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.

A non-canonical on-demand dopaminergic transmission underlying olfactory aversive learning.

Dopamine (DA) is involved in various brain functions including associative learning. However, it is unclear how a small number of DA neurons appropriately regulates various brain functions. DA neurons have a large number of release sites and release DA non-specifically to a large number of target neurons in the projection area in response to the activity of DA neurons. In contrast to this "broad transmission", recent studies in Drosophila ex vivo functional imaging studies have identified "on-demand transmission" that occurs independent on activity of DA neurons and releases DA specifically onto the target neurons that have produced carbon monoxide (CO) as a retrograde signal for DA release. Whereas broad transmission modulates the global function of the target area, on-demand transmission is suitable for modulating the function of specific circuits, neurons, or synapses. In Drosophila olfactory aversive conditioning, odor and shock information are associated in the brain region called mushroom body (MB) to form olfactory aversive memory. It has been suggested that DA neurons projecting to the MB mediate the transmission of shock information and reinforcement simultaneously. However, the circuit model based on on-demand transmission proposes that transmission of shock information and reinforcement are mediated by distinct neural mechanisms; while shock transmission is glutamatergic, DA neurons mediates reinforcement. On-demand transmission provides mechanical insights into how DA neurons regulate various brain functions.

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.

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.

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.

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.

Glial dysfunction causes age-related memory impairment in Drosophila.

Several aging phenotypes, including age-related memory impairment (AMI), are thought to be caused by cumulative oxidative damage. In Drosophila, age-related impairments in 1 hr memory can be suppressed by reducing activity of protein kinase A (PKA). However, the mechanism for this effect has been unclear. Here we show that decreasing PKA suppresses AMI by reducing activity of pyruvate carboxylase (PC), a glial metabolic enzyme whose amounts increase upon aging. Increased PC activity causes AMI through a mechanism independent of oxidative damage. Instead, increased PC activity is associated with decreases in D-serine, a glia-derived neuromodulator that regulates NMDA receptor activity. D-serine feeding suppresses both AMI and memory impairment caused by glial overexpression of dPC, indicating that an oxidative stress-independent dysregulation of glial modulation of neuronal activity contributes to AMI in Drosophila.

Fasting launches CRTC to facilitate long-term memory formation in Drosophila.

Canonical aversive long-term memory (LTM) formation in Drosophila requires multiple spaced trainings, whereas appetitive LTM can be formed after a single training. Appetitive LTM requires fasting prior to training, which increases motivation for food intake. However, we found that fasting facilitated LTM formation in general; aversive LTM formation also occurred after single-cycle training when mild fasting was applied before training. Both fasting-dependent LTM (fLTM) and spaced training-dependent LTM (spLTM) required protein synthesis and cyclic adenosine monophosphate response element-binding protein (CREB) activity. However, spLTM required CREB activity in two neural populations--mushroom body and DAL neurons--whereas fLTM required CREB activity only in mushroom body neurons. fLTM uses the CREB coactivator CRTC, whereas spLTM uses the coactivator CBP. Thus, flies use distinct LTM machinery depending on their hunger state.

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.

Mutations in the Drosophila insulin receptor substrate, CHICO, impair olfactory associative learning.

CHICO, the Drosophila homolog of vertebrate insulin receptor substrate (IRS), mediates insulin/insulin-like growth factor signaling (IIS), and reductions in chico severely disrupt cell growth and proliferation. We found extensive expression of chico in various Drosophila brain regions including the mushroom bodies (MBs), critical neural structures for olfactory learning. chico null mutants have significantly reduced brain sizes and perform poorly in an olfactory associative learning task, although their sensitivity to the odors and electric shocks used in this learning paradigm are normal. When initial memory is normalized by training for different amounts of time (short-duration training protocols), memory retention and retrieval in chico flies are indistinguishable from that of wild-type flies, demonstrating that chico mutants are defective specifically for memory formation. Inducing expression of a chico(+) transgene in neurons throughout development restores normal learning in a chico background, while inducing chico(+) specifically at the adult stage does not, suggesting that chico is required for development of a brain region required for forming olfactory associations. Significantly, expressing chico(+) in the MBs restores the number of MB neurons to wild-type amounts and also rescues chico learning defects. Our results suggest that chico-dependent growth of the MBs is essential for development of learning ability.

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.