fruit fly a remarkable model to decipher the sophisticated regulation of learning and memory by the quantity and quality of sleep. Research in  has stablished not only that sleep facilitates learning in wild-type and memory-impaired animals, but that sleep deprivation interferes with the acquisition of new memories. In addition, it is well-accepted that sleep is paramount in memory consolidation processes. Finally, studies in  have shown that that learning itself can promote sleep drive. Nevertheless, the molecular and network mechanisms underlying this intertwined relationship are still evasive. Recent remarkable work has shed light on the neural substrates that mediate sleep-dependent memory consolidation. In a similar way, the mechanistic insights of the neural switch control between sleep-dependent and sleep-independent consolidation strategies were recently described. This review will discuss the regulation of memory by sleep in , focusing on the most recent advances in the field and pointing out questions awaiting to be investigated.

Sleep benefits different stages of memory in .

Protein retention expansion microscopy (ExM) retains genetically encoded fluorescent proteins or antibody-conjugated fluorescent probes in fixed tissue and isotropically expands the tissue through a swellable polymer network to allow nanoscale (<70 nm) resolution on diffraction-limited confocal microscopes. Despite numerous advantages ExM brings to biological studies, the full protocol is time-consuming and can take multiple days to complete. Here, we adapted the ExM protocol to the vibratome-sectioned brain tissue of  tadpoles and implemented a microwave-assisted protocol to reduce the workflow from days to hours. In addition to the significantly accelerated processing time, our microwave-assisted ExM (ExM) protocol maintains the superior resolution and signal-to-noise ratio of the original ExM protocol. Furthermore, the ExM protocol yields higher magnitude of expansion, suggesting that in addition to accelerating the process through increased diffusion rate of reagents, microwave radiation may also facilitate the expansion process. To demonstrate the applicability of this method to other specimens and protocols, we adapted the microwave-accelerated protocol to whole mount adult brain tissue of  fruit flies, and successfully reduced the total processing time of a widely-used  IHC-ExM protocol from 6 days to 2 days. Our results demonstrate that with appropriate adjustment of the microwave parameters (wattage, pulse duration, interval, and number of cycles), this protocol can be readily adapted to different model organisms and tissue types to greatly increase the efficiency of ExM experiments.

Accelerated protein retention expansion microscopy using microwave radiation.

Protein retention expansion microscopy (ExM) retains fluorescent signals in fixed tissue and isotropically expands the tissue to allow nanoscale (<70 nm) resolution on diffraction-limited confocal microscopes. Despite the numerous advantages of ExM, the protocol is time-consuming. Here, we adapted an ExM protocol to vibratome-sectioned brain tissue of Xenopus laevis tadpoles and implemented a microwave (M/W)-assisted protocol (ExM) to reduce the workflow from days to hours. Our ExM protocol maintains the superior resolution of the original ExM protocol and yields a higher magnitude of expansion, suggesting that M/W radiation may also facilitate the expansion process. We then adapted the M/W protocol to the whole-mount brain of Drosophila melanogaster fruit flies, and successfully reduced the processing time of a widely used Drosophila IHC-ExM protocol from 6 to 2 days. This demonstrates that with appropriate adjustment of M/W parameters, this protocol can be readily adapted to different organisms and tissue types to greatly increase the efficiency of ExM experiments.

Article	Year
Rac1 Impairs Forgetting-Induced Cellular Plasticity in Mushroom Body Output Neurons. Frontiers in cellular neuroscience\| PubMed ID: 33061890	2020
The Making of Long-Lasting Memories: A Fruit Fly Perspective. Frontiers in behavioral neuroscience\| PubMed ID: 33859556	2021
Higher-order unimodal olfactory sensory preconditioning in . eLife\| PubMed ID: 36129180	2022
Sleep benefits different stages of memory in . Frontiers in physiology\| PubMed ID: 36744027	2023
Accelerated protein retention expansion microscopy using microwave radiation. bioRxiv : the preprint server for biology\| PubMed ID: 38766072	2024
Accelerated protein retention expansion microscopy using microwave radiation. Cell reports methods\| PubMed ID: 39579759	2024

Isaac Cervantes-Sandoval

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