Accelerated protein retention expansion microscopy using microwave radiation.

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 Xenopus laevis 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 ( M/W ExM) protocol maintains the superior resolution and signal-to-noise ratio of the original ExM protocol. Furthermore, the M/W 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 Drosophila melanogaster fruit flies, and successfully reduced the total processing time of a widely-used Drosophila 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.

Sleep benefits different stages of memory in Drosophila.

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. Drosophila 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 Drosophila 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 Drosophila 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 Drosophila, focusing on the most recent advances in the field and pointing out questions awaiting to be investigated.

Higher-order unimodal olfactory sensory preconditioning in Drosophila.

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 Rac1, 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 Rac1 lengthens the time window of the olfactory 'sensory buffer', allowing the establishment of associations between odors presented in sequence.

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

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 de novo 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 Drosophila melanogaster 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 Drosophila 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 de novo 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.

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

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 Drosophila, 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/ß. 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.

Reciprocal synapses between mushroom body and dopamine neurons form a positive feedback loop required for learning.

Current thought envisions dopamine neurons conveying the reinforcing effect of the unconditioned stimulus during associative learning to the axons of Drosophila mushroom body Kenyon cells for normal olfactory learning. Here, we show using functional GFP reconstitution experiments that Kenyon cells and dopamine neurons from axoaxonic reciprocal synapses. The dopamine neurons receive cholinergic input via nicotinic acetylcholine receptors from the Kenyon cells; knocking down these receptors impairs olfactory learning revealing the importance of these receptors at the synapse. Blocking the synaptic output of Kenyon cells during olfactory conditioning reduces presynaptic calcium transients in dopamine neurons, a finding consistent with reciprocal communication. Moreover, silencing Kenyon cells decreases the normal chronic activity of the dopamine neurons. Our results reveal a new and critical role for positive feedback onto dopamine neurons through reciprocal connections with Kenyon cells for normal olfactory learning.

Scribble Scaffolds a Signalosome for Active Forgetting.

Forgetting, one part of the brain's memory management system, provides balance to the encoding and consolidation of new information by removing unused or unwanted memories or by suppressing their expression. Recent studies identified the small G protein, Rac1, as a key player in the Drosophila mushroom bodies neurons (MBn) for active forgetting. We subsequently discovered that a few dopaminergic neurons (DAn) that innervate the MBn mediate forgetting. Here we show that Scribble, a scaffolding protein known primarily for its role as a cell polarity determinant, orchestrates the intracellular signaling for normal forgetting. Knocking down scribble expression in either MBn or DAn impairs normal memory loss. Scribble interacts physically and genetically with Rac1, Pak3, and Cofilin within MBn, nucleating a forgetting signalosome that is downstream of dopaminergic inputs that regulate forgetting. These results bind disparate molecular players in active forgetting into a single signaling pathway: Dopamine→ Dopamine Receptor→ Scribble→ Rac→ Cofilin.

Drosophila SLC22A Transporter Is a Memory Suppressor Gene that Influences Cholinergic Neurotransmission to the Mushroom Bodies.

The mechanisms that constrain memory formation are of special interest because they provide insights into the brain's memory management systems and potential avenues for correcting cognitive disorders. RNAi knockdown in the Drosophila mushroom body neurons (MBn) of a newly discovered memory suppressor gene, Solute Carrier DmSLC22A, a member of the organic cation transporter family, enhances olfactory memory expression, while overexpression inhibits it. The protein localizes to the dendrites of the MBn, surrounding the presynaptic terminals of cholinergic afferent fibers from projection neurons (Pn). Cell-based expression assays show that this plasma membrane protein transports cholinergic compounds with the highest affinity among several in vitro substrates. Feeding flies choline or inhibiting acetylcholinesterase in Pn enhances memory, an effect blocked by overexpression of the transporter in the MBn. The data argue that DmSLC22A is a memory suppressor protein that limits memory formation by helping to terminate cholinergic neurotransmission at the Pn:MBn synapse.

MiR-980 Is a Memory Suppressor MicroRNA that Regulates the Autism-Susceptibility Gene A2bp1.

MicroRNAs have been associated with many different biological functions, but little is known about their roles in conditioned behavior. We demonstrate that Drosophila miR-980 is a memory suppressor gene functioning in multiple regions of the adult brain. Memory acquisition and stability were both increased by miR-980 inhibition. Whole cell recordings and functional imaging experiments indicated that miR-980 regulates neuronal excitability. We identified the autism susceptibility gene, A2bp1, as an mRNA target for miR-980. A2bp1 levels varied inversely with miR-980 expression; memory performance was directly related to A2bp1 levels. In addition, A2bp1 knockdown reversed the memory gains produced by miR-980 inhibition, consistent with A2bp1 being a downstream target of miR-980 responsible for the memory phenotypes. Our results indicate that miR-980 represses A2bp1 expression to tune the excitable state of neurons, and the overall state of excitability translates to memory impairment or improvement.

Sleep Facilitates Memory by Blocking Dopamine Neuron-Mediated Forgetting.

Early studies from psychology suggest that sleep facilitates memory retention by stopping ongoing retroactive interference caused by mental activity or external sensory stimuli. Neuroscience research with animal models, on the other hand, suggests that sleep facilitates retention by enhancing memory consolidation. Recently, in Drosophila, the ongoing activity of specific dopamine neurons was shown to regulate the forgetting of olfactory memories. Here, we show this ongoing dopaminergic activity is modulated with behavioral state, increasing robustly with locomotor activity and decreasing with rest. Increasing sleep-drive, with either the sleep-promoting agent Gaboxadol or by genetic stimulation of the neural circuit for sleep, decreases ongoing dopaminergic activity, while enhancing memory retention. Conversely, increasing arousal stimulates ongoing dopaminergic activity and accelerates dopaminergic-based forgetting. Therefore, forgetting is regulated by the behavioral state modulation of dopaminergic-based plasticity. Our findings integrate psychological and neuroscience research on sleep and forgetting.

System-like consolidation of olfactory memories in Drosophila.

System consolidation, as opposed to cellular consolidation, is defined as the relatively slow process of reorganizing the brain circuits that maintain long-term memory. This concept is founded in part on observations made in mammals that recently formed memories become progressively independent of brain regions initially involved in their acquisition and retrieval and dependent on other brain regions for their long-term storage. Here we present evidence that olfactory appetitive and aversive memories in Drosophila evolve using a system-like consolidation process. We show that all three classes of mushroom body neurons (MBNs) are involved in the retrieval of short- and intermediate-term memory. With the passage of time, memory retrieval becomes independent of α'/ß' and γ MBNs, and long-term memory becomes completely dependent on α/ß MBNs. This shift in neuronal dependency for behavioral performance is paralleled by shifts in the activity of the relevant neurons during the retrieval of short-term versus long-term memories. Moreover, transient neuron inactivation experiments using flies trained to have both early and remote memories showed that the α'/ß' MBNs have a time-limited role in memory processing. These results argue that system consolidation is not a unique feature of the mammalian brain and memory systems, but rather a general and conserved feature of how different temporal memories are encoded from relatively simple to complex brains.

Distinct traces for appetitive versus aversive olfactory memories in DPM neurons of Drosophila.

The global logic used by the brain for differentially encoding positive and negative experiences remains unknown along with how such experiences are represented by collections of memory traces at the cellular level. Here we contrast the cellular memory traces that form in the dorsal paired medial (DPM) neurons of Drosophila after conditioning flies with odors associated with aversive or appetitive unconditioned stimuli (US). Our results show that the appetitive DPM neuron trace is distinguished from the aversive in three fundamental ways: (1) The DPM neurons do not respond to an appetitive US of sucrose by itself, in contrast to their robust response to an aversive US. (2) The appetitive trace persists for twice as long as the aversive trace. (3) The appetitive trace is expressed in both neurite branches of the neuron, rather than being confined to a single branch like the aversive trace. In addition, we demonstrate that training flies with nonnutritive sugars that elicit a behavioral memory that decays within 24 hr generates, like aversive conditioning, a short-lived and branch-restricted memory trace. These results indicate that the persistence and breadth of the DPM neuron memory trace influences the duration of behavioral memory.

Dopamine is required for learning and forgetting in Drosophila.

Psychological studies in humans and behavioral studies of model organisms suggest that forgetting is a common and biologically regulated process, but the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the bidirectional modulation of a small subset of dopamine neurons (DANs) after olfactory learning regulates the rate of forgetting of both punishing (aversive) and rewarding (appetitive) memories. Two of these DANs, MP1 and MV1, exhibit synchronized ongoing activity in the mushroom body neuropil in alive and awake flies before and after learning, as revealed by functional cellular imaging. Furthermore, while the mushroom-body-expressed dDA1 dopamine receptor is essential for the acquisition of memory, we show that the dopamine receptor DAMB, also highly expressed in mushroom body neurons, is required for forgetting. We propose a dual role for dopamine: memory acquisition through dDA1 signaling and forgetting through DAMB signaling in the mushroom body neurons.

Olfactory learning in Drosophila.

Studies of olfactory learning in Drosophila have provided key insights into the brain mechanisms underlying learning and memory. One type of olfactory learning, olfactory classical conditioning, consists of learning the contingency between an odor with an aversive or appetitive stimulus. This conditioning requires the activity of molecules that can integrate the two types of sensory information, the odorant as the conditioned stimulus and the aversive or appetitive stimulus as the unconditioned stimulus, in brain regions where the neural pathways for the two stimuli intersect. Compelling data indicate that a particular form of adenylyl cyclase functions as a molecular integrator of the sensory information in the mushroom body neurons. The neuronal pathway carrying the olfactory information from the antennal lobes to the mushroom body is well described. Accumulating data now show that some dopaminergic neurons provide information about aversive stimuli and octopaminergic neurons about appetitive stimuli to the mushroom body neurons. Inhibitory inputs from the GABAergic system appear to gate olfactory information to the mushroom bodies and thus control the ability to learn about odors. Emerging data obtained by functional imaging procedures indicate that distinct memory traces form in different brain regions and correlate with different phases of memory. The results from these and other experiments also indicate that cross talk between mushroom bodies and several other brain regions is critical for memory formation.

Differences between Naegleria fowleri and Naegleria gruberi in expression of mannose and fucose glycoconjugates.

Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis, a rapidly fatal parasitic disease of humans. The adherence of Naegleria trophozoites to the host cell is one of the most important steps in the establishment and invasiveness of this infectious disease. Currently, little is known about the surface molecules that may participate in the interaction of N. fowleri with their target cells. In the present study, we investigated the composition of glycoconjugates present on the surface of trophozoites of the pathogenic N. fowleri and the nonpathogenic Naegleria gruberi. With the use of biotinylated lectins in western blot and flow cytometric analysis, we showed that N. fowleri trophozoites present high levels of surface glycoconjugates that contain alpha-D-mannose, alpha-D-glucose, and terminal alpha-L-fucose residues. A significant difference in the expression of these glycoconjugates was observed between N. fowleri and the nonpathogenic N. gruberi. Furthermore, we suggest that glycoconjugates that contain D-mannose and L-fucose residues participate in the adhesion of N. fowleri and subsequent damage to MDCK cells.

Naegleria fowleri induces MUC5AC and pro-inflammatory cytokines in human epithelial cells via ROS production and EGFR activation.

Naegleria fowleri is an amoeboflagellate responsible for the fatal central nervous system (CNS) disease primary amoebic meningoencephalitis (PAM). This amoeba gains access to the CNS by invading the olfactory mucosa and crossing the cribriform plate. Studies using a mouse model of infection have shown that the host secretes mucus during the very early stages of infection, and this event is followed by an infiltration of neutrophils into the nasal cavity. In this study, we investigated the role of N. fowleri trophozoites in inducing the expression and secretion of airway mucin and pro-inflammatory mediators. Using the human mucoepidermal cell line NCI-H292, we demonstrated that N. fowleri induced the expression of the MUC5AC gene and protein and the pro-inflammatory mediators interleukin-8 (IL-8) and interleukin-1 beta (IL-1 beta), but not tumour necrosis factor-alpha or chemokine c-c motif ligand 11 (eotaxin). Since the production of reactive oxygen species (ROS) is a common phenomenon involved in the signalling pathways of these molecules, we analysed if trophozoites were capable of causing ROS production in NCI-H292 cells by detecting oxidation of the fluorescent probe 2,7-dichlorofluorescein diacetate. NCI-H292 cells generated ROS after 15-30 min of trophozoite stimulation. Furthermore, the expression of MUC5AC, IL-8 and IL-1 beta was inhibited in the presence of the ROS scavenger DMSO. In addition, the use of an epidermal growth factor receptor inhibitor decreased the expression of MUC5AC and IL-8, but not IL-1 beta. We conclude that N. fowleri induces the expression of some host innate defence mechanisms, such as mucin secretion (MUC5AC) and local inflammation (IL-8 and IL-1 beta) in respiratory epithelial cells via ROS production and suggest that these innate immune mechanisms probably prevent most PAM infections.

Mucins in the host defence against Naegleria fowleri and mucinolytic activity as a possible means of evasion.

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM). This parasite invades its host by penetrating the olfactory mucosa. During the initial stages of infection, the host response is initiated by the secretion of mucus that traps the trophozoites. Despite this response, some trophozoites are able to reach, adhere to and penetrate the epithelium. In the present work, we evaluated the effect of mucins on amoebic adherence and cytotoxicity to Madin-Darby canine kidney (MDCK) cells and the MUC5AC-inducing cell line NCI-H292. We showed that mucins inhibited the adhesion of amoebae to both cell lines; however, this inhibition was overcome in a time-dependent manner. N. fowleri re-established the capacity to adhere faster than N. gruberi. Moreover, mucins reduced the cytotoxicity to target cells and the progression of the illness in mice. In addition, we demonstrated mucinolytic activity in both Naegleria strains and identified a 37 kDa protein with mucinolytic activity. The activity of this protein was inhibited by cysteine protease inhibitors. Based on these results, we suggest that mucus, including its major mucin component, may act as an effective protective barrier that prevents most cases of PAM; however, when the number of amoebae is sufficient to overwhelm the innate immune response, the parasites may evade the mucus by degrading mucins via a proteolytic mechanism.

Characterization of brain inflammation during primary amoebic meningoencephalitis.

Naegleria fowleri is a free-living amoeba and the etiologic agent of primary amoebic meningoencephalitis (PAM). Trophozoites reach the brain by penetrating the olfactory epithelium, and invasion of the olfactory bulbs results in an intense inflammatory reaction. The contribution of the inflammatory response to brain damage in experimental PAM has not been delineated. Using both optical and electron microscopy, we analyzed the morphologic changes in the brain parenchyma due to inflammation during experimental PAM. Several N. fowleri trophozoites were observed in the olfactory bulbs 72 h post-inoculation, and the number of amoebae increased rapidly over the next 24 h. Eosinophils and neutrophils surrounding the amoebae were then noted at later times during infection. Electron microscopic examination of the increased numbers of neutrophils and the interactions with trophozoites indicated an active attempt to eliminate the amoebae. The extent of inflammation increased over time, with a predominant neutrophil response indicating important signs of damage and necrosis of the parenchyma. These data suggest a probable role of inflammation in tissue damage. To test the former hypothesis, we used CD38-/- knockout mice with deficiencies in chemotaxis to compare the rate of mortality with the parental strain, C57BL/6J. The results showed that inflammation and mortality were delayed in the knockout mice. Based on these results, we suggest that the host inflammatory response and polymorphonuclear cell lysis contribute to a great extent to the central nervous system tissue damage.

A biochemical comparison of proteases from pathogenic naegleria fowleri and non-pathogenic Naegleria gruberi.

Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis (PAM). Proteases have been suggested to be involved in tissue invasion and destruction during infection. We analyzed and compared the complete protease profiles of total crude extract and conditioned medium of both pathogenic N. fowleri and non-pathogenic Naegleria gruberi trophozoites. Using SDS-PAGE, we found differences in the number and molecular weight of proteolytic bands between the two strains. The proteases showed optimal activity at pH 7.0 and 35 degrees C for both strains. Inhibition assays showed that the main proteolytic activity in both strains is due to cysteine proteases although serine proteases were also detected. Both N. fowleri and N. gruberi have a variety of different protease activities at different pH levels and temperatures. These proteases may allow the amoebae to acquire nutrients from different sources, including those from the host. Although, the role of the amoebic proteases in the pathogenesis of PAM is not clearly defined, it seems that proteases and other molecules of the parasite as well as those from the host, could be participating in the damage to the human central nervous system.

Characterization of Naegleria fowleri strains isolated from human cases of primary amoebic meningoencephalitis in Mexico.

The protozoon Naegleria fowleri (N. fowleri) is a free-living amoeba that produces primary amoebic meningoencephalitis (PAM), which is an acute and frequently fatal infection of the central nervous system. We characterized the strains of N. fowleri isolated from the cerebrospinal fluid (CSF) of two cases presented in northwestern Mexico. The strains were isolated and cultured in 2% bactocasitone medium. Enflagellation assays, ultrastructural analysis, protein and protease electrophoresis patterns, and PCR were performed as confirmatory tests. Virulence tests were done using in Balb/c mice. Light microscopy analysis of brain tissue showed amoebae with abundant inflammatory reaction and extensive necrotic and hemorrhagic areas. The enflagellation assay was positive and the electron microscopy showed trophozoites with morphologic features typical of the genus. Protein and protease profiles of the isolated strains were identical to the reference strain. Finally, a 1500-bp PCR product was found in all three strains. Based on all the analyses performed, we concluded that the etiologic agent of both PAM cases was N. fowleri. The need for better epidemiological information and educational programs about basic clinical and pathological aspects of free-living amoebae provided by the health authorities are emphasized.