A subset of cholinergic mushroom body neurons blocks long-term memory formation in Drosophila.

Long-term memory (LTM) requires learning-induced synthesis of new proteins allocated to specific neurons and synapses in a neural circuit. Not all learned information, however, becomes permanent memory. How the brain gates relevant information into LTM remains unclear. In Drosophila adults, weak learning after a single training session in an olfactory aversive task typically does not induce protein-synthesis-dependent LTM. Instead, strong learning after multiple spaced training sessions is required. Here, we report that pre-synaptic active-zone protein synthesis and cholinergic signaling from the early α/ß subset of mushroom body (MB) neurons produce a downstream inhibitory effect on LTM formation. When we eliminated inhibitory signaling from these neurons, weak learning was then sufficient to form LTM. This bidirectional circuit mechanism modulates the transition between distinct memory phase functions in different subpopulations of MB neurons in the olfactory memory circuit.

CREBB repression of protein synthesis in mushroom body gates long-term memory formation in Drosophila.

Learned experiences are not necessarily consolidated into long-term memory (LTM) unless they are periodic and meaningful. LTM depends on de novo protein synthesis mediated by cyclic AMP response element-binding protein (CREB) activity. In Drosophila, two creb genes (crebA, crebB) and multiple CREB isoforms have reported influences on aversive olfactory LTM in response to multiple cycles of spaced conditioning. How CREB isoforms regulate LTM effector genes in various neural elements of the memory circuit is unclear, especially in the mushroom body (MB), a prominent associative center in the fly brain that has been shown to participate in LTM formation. Here, we report that i) spaced training induces crebB expression in MB α-lobe neurons and ii) elevating specific CREBB isoform levels in the early α/ß subpopulation of MB neurons enhances LTM formation. By contrast, learning from weak training iii) induces 5-HT1A serotonin receptor synthesis, iv) activates 5-HT1A in early α/ß neurons, and v) inhibits LTM formation. vi) LTM is enhanced when this inhibitory effect is relieved by down-regulating 5-HT1A or overexpressing CREBB. Our findings show that spaced training-induced CREBB antagonizes learning-induced 5-HT1A in early α/ß MB neurons to modulate LTM consolidation.

Neuropeptide F inhibits dopamine neuron interference of long-term memory consolidation in Drosophila.

Long-term memory (LTM) formation requires consolidation processes to overcome interfering signals that erode memory formation. Olfactory memory in Drosophila involves convergent projection neuron (PN; odor) and dopaminergic neuron (DAN; reinforcement) input to the mushroom body (MB). How post-training DAN activity in the posterior lateral protocerebrum (PPL1) continues to regulate memory consolidation remains unknown. Here we address this question using targeted transgenes in behavior and electrophysiology experiments to show that (1) persistent post-training activity of PPL1-α2α'2 and PPL1-α3 DANs interferes with aversive LTM formation; (2) neuropeptide F (NPF) signaling blocks this interference in PPL1-α2α'2 and PPL1-α3 DANs after spaced training to enable LTM formation; and (3) training-induced NPF release and neurotransmission from two upstream dorsal-anterior-lateral (DAL2) neurons are required to form LTM. Thus, NPF signals from DAL2 neurons to specific PPL1 DANs disinhibit the memory circuit, ensuring that periodic events are remembered as consolidated LTM.

CREBA and CREBB in two identified neurons gate long-term memory formation in Drosophila.

Episodic events are frequently consolidated into labile memory but are not necessarily transferred to persistent long-term memory (LTM). Regulatory mechanisms leading to LTM formation are poorly understood, however, especially at the resolution of identified neurons. Here, we demonstrate enhanced LTM following aversive olfactory conditioning in Drosophila when the transcription factor cyclic AMP response element binding protein A (CREBA) is induced in just two dorsal-anterior-lateral (DAL) neurons. Our experiments show that this process is regulated by protein-gene interactions in DAL neurons: (1) crebA transcription is induced by training and repressed by crebB overexpression, (2) CREBA bidirectionally modulates LTM formation, (3) crebA overexpression enhances training-induced gene transcription, and (4) increasing membrane excitability enhances LTM formation and gene expression. These findings suggest that activity-dependent gene expression in DAL neurons during LTM formation is regulated by CREB proteins.

Automated in situ brain imaging for mapping the Drosophila connectome.

Mapping the connectome, a wiring diagram of the entire brain, requires large-scale imaging of numerous single neurons with diverse morphology. It is a formidable challenge to reassemble these neurons into a virtual brain and correlate their structural networks with neuronal activities, which are measured in different experiments to analyze the informational flow in the brain. Here, we report an in situ brain imaging technique called Fly Head Array Slice Tomography (FHAST), which permits the reconstruction of structural and functional data to generate an integrative connectome in Drosophila. Using FHAST, the head capsules of an array of flies can be opened with a single vibratome sectioning to expose the brains, replacing the painstaking and inconsistent brain dissection process. FHAST can reveal in situ brain neuroanatomy with minimal distortion to neuronal morphology and maintain intact neuronal connections to peripheral sensory organs. Most importantly, it enables the automated 3D imaging of 100 intact fly brains in each experiment. The established head model with in situ brain neuroanatomy allows functional data to be accurately registered and associated with 3D images of single neurons. These integrative data can then be shared, searched, visualized, and analyzed for understanding how brain-wide activities in different neurons within the same circuit function together to control complex behaviors.

Visualizing long-term memory formation in two neurons of the Drosophila brain.

Long-term memory (LTM) depends on the synthesis of new proteins. Using a temperature-sensitive ribosome-inactivating toxin to acutely inhibit protein synthesis, we screened individual neurons making new proteins after olfactory associative conditioning in Drosophila. Surprisingly, LTM was impaired after inhibiting protein synthesis in two dorsal-anterior-lateral (DAL) neurons but not in the mushroom body (MB), which is considered the adult learning and memory center. Using a photoconvertible fluorescent protein KAEDE to report de novo protein synthesis, we have directly visualized cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-dependent transcriptional activation of calcium/calmodulin-dependent protein kinase II and period genes in the DAL neurons after spaced but not massed training. Memory retention was impaired by blocking neural output in DAL during retrieval but not during acquisition or consolidation. These findings suggest an extra-MB memory circuit in Drosophila: LTM consolidation (MB to DAL), storage (DAL), and retrieval (DAL to MB).

Serotonin-mushroom body circuit modulating the formation of anesthesia-resistant memory in Drosophila.

Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of intermediate-term memories: anesthesia-sensitive memory, which requires the amnesiac gene, and anesthesia-resistant memory (ARM), which requires the radish gene. Here, we report that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This result suggests that 5HT and Radish protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two dorsal paired medial (DPM) neurons onto the mushroom bodies (MBs), the olfactory learning and memory center in Drosophila: (i) DPM neurons were 5HT-antibody immunopositive; (ii) temporal inhibition of 5HT synthesis or release from DPM neurons, but not from other serotonergic neurons, impaired ARM formation; (iii) knocking down the expression of d5HT1A serotonin receptors in α/ß MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the Amnesiac peptide required for anesthesia-sensitive memory formation, the two DPM neurons also release 5HT acting on MB neurons for ARM formation.