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 down-regulation 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.

Keywords: CPEB; CREB; PUM; STAU; fragile X mental retardation.

Fig. 1.

Inhibiting protein synthesis in MB-V3 neurons impairs LTM formation. (A) Expression patterns of E0067-, E1132- and G0239-Gal4s. GFP labeled two pairs of MB-V3 neurons innervating α-lobe tips (arrow). Brain structures were immunostained with antidiscs large antibody (magenta). E0067- and E1132-Gal4s also expressed in brain surface glia (digitally removed). (Scale bars, 50 μm.) (B) Effects of RICIN^cs inhibition in E0067-, E1132-, and G0239-Gal4 neurons. One-day memory is impaired by inhibiting protein synthesis after spaced training (ST), but not massed training (MT), with active RICIN^cs (30 °C). One-day memory after spaced training was normal with inactive RICIN^cs (18 °C). Values are means ± SEM (***P < 0.001; n = 8 for each group). (C) The MB-V3 neuron projects Dscam-positive dendrites (green) exclusively within the entire α-lobe tip and syt-positive axons (green) at superior dorsofrontal protcerebrum outside of the MB. Fibers are labeled by a monomeric Kusabira Orange (mKO) protein (red). (D) Structural connections between MB-V3 dendrites and axons of MB neurons (L0124-LexA, dotted line) were visualized by GRASP labeling (green, Lower). (Scale bars, 50 μm.) For more details, please see SI Methods.

Fig. 2.

Neurotransmission and functional response of MB-V3 neurons during LTM formation. (A) Roles of MB-V3 neurotransmission on LTM. Blocking neurotransmission from MB-V3 neurons with temperature sensitive shi^ts1 protein (30 °C) during retrieval (P = 0.015; n = 12) or 8–16 h after training (P = 0.012; n = 8) impaired 1-d memory after spaced training, but not after massed training. Blocking neurotransmission during 0–8 or 16–24 h after spaced training or keeping the shi^ts1 flies in permissive temperature (18 °C) had no effect on 1-d memory. (B) Blocking neurotransmission from MB-V3 neurons during acquisition or 3 h after a single training session had no effects on memory retention. Values are means ± SEM (n = 8 for each group). (C) Neural activity in MB-V3 neurons in response to eight different odors (OCT, MCH, BA, EA, GA, MSC, EP, and IAA). Values are means ± SEM (n ≧ 8 for each odor). (D) Enhanced neural activity in MB-V3 neurons in response to conditioned odors after spaced, but not massed, training. Values are means ± SEM (P < 0.001; n ≧ 8 for each group). For more details, please see SI Methods.

Fig. 3.

Molecular machinery for LTM formation in the MB-V3 neurons. The G0239-Gal4-driven UAS-RNAi constructs (or CREB repressor) were inhibited by activated Gal80^ts (18 °C) throughout development and then were expressed by inactivating Gal80^ts (30 °C) 3 d before training. Control flies were kept constantly at 18 °C (activated Gal80^ts). (A–D) Induced knockdown of CREB with either UAS-dcreb2-b transgenic overexpression (A) or UAS-creb2^RNAi (B), of NMDAR1 and NMDAR2 with UAS-dsNR2;UAS-dsNR1 (C), or of ORB2 (D) with UAS-orb2^RNAi did not affect 1-d memory after spaced training. (E–K) In contrast, 1-d memory was impaired after spaced, but not massed, training with induced knockdown of ORB with UAS-orb^RNAi(R1) (P = 0.013) (E) or UAS-orb^RNAi(R5) (P = 0.01) (F), of CaMKII with UAS-CaMKII^hpn (P = 0.003) (G), of FMR with UAS-fmr^RNAi (1–7) (P < 0.001) (H) or UAS-fmr^RNAi (2–1) (P = 0.011) (I), of STUAFEN with UAS-stau^RNAi (P = 0.015) (J) or of PUMILIO with UAS-pum^RNAi (P = 0.009) (K). Values are means ± SEM (n ≧ 8 for each group). For more details, please see SI Methods.

Fig. 4.

A model of local mRNA translation in Drosophila olfactory memory. (A) Schematic representation of memory transformation from physiological coding in sparse α/β Kenyon cells (blue) to structural coding (red) in preexisting postsynaptic components of a MB-V3 neuron. (B) At activated MB-V3 synapses (red), presynaptic CS+US signals (blue), but not CS alone or US alone (gray), an CREB-independent mechanism relieves the translational block of RNA granules, which involves (i) STAUFEN detachment (38, 41), (ii) FMR and PUMILIO dephosphorylations (38, 42, 43), and (iii) ORB phosphorylation by activated CaMKII, leading to the poly-A tail elongation (–6). Consequently, these events recruit additional ribosomes to up-regulate mRNA translation of LTM-related proteins (42), which eventually lead to an enhanced response to the CS+. Note that the subcellular locations of proteins and their cofunctions in the model have not been experimentally determined.