The Drosophila Mushroom Body: From Architecture to Algorithm in a Learning Circuit.

The Drosophila brain contains a relatively simple circuit for forming Pavlovian associations, yet it achieves many operations common across memory systems. Recent advances have established a clear framework for Drosophila learning and revealed the following key operations: a) pattern separation, whereby dense combinatorial representations of odors are preprocessed to generate highly specific, nonoverlapping odor patterns used for learning; b) convergence, in which sensory information is funneled to a small set of output neurons that guide behavioral actions; c) plasticity, where changing the mapping of sensory input to behavioral output requires a strong reinforcement signal, which is also modulated by internal state and environmental context; and d) modularization, in which a memory consists of multiple parallel traces, which are distinct in stability and flexibility and exist in anatomically well-defined modules within the network. Cross-module interactions allow for higher-order effects where past experience influences future learning. Many of these operations have parallels with processes of memory formation and action selection in more complex brains.

A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging.

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca2+ concentration using fluorescence lifetime imaging microscopy (FLIM).

Forgetting is regulated through Rac activity in Drosophila.

Initially acquired memory dissipates rapidly if not consolidated. Such memory decay is thought to result either from the inherently labile nature of newly acquired memories or from interference by subsequently attained information. Here we report that a small G protein Rac-dependent forgetting mechanism contributes to both passive memory decay and interference-induced forgetting in Drosophila. Inhibition of Rac activity leads to slower decay of early memory, extending it from a few hours to more than one day, and to blockade of interference-induced forgetting. Conversely, elevated Rac activity in mushroom body neurons accelerates memory decay. This forgetting mechanism does not affect memory acquisition and is independent of Rutabaga adenylyl cyclase-mediated memory formation mechanisms. Endogenous Rac activation is evoked on different time scales during gradual memory loss in passive decay and during acute memory removal in reversal learning. We suggest that Rac's role in actin cytoskeleton remodeling may contribute to memory erasure.

A pair of dopamine neurons mediate chronic stress signals to induce learning deficit in Drosophila melanogaster.

Chronic stress could induce severe cognitive impairments. Despite extensive investigations in mammalian models, the underlying mechanisms remain obscure. Here, we show that chronic stress could induce dramatic learning and memory deficits in Drosophila melanogaster The chronic stress-induced learning deficit (CSLD) is long lasting and associated with other depression-like behaviors. We demonstrated that excessive dopaminergic activity provokes susceptibility to CSLD. Remarkably, a pair of PPL1-γ1pedc dopaminergic neurons that project to the mushroom body (MB) γ1pedc compartment play a key role in regulating susceptibility to CSLD so that stress-induced PPL1-γ1pedc hyperactivity facilitates the development of CSLD. Consistently, the mushroom body output neurons (MBON) of the γ1pedc compartment, MBON-γ1pedc>α/ß neurons, are important for modulating susceptibility to CSLD. Imaging studies showed that dopaminergic activity is necessary to provoke the development of chronic stress-induced maladaptations in the MB network. Together, our data support that PPL1-γ1pedc mediates chronic stress signals to drive allostatic maladaptations in the MB network that lead to CSLD.

Genetic dissection of active forgetting in labile and consolidated memories in Drosophila.

Different memory components are forgotten through distinct molecular mechanisms. In Drosophila, the activation of 2 Rho GTPases (Rac1 and Cdc42), respectively, underlies the forgetting of an early labile memory (anesthesia-sensitive memory, ASM) and a form of consolidated memory (anesthesia-resistant memory, ARM). Here, we dissected the molecular mechanisms that tie Rac1 and Cdc42 to the different types of memory forgetting. We found that 2 WASP family proteins, SCAR/WAVE and WASp, act downstream of Rac1 and Cdc42 separately to regulate ASM and ARM forgetting in mushroom body neurons. Arp2/3 complex, which organizes branched actin polymerization, is a canonical downstream effector of WASP family proteins. However, we found that Arp2/3 complex is required in Cdc42/WASp-mediated ARM forgetting but not in Rac1/SCAR-mediated ASM forgetting. Instead, we identified that Rac1/SCAR may function with formin Diaphanous (Dia), a nucleator that facilitates linear actin polymerization, in ASM forgetting. The present study, complementing the previously identified Rac1/cofilin pathway that regulates actin depolymerization, suggests that Rho GTPases regulate forgetting by recruiting both actin polymerization and depolymerization pathways. Moreover, Rac1 and Cdc42 may regulate different types of memory forgetting by tapping into different actin polymerization mechanisms.

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory.

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-ß'1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.

Distinct molecular underpinnings of Drosophila olfactory trace conditioning.

Trace conditioning is valued as a simple experimental model to assess how the brain associates events that are discrete in time. Here, we adapted an olfactory trace conditioning procedure in Drosophila melanogaster by training fruit flies to avoid an odor that is followed by foot shock many seconds later. The molecular underpinnings of the learning are distinct from the well-characterized simultaneous conditioning, where odor and punishment temporally overlap. First, Rutabaga adenylyl cyclase (Rut-AC), a putative molecular coincidence detector vital for simultaneous conditioning, is dispensable in trace conditioning. Second, dominant-negative Rac expression, thought to sustain early labile memory, significantly enhances learning of trace conditioning, but leaves simultaneous conditioning unaffected. We further show that targeting Rac inhibition to the mushroom body (MB) but not the antennal lobe (AL) suffices to achieve the enhancement effect. Moreover, the absence of trace conditioning learning in D1 dopamine receptor mutants is rescued by restoration of expression specifically in the adult MB. These results suggest the MB as a crucial neuroanatomical locus for trace conditioning, which may harbor a Rac activity-sensitive olfactory "sensory buffer" that later converges with the punishment signal carried by dopamine signaling. The distinct molecular signature of trace conditioning revealed here shall contribute to the understanding of how the brain overcomes a temporal gap in potentially related events.

Schizophrenia susceptibility gene dysbindin regulates glutamatergic and dopaminergic functions via distinctive mechanisms in Drosophila.

The dysfunction of multiple neurotransmitter systems is a striking pathophysiological feature of many mental disorders, schizophrenia in particular, but delineating the underlying mechanisms has been challenging. Here we show that manipulation of a single schizophrenia susceptibility gene, dysbindin, is capable of regulating both glutamatergic and dopaminergic functions through two independent mechanisms, consequently leading to two categories of clinically relevant behavioral phenotypes. Dysbindin has been reported to affect glutamatergic and dopaminergic functions as well as a range of clinically relevant behaviors in vertebrates and invertebrates but has been thought to have a mainly neuronal origin. We find that reduced expression of Drosophila dysbindin (Ddysb) in presynaptic neurons significantly suppresses glutamatergic synaptic transmission and that this glutamatergic defect is responsible for impaired memory. However, only the reduced expression of Ddysb in glial cells is the cause of hyperdopaminergic activities that lead to abnormal locomotion and altered mating orientation. This effect is attributable to the altered expression of a dopamine metabolic enzyme, Ebony, in glial cells. Thus, Ddysb regulates glutamatergic transmission through its neuronal function and regulates dopamine metabolism by regulating Ebony expression in glial cells.

Dexmedetomidine: a real-world safety analysis based on FDA adverse event reporting system database.

Objective: Using the FDA adverse event reporting system (FAERS) database to analyze the safety profile of Dexmedetomidine and provide guidance for clinical application. Methods: Data from the FAERS database from the first quarter of 2004 to the third quarter of 2023 were collected. Reporting odds ratio (ROR), the proportional reporting ratio (PRR), and the Bayesian confidence propagation neural network (BCPNN) were employed to detect and assess adverse events associated with Dexmedetomidine. Results: A total of 1910 reports of Dexmedetomidine as the primary suspect drug were obtained. After screening, 892 preferred terms were obtained, including 52 new preferred terms not mentioned in the drug insert. The common adverse events of Dexmedetomidine include bradycardia, cardiac arrest, hypotension, diabetes insipidus, arteriospasm coronary and agitation. Notably, cardiac disorders exhibited the highest number of reports and the highest signal intensity in the system organ class. Among the new preferred terms, those with high signal intensity include transcranial electrical motor evoked potential monitoring abnormal, acute motor axonal neuropathy, trigemino-cardiac reflex, glossoptosis, floppy iris syndrome, phaeochromocytoma crisis, postresuscitation encephalopathy and diabetes insipidus. Conclusion: This study mined and evaluated adverse events associated with Dexmedetomidine and also identified new adverse events. This could help alert clinicians to new adverse events not mentioned in the drug inserts, reducing the risk of drug.

Neural circuit mechanisms for transforming learned olfactory valences into wind-oriented movement.

How memories are used by the brain to guide future action is poorly understood. In olfactory associative learning in Drosophila, multiple compartments of the mushroom body act in parallel to assign a valence to a stimulus. Here, we show that appetitive memories stored in different compartments induce different levels of upwind locomotion. Using a photoactivation screen of a new collection of split-GAL4 drivers and EM connectomics, we identified a cluster of neurons postsynaptic to the mushroom body output neurons (MBONs) that can trigger robust upwind steering. These UpWind Neurons (UpWiNs) integrate inhibitory and excitatory synaptic inputs from MBONs of appetitive and aversive memory compartments, respectively. After formation of appetitive memory, UpWiNs acquire enhanced response to reward-predicting odors as the response of the inhibitory presynaptic MBON undergoes depression. Blocking UpWiNs impaired appetitive memory and reduced upwind locomotion during retrieval. Photoactivation of UpWiNs also increased the chance of returning to a location where activation was terminated, suggesting an additional role in olfactory navigation. Thus, our results provide insight into how learned abstract valences are gradually transformed into concrete memory-driven actions through divergent and convergent networks, a neuronal architecture that is commonly found in the vertebrate and invertebrate brains.

A modular chemigenetic calcium indicator enables in vivo functional imaging with near-infrared light.

Genetically encoded fluorescent calcium indicators have revolutionized neuroscience and other biological fields by allowing cellular-resolution recording of physiology during behavior. However, we currently lack bright, genetically targetable indicators in the near infrared that can be used in animals. Here, we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains that can be genetically targeted to specific cell populations. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several dye-ligands that efficiently label the central nervous system in animals. When bound to a near-infrared dye-ligand, WHaloCaMP1a is more than twice as bright as jGCaMP8s, and shows a 7× increase in fluorescence intensity and a 2.1 ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a with near-infrared fluorescence emission to image Ca2+ responses in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae, and to quantitate calcium concentration using fluorescence lifetime imaging microscopy (FLIM).

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator.

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Stress resistance conferred by neuronal expression of dominant-negative Rac in adult Drosophila melanogaster.

How does brain coordinate physiological and behavioral responses to achieve survival in adverse environment is intriguing yet complicated. During studies of the small G protein Rac's role in learning and memory, the authors unexpectedly observed that neuronal expression of dominant-negative Rac in adult Drosophila remarkably enhanced the survival of animals in various stress conditions, including oxidation, desiccation, starvation, and heat. The elevated stress resistance was not accompanied by a reduction in female fecundity or a change in whole-body lipid storage. The observation therefore implies the involvement of small G protein Rac in neuronal regulation of global stress responses.

Suppressor of Hairless is required for long-term memory formation in Drosophila.

Suppressor of Hairless [Su(H)] is a DNA-binding protein of the Notch-signaling pathway, which is important for developmental processes and has been implicated in behavior plasticity. It acts as a transcriptional activator in the Notch pathway, but also as a repressor in the absence of Notch signaling. Our previous work has shown that Notch signaling contributes to long-term memory formation in the Drosophila adult brain. In the present report, we show that Su(H) null heterozygous mutants perform normally for learning, early memory, and anesthesia-resistant memory, whereas long-term memory is impaired. Interestingly, we find overexpressing wild- type Su(H) also causes long-term memory defect in Drosophila. Significantly, induction of a heat-shock inducible Su(H)(+) transgene before training can fully rescue the memory defect of Su(H) mutants, thereby demonstrating an acute role for Su(H) in behavioral plasticity. We show that Su(H) is widely expressed in the adult brain. Transgenic expression of wild-type Su(H) in the Mushroom Bodies is sufficient to rescue the memory defect of Su(H) mutants. Our data clearly demonstrate that transcriptional activity of Su(H) in Notch signaling in the mushroom bodies is critical for the formation of long-term memory.

Bright and photostable chemigenetic indicators for extended in vivo voltage imaging.

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

Forgetting and small G protein Rac.

It is far from understood why we forget things that are known to us seconds ago. Emerging evidence emphasizes that small G protein Rac could be a key to understanding this type of rapid early memory forgetting. This current perspective article will first review these studies and then discuss their implications for the internal processes underlying forgetting.

The AKAP Yu is required for olfactory long-term memory formation in Drosophila.

Extensive neurogenetic analysis has shown that memory formation depends critically on cAMP-protein kinase A (PKA) signaling. Details of how this pathway is involved in memory formation, however, remain to be fully elucidated. From a large-scale behavioral screen in Drosophila, we identified the yu mutant to be defective in one-day memory after spaced training. The yu mutation disrupts a gene encoding an A-kinase anchoring protein (AKAP). AKAPs comprise a family of proteins, which determine the subcellular localization of PKAs and thereby critically restrict cAMP signaling within a cell. Further behavioral characterizations revealed that long-term memory (LTM) was disrupted specifically in the yu mutant, whereas learning, short-term memory and anesthesia-resistant memory all appeared normal. Another independently isolated mutation of the yu gene failed to complement the LTM defect associated with the yu mutation, and this phenotypic defect could be rescued by induced acute expression of a yu(+) transgene, suggesting that yu functions physiologically during memory formation. AKAP Yu is expressed preferentially in the mushroom body (MB) neuroanatomical structure, and expression of a yu(+) transgene to the MB, but not to other brain regions, is sufficient to rescue the LTM defect of the yu mutant. These observations lead us to conclude that proper localization of PKA by Yu AKAP in MB neurons is required for the formation of LTM.