The neural circuit linking mushroom body parallel circuits induces memory consolidation in Drosophila

Abstract

Memory consolidation is augmented by repeated learning following rest intervals, which is known as the spacing effect. Although the spacing effect has been associated with cumulative cellular responses in the neurons engaged in memory, here, we report the neural circuit-based mechanism for generating the spacing effect in the memory-related mushroom body (MB) parallel circuits in Drosophila To investigate the neurons activated during the training, we monitored expression of phosphorylation of mitogen-activated protein kinase (MAPK), ERK [phosphorylation of extracellular signal-related kinase (pERK)]. In an olfactory spaced training paradigm, pERK expression in one of the parallel circuits, consisting of γm neurons, was progressively inhibited via dopamine. This inhibition resulted in reduced pERK expression in a postsynaptic GABAergic neuron that, in turn, led to an increase in pERK expression in a dopaminergic neuron specifically in the later session during spaced training, suggesting that disinhibition of the dopaminergic neuron occurs during spaced training. The dopaminergic neuron was significant for gene expression in the different MB parallel circuits consisting of α/βs neurons for memory consolidation. Our results suggest that the spacing effect-generating neurons and the neurons engaged in memory reside in the distinct MB parallel circuits and that the spacing effect can be a consequence of evolved neural circuit architecture.

Keywords: Drosophila; gene expression; long-term memory; mushroom body; spaced learning.

Fig. 1.

Expression of pERK is decreased during spaced training. (A and B) Nuclear pERK in subsets of MB neurons following single (A, Upper) or spaced training (A, Lower). GFP fused to the nuclear localization signal (nlsGFP) was expressed using the split-GAL4 drivers (SI Appendix, Fig. S2): MB185B for α/βs (P = 0.0087; n = 5 to 6), MB594B for α/βc (P = 0.3602; n = 5–6), and MB131B for γm (P = 0.0184; n = 4 to 5) (Scale bar, 10 μm). (C and D) Activation of γm neurons by pulsed red light (5 Hz, 1 min) during the shock periods in the last three sessions of spaced training impaired 1-d memory (Kruskal–Wallis test, P = 0.0005; n = 8–12) (D) without affecting 1-h memory after spaced training (P = 0.1320; n = 6) (C). Light was illuminated during paring of CS+ odor with electric shock. CsChrimson was expressed in γm neurons using MB131B. (E–H) Dopamine signaling was required for the decrease in pERK expression in γm neurons. Dumb2 mutant flies carry an upstream activating sequence (UAS) insertion in the first intron, which disrupts the expression of DopR1 but allows expression of DopR1 by crossing with the GAL4 driver. GFP was expressed by γm-LexA (R16A06-LexA) (SI Appendix, Fig. S2F). (E and F, Upper) Single training. (E and F, Lower) Spaced training. (G) (Kruskal–Wallis test, P = 0.0001; n = 6). (F and H) DopR1 was rescued in α/βs neurons using MB477B (P = 0.3939; n = 6) and in γm neurons using MB131B (P = 0.0014; n = 6) (Scale bar, 10 μm). The arrowheads indicate neurons expressing pERK. Data are represented as a mean ± SEM. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01.

Fig. 2.

Artificial activation of γm neurons impairs Arc2 expression in LTM formation. (A–C) Arc2 mRNA was specifically induced by spaced training. The flies were subjected to spaced training (Kruskal–Wallis test, P = 0.0048; n = 6) (A), single training (Kruskal–Wallis test, P = 0.6700; n = 6) (B), or massed training (Kruskal–Wallis test, P = 0.3313; n = 6) (C) (SI Appendix, Fig. S4A for the experimental schedules). RNA extracted from the fly heads was analyzed via RT-qPCR. (D) Knockdown of Arc2 impaired 1-d memory after spaced training. RNAi-based knockdown of Arc2 (Arc2-IR) in the whole MBs was performed using MBsw (38) by feeding the flies RU486 for 3 d (P = 0.0003; n = 8). (E) Arc2 protein was expressed in α/βs neurons at 2 h after spaced training. HA tags were inserted at the C terminus of Arc2 (Arc2::HA). NlsGFP was expressed in α/βs neurons using MB477B and in γm neurons using MB131B (Scale bar, 10 μm). The arrowheads indicate neurons expressing Arc2. (F and G) Arc2 mRNA expression at 1 h after spaced training was inhibited by expressing CREB2-b in α/βs neurons using MB477B (Kruskal–Wallis test, P < 0.0001; n = 9) (F) and by activation of γm neurons during the last three sessions of spaced training (Kruskal–Wallis test, P = 0.0039; n = 6) (G). Pulsed red light (5 Hz, 1 min) was delivered to flies expressing CsChrimson using MB131B (γm GAL4) as they received electric shocks during the last three sessions (G). Data are represented as a mean ± SEM. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

A GABAergic neuron (MBON-γ1pedc) postsynaptic to γm neurons mediates gene expression required for LTM formation. (A and B) Activation of the MBON-γ1pedc neuron by pulsed red light (40 Hz, 1 min) during the shock periods of the last three sessions of spaced training impaired 1-d memory (Kruskal–Wallis test, P = 0.0001; n = 8–10) (A), and Arc2 mRNA expression at 1 h after spaced training (Kruskal–Wallis test, P = 0.0025; n = 6) (B). CsChrimson was expressed in the MBON-γ1pedc neuron using MB112C (SI Appendix, Fig. S6C). (C) Nuclear pERK expression was decreased in the MBON-γ1pedc neuron after spaced training. The MBON-γ1pedc neuron was labeled with nlsGFP using MB112C (Scale bar, 2 μm). n = 7–14 for all data. Data are represented as a mean ± SEM. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

The postsynaptic neuron to the GABAergic MBON-γ1pedc neuron (PPL1-α′2α2) is required for gene expression in LTM formation. (A and B) Nuclear pERK expression was increased in the PPL1-α′2α2 neuron following spaced training. The PPL1-α′2α2 neuron was labeled with nlsGFP using MB058B (SI Appendix, Fig. S6E) (Kruskal–Wallis test, P < 0.0001; n = 15–20). (C and D) Inactivation of the PPL1-α′2α2 neuron impaired Arc2 mRNA expression at 1 h after spaced training (Kruskal–Wallis test, P < 0.0001; n = 9 to 10) (C) and 1-d memory after spaced training (Kruskal–Wallis test, P = 0.0109; n = 8) (D). Kir2.1 was expressed in the PPL1-α′2α2 neuron using MB058B. (E and F) Optogenetic inactivation of the PPL1-α′2α2 neuron impaired 1-d memory after spaced training (Kruskal–Wallis test, P < 0.0001; n = 8) (E), and Arc2 mRNA expression at 1 h after spaced training (Kruskal–Wallis test, P = 0.0128; n = 6) (F). MB058B was used to express eNpHR3.0 in the PPL1-α′2α2 neuron. Flies were illuminated by red light at 40 Hz during the shock periods of the indicated sessions of spaced training. (G) Model: Arc2 expression is induced in α/βs neurons via simultaneous activation of α/βs and PPL1-α′2α2 neurons. Spaced training allows activation of the PPL1-α′2α2 neuron due to reduced activity in γm and GABAergic MBON-γ1pedc neurons. Data are represented as a mean ± SEM. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.