Drosophila ORB protein in two mushroom body output neurons is necessary for long-term memory formation

Abstract

Memory is initially labile and gradually consolidated over time through new protein synthesis into a long-lasting stable form. Studies of odor-shock associative learning in Drosophila have established the mushroom body (MB) as a key brain structure involved in olfactory long-term memory (LTM) formation. Exactly how early neural activity encoded in thousands of MB neurons is consolidated into protein-synthesis-dependent LTM remains unclear. Here, several independent lines of evidence indicate that changes in two MB vertical lobe V3 (MB-V3) extrinsic neurons are required and contribute to an extended neural network involved in olfactory LTM: (i) inhibiting protein synthesis in MB-V3 neurons impairs LTM; (ii) MB-V3 neurons show enhanced neural activity after spaced but not massed training; (iii) MB-V3 dendrites, synapsing with hundreds of MB α/β neurons, exhibit dramatic structural plasticity after removal of olfactory inputs; (iv) neurotransmission from MB-V3 neurons is necessary for LTM retrieval; and (v) RNAi-mediated downregulation of oo18 RNA-binding protein (involved in local regulation of protein translation) in MB-V3 neurons impairs LTM. Our results suggest a model of long-term memory formation that includes a systems-level consolidation process, wherein an early, labile olfactory memory represented by neural activity in a sparse subset of MB neurons is converted into a stable LTM through protein synthesis in dendrites of MB-V3 neurons synapsed onto MB α lobes.L ong-term memory (LTM) and long-term synaptic plasticity require de novo protein synthesis, which is regulated at transcriptional and/or translational levels in a synapse-specific manner (1-3). Synapse-specific plasticity during LTM formation in some contexts may involve local regulation of protein translation by a family of RNA-binding proteins, the cytoplasmic polyadenylation element-binding proteins (CPEBs) (2). Neuronal CPEBs have two conformational states. The inactive state predominates at low levels of CPEB expression and represses translation from nascent mRNAs. The active state is achieved either via a self-perpetuating prion-like state when expression levels surpass a threshold or via Ca 2+ /calmoduline-dependent protein kinase II (CaMKII)-mediated phosphorylation, and translation is initiated by elongation of an mRNA's poly-A tail (4-6). In other species, CPEB1 has been shown to contribute to long-term facilitation or potentiation (5, 7). In Drosophila, oo18 RNA-binding protein 2 (ORB2) appears required for long-term memory formation after courtship conditioning (8, 9). Any role for ORB in fruit fly memory formation, however, remains unclear.Drosophila can learn to associate an odor (conditioned stimulus, CS) with foot-shock punishment (unconditioned stimulus, US). This odor-shock association initially is labile, lasting for only about a day after one training session. With repetitive, spaced training (ST) sessions (rest intervals between each session), a protein synthesis-dependent, LTM is formed. With repetitive, massed training (MT) ses...

RNA-binding profiles of Drosophila CPEB proteins Orb and Orb2

“…Existing expression data suggest that the localization of Orb and Orb2 does not overlap substantially, and they may in fact down-regulate the expression of the respective paralogue (13,19). Indeed, both Orb and Orb2 are involved in LTM, albeit in distinct neuronal classes (12,15). A mechanism of acquiring functional divergence in which changes in protein expression patterns are faster than changes in target sequence specificity was suggested for DNA-binding proteins and constitutes a likely pattern in the evolution of nucleic acid-binding protein families (58).…”

“…The molecular machinery controlling local protein synthesis has been best studied in vertebrate oocyte development during which members of the cytoplasmic polyadenylation element binding (CPEB) protein family, in concert with other RNA-binding proteins, control the temporal expression of maternal RNAs (8,9). More recently, CPEB proteins also have been found to be expressed in adult nervous systems (10)(11)(12)(13) where they are thought to be involved in synaptic plasticity and LTM (12,(14)(15)(16)(17), suggesting evolutionary conservation of the mechanism of local translation across species, tissues, and developmental stages.…”

“…Although both proteins play critical and nonredundant roles in germline formation (18,19), Orb2 is also essential for nervous system development (13) and is acutely required for LTM (12,20), as is Orb (15). Both proteins contain an unstructured N-terminal poly-glutamine (poly-Q) stretch and a well-conserved C-terminal RNA-binding domain (RBD) consisting of two RNA recognition motifs (RRMs) and a zinc finger (Znf) region (12).…”

The differential requirement of mushroom body α/β subdivisions in long-term memory retrieval in Drosophila

“…Also, MB α/β subdivisions might communicate with each other through feedback loops constructed by various MB extrinsic neurons, such as DPM neurons and APL neurons (Lee et al, 1999;Keene et al, 2006;Wu et al, 2011;Pitman et al, 2011). Another possibility is that MB α/β subdivisions, being parallel with each other, release neurotransmitters to their common downstream neurons suggested for LTM storage, such as the DAL neurons (Chen et al, 2012) MB-V2 neurons (Séjourné et al, 2011), MB-V3 neurons (Pai et al, 2013) and the ellipsoid body (Wu et al, 2007), to regulate formation, consolidation and retrieval of LTM separately.…”

Histone deacetylase inhibition facilitates massed pattern-induced synaptic plasticity and memory

“…Studies have shown pattern sensitivity of different molecular processes relevant for these events (Ye et al 2008;Akalal et al 2010;Kim et al 2010;Pai et al 2013). Wu et al (2001) showed that spaced depolarizationinduced activation of extracellular signal-regulated kinase (ERK) is longer lasting than the ERK activation induced by massed pattern of stimulation.…”