Dopamine activity in projection neurons regulates short-lasting olfactory approach memory in Drosophila.

Survival in many animals requires the ability to associate certain cues with danger and others with safety. In a Drosophila melanogaster aversive olfactory conditioning paradigm, flies are exposed to two odours, one presented coincidentally with electrical shocks, and a second presented 45 s after shock cessation. When flies are later given a choice between these two odours, they avoid the shock-paired odour and prefer the unpaired odour. While many studies have examined how flies learn to avoid the shock-paired odour through formation of odour-fear associations, here we demonstrate that conditioning also causes flies to actively approach the second odour. In contrast to fear memories, which are longer lasting and requires activity of D1-like dopamine receptors only in the mushroom bodies, approach memory is short-lasting and requires activity of D1-like dopamine receptors in projection neurons originating from the antennal lobes, primary olfactory centers. Further, while recall of fear memories requires activity of the mushroom bodies, recall of approach memories does not. Our data suggest that olfactory approach memory is formed using different mechanisms in different brain locations compared to aversive and appetitive olfactory memories.

Carbon Monoxide, a Retrograde Messenger Generated in Postsynaptic Mushroom Body Neurons, Evokes Noncanonical Dopamine Release.

Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated. In Drosophila melanogaster brain explant preparations, DA is released specifically onto α3/α'3 compartments of mushroom body (MB) neurons that have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. Here we found that coincidentally activated MB neurons generate carbon monoxide (CO), which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in postsynaptic MB neurons, and CO-evoked DA release requires Ca2+ efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. We propose that DA neurons use two distinct modes of transmission to produce global and local DA signaling.SIGNIFICANCE STATEMENT Dopamine (DA) is needed for various higher brain functions, including memory formation. However, DA neurons form extensive synaptic connections, while memory formation requires highly specific and localized DA release. Here we identify a mechanism through which DA release from presynaptic terminals is controlled by postsynaptic activity. Postsynaptic neurons activated by cholinergic and glutamatergic inputs generate carbon monoxide, which acts as a retrograde messenger inducing presynaptic DA release. Released DA is required for memory-associated plasticity. Our work identifies a novel mechanism that restricts DA release to the specific postsynaptic sites that require DA during memory formation.

Long-term memory formation in Drosophila requires training-dependent glial transcription.

Long-term memory (LTM) formation requires de novo gene expression in neurons, and subsequent structural and functional modification of synapses. However, the importance of gene expression in glia during this process has not been well studied. In this report, we characterize a cell adhesion molecule, Klingon (Klg), which is required for LTM formation in Drosophila. We found that Klg localizes to the juncture between neurons and glia, and expression in both cell types is required for LTM. We further found that expression of a glial gene, repo, is reduced in klg mutants and knockdown lines. repo expression is required for LTM, and expression increases upon LTM induction. In addition, increasing repo expression in glia is sufficient to restore LTM in klg knockdown lines. These data indicate that neuronal activity enhances Klg-mediated neuron-glia interactions, causing an increase in glial expression of repo. Repo is a homeodomain transcription factor, suggesting that further downstream glial gene expression is also required for LTM.

Identification of a novel role for Drosophila MESR4 in lipid metabolism.

Drosophila provides a powerful genetic model to analyze lipid metabolism. Drosophila has an adipose-like organ called the fat body, which plays a crucial role in energy homeostasis. Here, we conducted a fat body-specific misexpression screen to identify genes involved in lipid metabolism. We found that over-expression of a nuclear protein with nine C2 H2 type zinc-finger motifs and a PHD-finger, Misexpression suppressor of ras 4 (MESR4), reduces lipid accumulation in the fat body, whereas MESR4 knockdown increases it. We further show that MESR4 up-regulates the expression of major lipases, which may account for the reduction in lipid storage in the fat body and the release of free fatty acids (FFAs) in the body. These results suggest that MESR4 acts as an important upstream regulator of energy homeostasis.

Long-term enhancement of synaptic transmission between antennal lobe and mushroom body in cultured Drosophila brain.

In Drosophila, the mushroom body (MB) is a critical brain structure for olfactory associative learning. During aversive conditioning, the MBs are thought to associate odour signals, conveyed by projection neurons (PNs) from the antennal lobe (AL), with shock signals conveyed through ascending fibres of the ventral nerve cord (AFV). Although synaptic transmission between AL and MB might play a crucial role for olfactory associative learning, its physiological properties have not been examined directly. Using a cultured Drosophila brain expressing a Ca(2+) indicator in the MBs, we investigated synaptic transmission and plasticity at the AL-MB synapse. Following stimulation with a glass micro-electrode, AL-induced Ca(2+) responses in the MBs were mediated through Drosophila nicotinic acetylcholine receptors (dnAChRs), while AFV-induced Ca(2+) responses were mediated through Drosophila NMDA receptors (dNRs). AL-MB synaptic transmission was enhanced more than 2 h after the simultaneous 'associative-stimulation' of AL and AFV, and such long-term enhancement (LTE) was specifically formed at the AL-MB synapses but not at the AFV-MB synapses. AL-MB LTE was not induced by intense stimulation of the AL alone, and the LTE decays within 60 min after subsequent repetitive AL stimulation. These phenotypes of associativity, input specificity and persistence of AL-MB LTE are highly reminiscent of olfactory memory. Furthermore, similar to olfactory aversive memory, AL-MB LTE formation required activation of the Drosophila D1 dopamine receptor, DopR, along with dnAChR and dNR during associative stimulations. These physiological and genetic analogies indicate that AL-MB LTE might be a relevant cellular model for olfactory memory.

Glia transmit negative valence information during aversive learning in Drosophila.

During Drosophila aversive olfactory conditioning, aversive shock information needs to be transmitted to the mushroom bodies (MBs) to associate with odor information. We report that aversive information is transmitted by ensheathing glia (EG) that surround the MBs. Shock induces vesicular exocytosis of glutamate from EG. Blocking exocytosis impairs aversive learning, whereas activation of EG can replace aversive stimuli during conditioning. Glutamate released from EG binds to N-methyl-d-aspartate receptors in the MBs, but because of Mg2+ block, Ca2+ influx occurs only when flies are simultaneously exposed to an odor. Vesicular exocytosis from EG also induces shock-associated dopamine release, which plays a role in preventing formation of inappropriate associations. These results demonstrate that vesicular glutamate released from EG transmits negative valence information required for associative learning.

Calcineurin and its regulator sra/DSCR1 are essential for sleep in Drosophila.

Sleep is a fundamental biological process for all animals. However, the molecular mechanisms that regulate sleep are still poorly understood. Here we report that sleep-like behavior in Drosophila is severely impaired by mutations in sarah (sra), a member of the Regulator of Calcineurin (RCAN) family of genes. Sleep reduction in sra mutants is highly correlated with decreases in Sra protein levels. Pan-neural expression of sra rescues this behavioral phenotype, indicating that neuronal sra function is required for normal sleep. Since Sra regulates calcineurin (CN), we generated and examined the behavior of knock-out mutants for all Drosophila CN genes: CanA-14F, Pp2B-14D, and CanA1 (catalytic subunits), and CanB and CanB2 (regulatory subunits). While all mutants show at least minor changes in sleep, CanA-14F(KO) and CanB(KO) have striking reductions, suggesting that these are the major CN subunits regulating sleep. In addition, neuronal expression of constitutively active forms of CN catalytic subunits also significantly reduces sleep, demonstrating that both increases and decreases in CN activity inhibit sleep. sra sleep defects are suppressed by CN mutations, indicating that sra and CN affect sleep through a common mechanism. Our results demonstrate that CN and its regulation by Sra are required for normal sleep in Drosophila and identify a critical role of Ca(2+)/calmodulin-dependent signaling in sleep regulation.

The Drosophila cell adhesion molecule klingon is required for long-term memory formation and is regulated by Notch.

The ruslan (rus) mutant was previously identified in a behavioral screen for mutants defective in long-lasting memory, which consists of two consolidated memory types, anesthesia-resistant memory, and protein synthesis-dependent long-term memory (LTM). We demonstrate here that rus is a new allele of klingon (klg), which encodes a homophilic cell adhesion molecule. Klg is acutely required for LTM but not anesthesia-resistant memory formation, and Klg expression increases upon LTM induction. LTM formation also requires activity of the Notch cell-surface receptor. Although defects in Notch have been implicated in memory loss because of Alzheimer's disease, downstream signaling linking Notch to memory have not been determined. Strikingly, we found that Notch activity increases upon LTM induction and regulates Klg expression. Furthermore, Notch-induced enhancement of LTM is disrupted by a klg mutation. We propose that Klg is a downstream effector of Notch signaling that links Notch activity to memory.

Acute inhibition of PKA activity at old ages ameliorates age-related memory impairment in Drosophila.

Age-related memory impairment (AMI) is a critical and debilitating phenotype of brain aging, but its underlying molecular mechanisms are largely unknown. In Drosophila, AMI is highly correlated with PKA activity in the mushroom bodies, neural centers essential for forming associative olfactory memories. Heterozygous mutations in DC0 (DC0/+), which encodes the major catalytic subunit of PKA (PKAc), significantly suppress AMI, while overexpression of a DC0 transgene (DC0(+)) impairs memory and occludes AMI. PKA activity does not increase upon aging, and it is not clear whether AMI is caused by continual PKA activity throughout aging or by an acute increase in PKA signaling at old ages. Likewise, it is not clear whether AMI can be ameliorated by acute interventions at old ages or whether continuous intervention throughout aging is necessary. We show here that an acute increase in PKA activity at old ages is sufficient to restore normal AMI in DC0/+ flies. Conversely, acute expression of a PKA inhibitory peptide at old ages is sufficient to reverse AMI in a wild-type background. These results indicate that AMI in Drosophila is caused by an age-dependent change in PKA-dependent signaling that can be reversed by acute interventions at old ages.

Protein kinase A inhibits a consolidated form of memory in Drosophila.

Increasing activity of the cAMP/protein kinase A (PKA) pathway has often been proposed as an approach to improve memory in various organisms. However, here we demonstrate that single-point mutations, which decrease PKA activity, dramatically improve aversive olfactory memory in Drosophila. These mutations do not affect formation of early memory phases or of protein synthesis-dependent long-term memory but do cause a significant increase in a specific consolidated form of memory, anesthesia-resistant memory. Significantly, heterozygotes of null mutations in PKA are sufficient to cause this memory increase. Expressing a PKA transgene in the mushroom bodies, brain structures critical for memory formation in Drosophila, reduces memory back to wild-type levels. These results indicate that although PKA is critical for formation of several memory phases, it also functions to inhibit at least one memory phase.

The Drosophila DCO mutation suppresses age-related memory impairment without affecting lifespan.

The study of age-related memory impairment (AMI) has been hindered by a lack of AMI-specific mutants. In a screen for such mutants in Drosophila melanogaster, we found that heterozygous mutations of DCO (DCO/+), which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), delay AMI more than twofold without affecting lifespan or memory at early ages. AMI is restored when a DCO transgene is expressed in mushroom bodies, structures important for olfactory memory formation. Furthermore, increasing cAMP and PKA activity in mushroom bodies causes premature AMI, whereas reducing activity suppresses AMI. In Drosophila AMI consists of a specific reduction in memory dependent on the amnesiac (amn) gene. amn encodes putative neuropeptides that have been proposed to regulate cAMP levels in mushroom bodies. Notably, both the memory and AMI defects of amn mutants are restored in amn;DCO/+ double mutants, suggesting that AMI is caused by an age-related disruption of amn-dependent memory via PKA activity in mushroom bodies.

High-sucrose diets contribute to brain angiopathy with impaired glucose uptake and psychosis-related higher brain dysfunctions in mice.

Metabolic dysfunction is thought to contribute to the severity of psychiatric disorders; however, it has been unclear whether current high­simple sugar diets contribute to pathogenesis of these diseases. Here, we demonstrate that a high-sucrose diet during adolescence induces psychosis-related behavioral endophenotypes, including hyperactivity, poor working memory, impaired sensory gating, and disrupted interneuron function in mice deficient for glyoxalase-1 (GLO1), an enzyme involved in detoxification of sucrose metabolites. Furthermore, the high-sucrose diet induced microcapillary impairments and reduced brain glucose uptake in brains of Glo1-deficient mice. Aspirin protected against this angiopathy, enhancing brain glucose uptake and preventing abnormal behavioral phenotypes. Similar vascular damage to our model mice was found in the brains of randomly collected schizophrenia and bipolar disorder patients, suggesting that psychiatric disorders are associated with angiopathy in the brain caused by various environmental stresses, including metabolic stress.

Recurrent loops: Incorporating prediction error and semantic/episodic theories into Drosophila associative memory models.

In 2003, Martin Heisenberg et al. presented a model of how associative memories could be encoded and stored in the insect brain. This model was extremely influential in the Drosophila memory field, but did not incorporate several important mammalian concepts, including ideas of separate episodic and semantic types of memory and prediction error hypotheses. In addition, at that time, the concept of memory traces recurrently entering and exiting the mushroom bodies, brain areas where associative memories are formed and stored, was unknown. In this review, I present a simple updated model incorporating these ideas, which may be useful for future studies.

Inhibiting Glutamate Activity during Consolidation Suppresses Age-Related Long-Term Memory Impairment in Drosophila.

In Drosophila, long-term memory (LTM) formation requires increases in glial gene expression. Klingon (Klg), a cell adhesion molecule expressed in both neurons and glia, induces expression of the glial transcription factor, Repo. However, glial signaling downstream of Repo has been unclear. Here we demonstrate that Repo increases expression of the glutamate transporter, EAAT1, and EAAT1 is required during consolidation of LTM. The expressions of Klg, Repo, and EAAT1 decrease upon aging, suggesting that age-related impairments in LTM are caused by dysfunction of the Klg-Repo-EAAT1 pathway. Supporting this idea, overexpression of Repo or EAAT1 rescues age-associated impairments in LTM. Pharmacological inhibition of glutamate activity during consolidation improves LTM in klg mutants and aged flies. Altogether, our results indicate that LTM formation requires glial-dependent inhibition of glutamate signaling during memory consolidation, and aging disrupts this process by inhibiting the Klg-Repo-EAAT1 pathway.

Aging specifically impairs amnesiac-dependent memory in Drosophila.

Age-related memory impairment (AMI) is observed in many species. However, it is uncertain whether AMI results from a specific or a nonspecific decay in memory processing. In Drosophila, memory acquired after a single olfactory conditioning paradigm has three distinct phases: short-term memory (STM), middle-term memory (MTM), and longer-lasting anesthesia-resistant memory (ARM). Here, we demonstrate that age-related defects in olfactory memory are identical to those of the MTM mutant amnesiac (amn). Furthermore, amn flies do not exhibit an age-dependent decrease in memory, in contrast to other memory mutants. The absence of AMI in amn flies is restored by expression of an amn transgene predominantly in DPM cells. Thus, we propose that AMI in flies results from a specific decrease in amn-dependent MTM.

Long-Term Memory Engram Cells Are Established by c-Fos/CREB Transcriptional Cycling.

Training-dependent increases in c-fos have been used to identify engram cells encoding long-term memories (LTMs). However, the interaction between transcription factors required for LTM, including CREB and c-Fos, and activating kinases such as phosphorylated ERK (pERK) in the establishment of memory engrams has been unclear. Formation of LTM of an aversive olfactory association in flies requires repeated training trials with rest intervals between trainings. Here, we find that prolonged rest interval-dependent increases in pERK induce transcriptional cycling between c-Fos and CREB in a subset of KCs in the mushroom bodies, where olfactory associations are made and stored. Preexisting CREB is required for initial c-fos induction, while c-Fos is required later to increase CREB expression. Blocking or activating c-fos-positive engram neurons inhibits memory recall or induces memory-associated behaviors. Our results suggest that c-Fos/CREB cycling defines LTM engram cells required for LTM.

Two Parallel Pathways Assign Opposing Odor Valences during Drosophila Memory Formation.

During olfactory associative learning in Drosophila, odors activate specific subsets of intrinsic mushroom body (MB) neurons. Coincident exposure to either rewards or punishments is thought to activate extrinsic dopaminergic neurons, which modulate synaptic connections between odor-encoding MB neurons and MB output neurons to alter behaviors. However, here we identify two classes of intrinsic MB γ neurons based on cAMP response element (CRE)-dependent expression, γCRE-p and γCRE-n, which encode aversive and appetitive valences. γCRE-p and γCRE-n neurons act antagonistically to maintain neutral valences for neutral odors. Activation or inhibition of either cell type upsets this balance, toggling odor preferences to either positive or negative values. The mushroom body output neurons, MBON-γ5ß'2a/ß'2mp and MBON-γ2α'1, mediate the actions of γCRE-p and γCRE-n neurons. Our data indicate that MB neurons encode valence information, as well as odor information, and this information is integrated through a process involving MBONs to regulate learning and memory.

Coincident postsynaptic activity gates presynaptic dopamine release to induce plasticity in Drosophila mushroom bodies.

Simultaneous stimulation of the antennal lobes (ALs) and the ascending fibers of the ventral nerve cord (AFV), two sensory inputs to the mushroom bodies (MBs), induces long-term enhancement (LTE) of subsequent AL-evoked MB responses. LTE induction requires activation of at least three signaling pathways to the MBs, mediated by nicotinic acetylcholine receptors (nAChRs), NMDA receptors (NRs), and D1 dopamine receptors (D1Rs). Here, we demonstrate that inputs from the AL are transmitted to the MBs through nAChRs, and inputs from the AFV are transmitted by NRs. Dopamine signaling occurs downstream of both nAChR and NR activation, and requires simultaneous stimulation of both pathways. Dopamine release requires the activity of the rutabaga adenylyl cyclase in postsynaptic MB neurons, and release is restricted to MB neurons that receive coincident stimulation. Our results indicate that postsynaptic activity can gate presynaptic dopamine release to regulate plasticity.

A Drosophila ex vivo model of olfactory appetitive learning.

During olfactory appetitive learning, animals associate an odor, or conditioned stimulus (CS), with an unconditioned stimulus (US), often a sugar reward. This association induces feeding behavior, a conditioned response (CR), upon subsequent exposure to the CS. In this study, we developed a model of this behavior in isolated Drosophila brains. Artificial activation of neurons expressing the Gr5a sugar-responsive gustatory receptor (Gr5a GRNs) induces feeding behavior in starved flies. Consistent with this, we find that in dissected brains, activation of Gr5a GRNs induces Ca2+ transients in motor neurons, MN11 + 12, required for ingestion. Significantly, activation of Gr5a GRNs can substitute for presentation of sugar rewards during olfactory appetitive learning. Similarly, in dissected brains, coincident stimulation of Gr5a GRNs and the antennal lobe (AL), which processes olfactory information, results in increased Ca2+ influx into MN11 + 12 cells upon subsequent AL stimulation. Importantly, olfactory appetitive associations are not formed in satiated flies. Likewise, AL-evoked Ca2+ transients in MN11 + 12 are not produced in ex vivo brains from satiated flies. Our results suggest that a starved/satiated state is maintained in dissected brains, and that this ex vivo system will be useful for identification of neural networks involved in olfactory appetitive learning.

Learning defects in Drosophila growth restricted chico mutants are caused by attenuated adenylyl cyclase activity.

BACKGROUND: Reduced insulin/insulin-like growth factor signaling (IIS) is a major cause of symmetrical intrauterine growth retardation (IUGR), an impairment in cell proliferation during prenatal development that results in global growth defects and mental retardation. In Drosophila, chico encodes the only insulin receptor substrate. Similar to other animal models of IUGR, chico mutants have defects in global growth and associative learning. However, the physiological and molecular bases of learning defects caused by chico mutations, and by symmetrical IUGR, are not clear. RESULTS: In this study, we found that chico mutations impair memory-associated synaptic plasticity in the mushroom bodies (MBs), neural centers for olfactory learning. Mutations in chico reduce expression of the rutabaga-type adenylyl cyclase (rut), leading to decreased cAMP synthesis in the MBs. Expressing a rut (+) transgene in the MBs restores memory-associated plasticity and olfactory associative learning in chico mutants, without affecting growth. Thus chico mutations disrupt olfactory learning, at least in part, by reducing cAMP signaling in the MBs. CONCLUSIONS: Our results suggest that some cognitive defects associated with reduced IIS may occur, independently of developmental defects, from acute reductions in cAMP signaling.