SnapShot: Olfactory Classical Conditioning of Drosophila.

This SnapShot summarizes current knowledge about the molecules and circuitry that mediate olfactory memory formation in Drosophila, with emphasis on neural circuits carrying the learned sensory information; the molecular mechanisms for acquisition, memory storage, and forgetting; and the output pathways for memory expression. To view this SnapShot, open or download the PDF.

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.

Dopamine-based mechanism for transient forgetting.

Active forgetting is an essential component of the memory management system of the brain1. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. Here we identify a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval.

Active forgetting and neuropsychiatric diseases.

Recent and pioneering animal research has revealed the brain utilizes a variety of molecular, cellular, and network-level mechanisms used to forget memories in a process referred to as "active forgetting". Active forgetting increases behavioral flexibility and removes irrelevant information. Individuals with impaired active forgetting mechanisms can experience intrusive memories, distressing thoughts, and unwanted impulses that occur in neuropsychiatric diseases. The current evidence indicates that active forgetting mechanisms degrade, or mask, molecular and cellular memory traces created in synaptic connections of "engram cells" that are specific for a given memory. Combined molecular genetic/behavioral studies using Drosophila have uncovered a complex system of cellular active-forgetting pathways within engram cells that is regulated by dopamine neurons and involves dopamine-nitric oxide co-transmission and reception, endoplasmic reticulum Ca2+ signaling, and cytoskeletal remodeling machinery regulated by small GTPases. Some of these molecular cellular mechanisms have already been found to be conserved in mammals. Interestingly, some pathways independently regulate forgetting of distinct memory types and temporal phases, suggesting a multi-layering organization of forgetting systems. In mammals, active forgetting also involves modulation of memory trace synaptic strength by altering AMPA receptor trafficking. Furthermore, active-forgetting employs network level mechanisms wherein non-engram neurons, newly born-engram neurons, and glial cells regulate engram synapses in a state and experience dependent manner. Remarkably, there is evidence for potential coordination between the network and cellular level forgetting mechanisms. Finally, subjects with several neuropsychiatric diseases have been tested and shown to be impaired in active forgetting. Insights obtained from research on active forgetting in animal models will continue to enrich our understanding of the brain dysfunctions that occur in neuropsychiatric diseases.

Rac in the act of forgetting.

Forgetting has been thought to occur as a result of the natural decay of the neuronal changes induced by learning or because of interference from other cognitive functions. In this issue, Shuai et al. (2010) find that the small G protein Rac may function as a switch for remembering versus forgetting.

Active forgetting requires Sickie function in a dedicated dopamine circuit in Drosophila.

Forgetting is an essential component of the brain's memory management system, providing a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the memory suppressor gene, sickie, functions in a single dopamine neuron (DAn) by supporting the process of active forgetting in Drosophila. RNAi knockdown (KD) of sickie impairs forgetting by reducing the Ca2+ influx and DA release from the DAn that promotes forgetting. Coimmunoprecipitation/mass spectrometry analyses identified cytoskeletal and presynaptic active zone (AZ) proteins as candidates that physically interact with Sickie, and a focused RNAi screen of the candidates showed that Bruchpilot (Brp)-a presynaptic AZ protein that regulates calcium channel clustering and neurotransmitter release-impairs active forgetting like sickie KD. In addition, overexpression of brp rescued the impaired forgetting of sickie KD, providing evidence that they function in the same process. Moreover, we show that sickie KD in the DAn reduces the abundance and size of AZ markers but increases their number, suggesting that Sickie controls DAn activity for forgetting by modulating the presynaptic AZ structure. Our results identify a molecular and circuit mechanism for normal levels of active forgetting and reveal a surprising role of Sickie in maintaining presynaptic AZ structure for neurotransmitter release.

Ras acts as a molecular switch between two forms of consolidated memory in Drosophila.

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. Here, we demonstrate that the Ras→Raf→rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. Our initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. We found that these effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. We conclude that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM.

Aversive Training Induces Both Presynaptic and Postsynaptic Suppression in Drosophila.

The α'ß' subtype of Drosophila mushroom body neurons (MBn) is required for memory acquisition, consolidation and early memory retrieval after aversive olfactory conditioning. However, in vivo functional imaging studies have failed to detect an early forming memory trace in these neurons as reflected by an enhanced G-CaMP signal in response to presentation of the learned odor. Moreover, whether cellular memory traces form early after conditioning in the mushroom body output neurons (MBOn) downstream of the α'ß' MBn remains unknown. Here, we show that aversive olfactory conditioning suppresses the calcium responses to the learned odor in both α'3 and α'2 axon segments of α'ß' MBn and in the dendrites of α'3 MBOn immediately after conditioning using female flies. Notably, the cellular memory traces in both α'3 MBn and α'3 MBOn are short-lived and persist for <30 min. The suppressed response in α'3 MBn is accompanied by a reduction of acetylcholine (ACh) release, suggesting that the memory trace in postsynaptic α'3 MBOn may simply reflect the suppression in presynaptic α'3 MBn. Furthermore, we show that the α'3 MBn memory trace does not occur from the inhibition of GABAergic neurons via GABAA receptor activation. Because activation of the α'3 MBOn drives approach behavior of adult flies, our results demonstrate that aversive conditioning promotes avoidance behavior through suppression of the α'3 MBn-MBOn circuit.SIGNIFICANCE STATEMENTDrosophila learn to avoid an odor if that odor is repeatedly paired with electric shock. Mushroom body neurons (MBns) are known to be major cell types that mediate this form of aversive conditioning. Here we show that aversive conditioning causes a reduced response to the conditioned odor in an axon branch of one subtype of the MBn for no more than 30 min after conditioning, and in the dendrites of postsynaptic, MB output neurons (MBOns). Because experimenter-induced activation of the MBOn induces approach behavior by the fly, our data support a model that aversive learning promotes avoidance by suppressing the MBn-MBOn synapses that normally promote attraction.

Elongator complex is required for long-term olfactory memory formation in Drosophila.

The evolutionarily conserved Elongator Complex associates with RNA polymerase II for transcriptional elongation. Elp3 is the catalytic subunit, contains histone acetyltransferase activity, and is associated with neurodegeneration in humans. Elp1 is a scaffolding subunit and when mutated causes familial dysautonomia. Here, we show that elp3 and elp1 are required for aversive long-term olfactory memory in Drosophila RNAi knockdown of elp3 in adult mushroom bodies impairs long-term memory (LTM) without affecting earlier forms of memory. RNAi knockdown with coexpression of elp3 cDNA reverses the impairment. Similarly, RNAi knockdown of elp1 impairs LTM and coexpression of elp1 cDNA reverses this phenotype. The LTM deficit in elp3 and elp1 knockdown flies is accompanied by the abolishment of a LTM trace, which is registered as increased calcium influx in response to the CS+ odor in the α-branch of mushroom body neurons. Coexpression of elp1 or elp3 cDNA rescues the memory trace in parallel with LTM. These data show that the Elongator complex is required in adult mushroom body neurons for long-term behavioral memory and the associated long-term memory trace.

Developmental inhibition of miR-iab8-3p disrupts mushroom body neuron structure and adult learning ability.

MicroRNAs are small non-coding RNAs that inhibit protein expression post-transcriptionally. They have been implicated in many different physiological processes, but little is known about their individual involvement in learning and memory. We recently identified several miRNAs that either increased or decreased intermediate-term memory when inhibited in the central nervous system, including miR-iab8-3p. We report here a new developmental role for this miRNA. Blocking the expression of miR-iab8-3p during the development of the organism leads to hypertrophy of individual mushroom body neuron soma, a reduction in the field size occupied by axonal projections, and adult intellectual disability. We further identified four potential mRNA targets of miR-iab8-3p whose inhibition modulates intermediate-term memory including ceramide phosphoethanolamine synthase, which may account for the behavioral effects produced by miR-iab8-3p inhibition. Our results offer important new information on a microRNA required for normal neurodevelopment and the capacity to learn and remember normally.

Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila.

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

Aging impairs protein-synthesis-dependent long-term memory in Drosophila.

Although aging is known to impair intermediate-term memory in Drosophila, its effect on protein-synthesis-dependent long-term memory (LTM) is unknown. We show here that LTM is impaired with age, not due to functional defects in synaptic output of mushroom body (MB) neurons, but due to connectivity defects of dorsal paired medial (DPM) neurons with their postsynaptic MB neurons. GFP reconstitution across synaptic partners (GRASP) experiments revealed structural connectivity defects in aged animals of DPM neurons with MB axons in the α lobe neuropil. As a consequence, a protein-synthesis-dependent LTM trace in the α/ß MB neurons fails to form. Aging thus impairs protein-synthesis-dependent LTM along with the α/ß MB neuron LTM trace by lessening the connectivity of DPM and α/ß MB neurons.

Interactions between ICAM-5 and ß1 integrins regulate neuronal synapse formation.

Intercellular adhesion molecule-5 (ICAM-5) is a dendrite-specific adhesion molecule, which functions in both the immune and nervous systems. ICAM-5 is the only negative regulator that has been identified for maturation of dendritic spines so far. Shedding of the ICAM-5 ectodomain promotes spine maturation and enhances synaptic activity. However, the mechanism by which ICAM-5 regulates spine development remains poorly understood. In this study, we found that ablation of ICAM5 expression resulted in a significant increase in the formation of synaptic contacts and the frequency of miniature excitatory post-synaptic currents, an indicator of pre-synaptic release probability. Antibodies against ICAM-5 and ß1 integrins altered spine maturation. Furthermore, we found that ß1 integrins serve as binding partners for ICAM-5. ß1 integrins were immunoprecipitated with ICAM-5 from mouse brain and the binding region in ICAM-5 was localized to the two first Ig domains. ß1 integrins were juxtaposed to filopodia tips at the early stage of synaptic formation, but as synapses matured, ß1 integrins covered the mushroom spines. Loss of ß1 integrins from the pre-synaptic sites affected the morphology of the post-synaptic structures. ICAM-5 ectodomain cleavage decreased or increased when the interaction between ICAM-5 and ß1 integrins was potentiated or weakened, respectively, using antibodies. These results suggest that the interaction between ICAM-5 and ß1 integrins is important in formation of functional synapses.

Aging impairs intermediate-term behavioral memory by disrupting the dorsal paired medial neuron memory trace.

How the functional activity of the brain is altered during aging to cause age-related memory impairments is unknown. We used functional cellular imaging to monitor two different calcium-based memory traces that underlie olfactory classical conditioning in young and aged Drosophila. Functional imaging of neural activity in the processes of the dorsal paired medial (DPM) and mushroom body neurons revealed that the capacity to form an intermediate-term memory (ITM) trace in the DPM neurons after learning is lost with age, whereas the capacity to form a short-term memory trace in the α'/ß' mushroom body neurons remains unaffected by age. Stimulation of the DPM neurons by activation of a temperature-sensitive cation channel between acquisition and retrieval enhanced ITM in aged but not young flies. These data indicate that the functional state of the DPM neurons is selectively altered with age to cause an age-related impairment of ITM, and demonstrate that altering the excitability of DPM neurons can restore age-related memory impairments.

Functional neuroanatomy of Drosophila olfactory memory formation.

New approaches, techniques and tools invented over the last decade and a half have revolutionized the functional dissection of neural circuitry underlying Drosophila learning. The new methodologies have been used aggressively by researchers attempting to answer three critical questions about olfactory memories formed with appetitive and aversive reinforcers: (1) Which neurons within the olfactory nervous system mediate the acquisition of memory? (2) What is the complete neural circuitry extending from the site(s) of acquisition to the site(s) controlling memory expression? (3) How is information processed across this circuit to consolidate early-forming, disruptable memories to stable, late memories? Much progress has been made and a few strong conclusions have emerged: (1) Acquisition occurs at multiple sites within the olfactory nervous system but is mediated predominantly by the γ mushroom body neurons. (2) The expression of long-term memory is completely dependent on the synaptic output of α/ß mushroom body neurons. (3) Consolidation occurs, in part, through circuit interactions between mushroom body and dorsal paired medial neurons. Despite this progress, a complete and unified model that details the pathway from acquisition to memory expression remains elusive.

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.

Learning and memory using Drosophila melanogaster: a focus on advances made in the fifth decade of research.

In the last decade, researchers using Drosophila melanogaster have made extraordinary progress in uncovering the mysteries underlying learning and memory. This progress has been propelled by the amazing toolkit available that affords combined behavioral, molecular, electrophysiological, and systems neuroscience approaches. The arduous reconstruction of electron microscopic images resulted in a first-generation connectome of the adult and larval brain, revealing complex structural interconnections between memory-related neurons. This serves as substrate for future investigations on these connections and for building complete circuits from sensory cue detection to changes in motor behavior. Mushroom body output neurons (MBOn) were discovered, which individually forward information from discrete and non-overlapping compartments of the axons of mushroom body neurons (MBn). These neurons mirror the previously discovered tiling of mushroom body axons by inputs from dopamine neurons and have led to a model that ascribes the valence of the learning event, either appetitive or aversive, to the activity of different populations of dopamine neurons and the balance of MBOn activity in promoting avoidance or approach behavior. Studies of the calyx, which houses the MBn dendrites, have revealed a beautiful microglomeruluar organization and structural changes of synapses that occur with long-term memory (LTM) formation. Larval learning has advanced, positioning it to possibly lead in producing new conceptual insights due to its markedly simpler structure over the adult brain. Advances were made in how cAMP response element-binding protein interacts with protein kinases and other transcription factors to promote the formation of LTM. New insights were made on Orb2, a prion-like protein that forms oligomers to enhance synaptic protein synthesis required for LTM formation. Finally, Drosophila research has pioneered our understanding of the mechanisms that mediate permanent and transient active forgetting, an important function of the brain along with acquisition, consolidation, and retrieval. This was catalyzed partly by the identification of memory suppressor genes-genes whose normal function is to limit memory formation.

Innate and learned odor-guided behaviors utilize distinct molecular signaling pathways in a shared dopaminergic circuit.

Odor-based learning and innate odor-driven behavior have been hypothesized to require separate neuronal circuitry. Contrary to this notion, innate behavior and olfactory learning were recently shown to share circuitry that includes the Drosophila mushroom body (MB). But how a single circuit drives two discrete behaviors remains unknown. Here, we define an MB circuit responsible for both olfactory learning and innate odor avoidance and the distinct dDA1 dopamine receptor-dependent signaling pathways that mediate these behaviors. Associative learning and learning-induced MB plasticity require rutabaga-encoded adenylyl cyclase activity in the MB. In contrast, innate odor preferences driven by naive MB neurotransmission are rutabaga independent, requiring the adenylyl cyclase ACXD. Both learning and innate odor preferences converge on PKA and the downstream MBON-γ2α'1. Importantly, the utilization of this shared circuitry for innate behavior only becomes apparent with hunger, indicating that hardwired innate behavior becomes more flexible during states of stress.

Early Mitochondrial Defects in the 5xFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: Mitochondrial (MT) dysfunction is a hallmark of Alzheimer's disease (AD). Amyloid-ß protein precursor and amyloid-ß peptides localize to MT and lead to MT dysfunction in familial forms of AD. This dysfunction may trigger subsequent types of pathology. OBJECTIVE: To identify the MT phenotypes that occur early in order to help understand the cascade of AD pathophysiology. METHODS: The 5xFAD mouse model was used to explore the time course of MT pathologies in both sexes. Protein biomarkers for MT dynamics were measured biochemically and MT function was measured using oxygen consumption and ATP assays. RESULTS: We discovered progressive alterations in mitochondrial dynamics (biogenesis, fission, fusion, and mitophagy) and function (O2 consumption, ATP generation, and Ca2+ import) in the hippocampus of 5xFAD mice in both sexes as early as 2 months of age. Thus, mitochondrial dynamics and function become altered at young ages, consistent with an early role for mitochondria in the AD pathological cascade. CONCLUSION: Our study offers the baseline information required to understand the hierarchical relationship between the multiple pathologies that develop in this mouse model and provides early biomarkers for MT dysfunction. This will aid in dissecting the temporal cascade of pathologies, understanding sex-specific differences, and in testing the efficacy of putative mitochondrial therapeutics.

The long-term memory trace formed in the Drosophila α/ß mushroom body neurons is abolished in long-term memory mutants.

A prior screen identified dozens of Drosophila melanogaster mutants that possess defective long-term memory (LTM). Using spaced olfactory conditioning, we trained 26 of these mutant lines to associate an odor cue with electric shock and then examined the memory of this conditioning 24 h later. All of the mutants tested revealed a deficit in LTM compared to the robust LTM observed in control flies. We used in vivo functional optical imaging to measure the magnitude of a previously characterized LTM trace, which is manifested as increased calcium influx into the axons of α/ß mushroom body neurons in response to the conditioned odor. This memory trace was defective in all 26 of the LTM mutants. These observations elevate the significance of this LTM trace given that 26 independent mutants all exhibit a defect in the trace, and further suggest that the calcium trace is a fundamental mechanism underlying Drosophila LTM.