Skip to main content
NCBI home page
Learning & Memory logoLink to Learning & Memory
. 2024 May;31(5):a054013. doi: 10.1101/lm.054013.124

Presynaptic regulators in memory formation

Oriane Turrel 1, Lili Gao 1, Stephan J Sigrist 1,2,✉
PMCID: PMC11199941  PMID: 38862173

Abstract

The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we investigate the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the Drosophila mushroom body Kenyon cells. Our findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.

Synaptic plasticity in the brain is widely acknowledged to play a crucial role in learning and memory formation. However, understanding how plastic changes affect synapse performance and consequently circuit operation during these processes remains a significant challenge in contemporary neuroscience research. The rapid formation of memory engrams and subsequent refinement processes likely involve alterations in both postsynaptic and presynaptic compartments, and encompass both Hebbian and homeostatic forms of synaptic plasticity covering a timescale from milliseconds to hours and days.

The mushroom body (MB) of Drosophila melanogaster, commonly known as the fruit fly, has been worked out as a central hub for associative-memory formation and storage. After a single cycle of conditioning, flies form both short-term memory (STM) and middle-term memory (MTM), which can be assessed immediately or 1–3 h postconditioning, respectively. Long-term memory (LTM) is robustly formed after three spaced conditioning cycles. The MB-intrinsic Kenyon cells (KCs) form a neuropile comprising both axons and dendrites. Dendrites are grouped together to form the calyx, while the extended axons create the MB lobes, consisting of two verticals (α/α′) and three horizontals (β/β′ and γ) (Crittenden et al. 1998; Gerber et al. 2004; Krashes et al. 2007). Extensive research has elucidated the circuitry within the MB and allowed for the identification of specific subpopulations of KCs responsible for each memory phase (Isabel and Preat 2008; Bouzaiane et al. 2015).

The presynaptic specialization of chemical synapses triggers synaptic transmission by incoming action potentials activating presynaptic Ca2+ influx through voltage-gated channels. In turn, this triggers the fusion and exocytosis of synaptic vesicles (SVs) containing neurotransmitters at dedicated sites called active zones (AZs) (Rizo and Rosenmund 2008; Südhof 2012). The molecular mechanisms of presynaptic SV release and AZ organization in D. melanogaster are generally similar to those in mammals. Over the past few years, significant progress has been made in describing the developmental assembly and resulting macromolecular organization of Drosophila AZs, particularly using the larval neuromuscular junction (NMJ) (Liu et al. 2011; Böhme et al. 2016). NMJ synapses allow for a combination of genetics, pharmacology, electrophysiology, and super-resolution imaging techniques. Here, the master scaffold protein Bruchpilot (BRP), a member of the ELKS/CAST protein family of AZ proteins, has been demonstrated to localize Unc13A (a member of the Munc13 family of release factors) in defined nanoscopic modules proximal to voltage-gated Ca2+ channels Cacophony (Böhme et al. 2016; Reddy-Alla et al. 2017; Ghelani et al. 2023).

Presynaptic changes in SV release are believed to contribute to memory processes (Citri and Malenka 2008; Kittel and Heckmann 2016) including in Drosophila’s KCs (Dubnau et al. 2001; Woitkuhn et al. 2020). When exploring potential plasticity mechanisms at Drosophila presynaptic terminals, homeostatic plasticity processes at NMJ synapses appear relevant (Böhme et al. 2019; Turrel et al. 2022; Ramesh et al. 2023). Following the pharmacological blockade of postsynaptic glutamate receptors with Philanthotoxin, a robust presynaptic homeostatic potentiation (PHP) occurs, characterized by increased SV release per action potential or quantal content. Philanthotoxin triggers rapid presynaptic AZ remodeling, evidenced by augmented levels of AZ cytomatrix and SV release proteins, notably BRP, Unc13A, and Cacophony, leading to enhanced Ca2+ transients and increased release-ready SVs. BRP-null mutant studies delineated two different phases (Böhme et al. 2019): a rapid induction phase of SV release potentiation (measured at 10 min Philanthotoxin treatment; BRP-independent), followed by a sustained phase (measured after 30 min Philanthotoxin treatment; BRP-dependent). Linking NMJ AZ plasticity with memory-related plasticity in the MB, paired conditioning was found to augment BRP and Unc13A levels within KCs over several hours, reversing within a day. Genetic evidence suggests that this conditioning-triggered AZ remodeling in KCs is crucial for aversive olfactory MTM (Turrel et al. 2022). Furthermore, mutants of Aplip-1, Arl8, and IMAC, involved in BRP trafficking and AZ assembly, display impaired AZ scaffold remodeling. While these mutants exhibit PHP at NMJs, they fail to sustain it. Intriguingly, MB knockdown of these AZ-plasticity-promoting factors supports STM formation but not MTM formation (Turrel et al. 2022).

In this study, we extend our analysis to presynaptic regulatory processes significant for specific memory phases in the KCs. Specifically, we selected two proteins that per se have distinct roles based on NMJ analysis. The small GTPase Rab3 is involved in acute SV cycling, docking, and exocytosis (Takamori et al. 2006; Binotti et al. 2016). In contrast, Neurexin-1 (Nrx) is a presynaptic cell adhesion membrane protein that signals to advance the postsynaptic specialization of NMJ synapses (Owald et al. 2012; Ramesh et al. 2021). However, both proteins share the characteristic of altering the distribution of BRP over developmentally forming AZs at NMJs (Graf et al. 2009; Chen et al. 2015): Indeed, both Rab3 and Nrx mutants exhibit NMJs where BRP is sequestered to fewer but enlarged AZs.

To address the role of both factors in postmaturation neurons of adult fruit flies, we used the TARGET system to induce the expression of RNA interference (RNAi) only in the adult posthatching brain, in order to avoid compensatory effects due to the absence of those proteins during development. This expression system exploits the inhibition of the GAL4 transcription factor activity by expressing a temperature-sensitive Gal80 inhibitor (Gal80ts) at low temperature (18°C) (McGuire et al. 2003). This inhibition can then be lifted by shifting the animals to a higher temperature (30°C). The tubulin-Gal80ts;OK107 combination (Gal80ts;OK107) was used to allow for conditional expression in all KCs, while tubulin-Gal80ts;c739 (Gal80ts;c739) was used to conditionally express in the adult α/β-KCs only. In our experiments, adult flies, both sexes, were raised at 18°C until 2–3 d after hatching, and then either shifted for 5 d to 30°C before conditioning to induce RNAi expression or kept at 18°C for noninduced controls before training and testing.

To assess memory, flies underwent classical olfactory aversive conditioning following the protocol outlined by Tully and Quinn (1985). Conditioning involved groups of ∼30–40 flies exposed to 3-octanol and 4-methylcyclohexanol odors (diluted at 1:100) paired with electrical shocks as a behavioral reinforcer, using a 120 AC current. Memory conditioning and tests were conducted using a T-maze apparatus (Tully and Quinn 1985; Turrel et al. 2022). During single-cycle training, flies were exposed to one odor (conditioned stimulus, CS+) paired with electrical shocks (unconditioned stimulus, US, 12 shocks of 1.30 sec and an ITI of 3.70 sec) for 1 min, followed by 1 min of pure air flow, and then the presentation of a second odor (CS−) without shock for another minute. In the test phase, directly after or 1–3 h after conditioning for STM and MTM, respectively, flies were given 1 min to choose between two arms, each with a distinct odor. A performance index (PI) was calculated as the difference between the number of flies in each arm divided by the sum of flies in both arms. The PI values ranged from 0 to 1, where 0 indicated no associative learning (50:50 distribution of flies) and a value of 1 indicated complete learning (all flies avoided the conditioned odor). To observe LTM, flies were tested 24 h after three cycles of conditioning spaced by 15 min of rest. For olfactory acuity and shock reactivity assessments, ∼40 flies were placed in a choice position between either one odor and air for 1 min or electric shocks and no shock, respectively.

We initiated our investigation by assessing the efficacy of acute RNAi expression against Rab3 and Nrx in KCs, while concurrently examining whether such knockdown would interfere with the overall architecture of AZs in the KCs. First, we generated a polyclonal antibody against Rab3 by performing immunization of rabbits with the purified full-length Rab3 protein (immunization and affinity purification of the antibody-containing serum was performed by the company BioGENES). We next assessed and confirmed its specificity in control w1118 and mutant rab3rup (Graf et al. 2009; Chen et al. 2015) flies by western blot (Supplemental Fig. S1A; western blot analysis performed as in Huang et al. [2020]) and by immunostaining in adult brains (Supplemental Fig. S1B; brains processed as in Turrel et al. [2022]). Then, after 5 d of RNAi induction, we stained the brains of Gal80ts;OK107/RNAi-Rab3 flies and observed a reduction of ∼40% of Rab3 antibody staining intensity within the MB lobes, while Gal80ts;OK107/RNAi-Nrx flies showed a reduction of ∼30% after staining with an Nrx antibody (diluted 1:250) (described in Owald et al. [2012]) (Fig. 1A). To investigate the AZ architecture, in a second experiment, we stained against BRP, Syd1, and Unc13A. Flies with an acute knockdown of Rab3 or Nrx showed normal BRP (BRPnc82, diluted 1:50; DSHB), Syd1 (diluted 1:250) (Owald et al. 2010), and Unc13A (diluted 1:250) (Böhme et al. 2016) intensity (Fig. 1B), indicating the gross AZ architecture is not affected by those knockdowns. Given that our behavioral data do not show any trend for leakiness (see Fig. 2), as driver and RNAi controls behaved similarly, we did not perform similar immunostainings for the RNAi-only controls.

Figure 1.

Figure 1.

Open in a new tab

Rab3 and Nrx adult knockdown in all KCs do not alter the gross architecture of AZs. (A) Quantification of Rab3 (F(2,35) = 4.829, P = 0.0145, n = 12, post hoc Tukey's test Gal80ts;OK107/+ vs. Gal80ts;OK107/RNAi-Rab3, [*] P < 0.05) and Nrx (F(2,35) = 6.353, P = 0.0046, n = 12, post hoc Tukey's test Gal80ts;OK107/+ vs. Gal80ts;OK107/RNAi-Rab3, [**] P < 0.01) staining intensity in MB lobes of flies presenting knockdown of either Rab3 or Nrx in adult MB lobes. Representative confocal images are shown with quantitative graphics. Scale bar, 50 µm. (B) Quantification of BRPnc82 (F(2,41) = 1.704, P = 0.1953, n = 14), Syd1 (F(2,41) = 3.154, P = 0.0538, n = 14), and Unc13A (F(2,41) = 0.5814, P = 0.5639, n = 14) staining intensity in MB lobes of Gal80ts;OK107/RNAi-Rab3 flies and Gal80ts;OK107/RNAi-Nrx flies compared to control flies. Representative confocal images are shown with quantitative graphics. Scale bar, 50 µm. Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad Software). Differences were tested by one-way ANOVA with Tukey's post hoc test. Asterisks denote the least significant of the pairwise post hoc comparisons between the genotype of interest and its controls following the usual nomenclature. P > 0.05, (*) P ≤ 0.05, (**) P ≤ 0.01.

Figure 2.

Figure 2.

Open in a new tab

Memory after postdevelopmental KCs knockdown of Rab3 and Nrx. (A) Flies expressing RNAi-Rab3 in the adult MB lobes present defect of STM (F(2,23) = 6.701, P = 0.0056, n = 8, post hoc Tukey's multiple comparisons test, Gal80ts;OK107/+ vs. Gal80ts;OK107/RNAi-Rab3, [**] P < 0.01, Gal80ts;OK107/RNAi-Rab3 vs. +/RNAi-Rab3, [*] P < 0.05), MTM 3 h (F(2,38) = 12.32, P < 0.0001, n ≥ 11, post hoc Tukey's multiple comparisons test, Gal80ts;OK107/+ vs. Gal80ts;OK107/RNAi-Rab3, [***] P < 0.001, Gal80ts;OK107/RNAi-Rab3 vs. +/RNAi-Rab3, [**] P < 0.01), and LTM (F(2,17) = 6.895, P = 0.0075, n = 6, post hoc Tukey's multiple comparisons test, Gal80ts;OK107/+ vs. Gal80ts;OK107/RNAi-Rab3, [**] P < 0.01, Gal80ts;OK107/RNAi-Rab3 vs. +/RNAi-Rab3, [*] P < 0.05) but normal MTM 1 h (F(2,28) = 0.2027, P = 0.7406, n ≥ 9) after 5 d of induction. Without induction, those flies have normal STM (F(2,19) = 0.1396, P = 0.8707, n ≥ 4), MTM 3 h (F(2,23) = 0.7502, P = 0.4845, n = 8), and LTM (F(2,17) = 0.6034, P = 0.5597, n = 6). Half-scores are presented in Supplemental Table S1. (B) Flies expressing RNAi-Rab3 in the adult αβ lobes present normal STM (F(2,17) = 2.385, P = 0.1260, n = 6) and MTM 1 h (F(2,15) = 0.3770, P = 0.6932, n ≥ 5), whereas MTM 3 h (F(2,17) = 6.035, P = 0.0119, n = 6, post hoc Tukey's multiple comparisons test, Gal80ts;c739/+ vs. Gal80ts;c739/RNAi-Rab3, [*] P < 0.05, Gal80ts;c739/RNAi-Rab3 vs. +/RNAi-Rab3, [*] P < 0.05) and LTM (F(2,17) = 31.42, P < 0.0001, n = 6, post hoc Tukey's multiple comparisons test, Gal80ts;c739/+ vs. Gal80ts;c739/RNAi-Rab3, [***] P < 0.001, Gal80ts;c739/RNAi-Rab3 vs. +/RNAi-Rab3, [***] P < 0.001) are decreased after 5 d of induction. Without induction, those flies have normal MTM 3 h (F(2,17) = 2.615, P = 0.1061, n = 6) and LTM (F(2,17) = 0.7554, P = 0.4869, n = 6). Half-scores are presented in Supplemental Table S2. (C) Flies expressing RNAi-Nrx in the adult MB lobes exhibit defect of STM (F(2,17) = 5.671, P = 0.0146, n = 6, post hoc Tukey's multiple comparisons test, Gal80ts;OK107/+ vs. Gal80ts;OK107/RNAi-Nrx, [*] P < 0.05, Gal80ts;OK107/RNAi-Nrx vs. +/RNAi-Nrx, [*] P < 0.05), MTM 1 h (F(2,42) = 14.80, P < 0.0001, n ≥ 12, post hoc Tukey's multiple comparisons tests, Gal80ts;OK107/+ vs. Gal80ts;OK107/RNAi-Nrx, [***] P < 0.001, Gal80ts;OK107/RNAi-Nrx vs. +/RNAi-Nrx, [***] P < 0.001), and 3 h (F(2,26) = 6.363, P = 0.0061, n ≥ 8, post hoc Tukey's multiple comparisons test, Gal80ts;OK107/+ vs. Gal80ts;OK107/RNAi-Nrx, [*] P < 0.05, Gal80ts;OK107/RNAi-Nrx vs. +/RNAi-Nrx, [*] P < 0.05). Those flies present normal LTM (F(2,17) = 1.066, P = 0.3692, n = 6). After staying at 18°C, the STM (F(2,30) = 0.8680, P = 0.4308, n ≥ 9), MTM 1 h (F(2,26) = 0.5975, P = 0.5582, n ≥ 8), and 3 h (F(2,23) = 0.5992, P = 0.5584, n = 8) of those flies appear normal. Half-scores are presented in Supplemental Table S3. Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad Software). Differences were tested by one-way ANOVA with Tukey's post hoc test. Asterisks denote the least significant of the pairwise post hoc comparisons between the genotype of interest and its controls following the usual nomenclature. P > 0.05, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001. The gray squares indicate experiments in which noninduced controls were not needed.

Before assessing memory function, it was imperative to ensure that the expression of the RNAi constructs within KCs did not affect the sensory capabilities of the tested flies. Therefore, we initially evaluated olfactory acuity and shock sensitivity in the knockdown flies following 5 d of induction at 30°C. Even with natural variation due to testing at different periods of the year between each set of genotypes, we observed normal olfactory acuity to both odors used in the study, as well as normal shock sensitivity in all genotypes and no visible changes between each mutant line and their genetic controls (Supplemental Fig. S1C–E).

After 5 d of induction, we conditioned flies expressing RNAi against Rab3 in all adult KCs. After one cycle of aversive olfactory conditioning, for STM and MTM, or three spaced cycles, for LTM, Gal80ts;OK107/RNAi-Rab3 flies presented a defect of STM, 3-h MTM, and LTM, while surprisingly 1-h MTM was normal (Fig. 2A). To verify that the defects observed were indeed due to the effective knockdown of Rab3, we also tested the memory of flies after 5 d at 18°C. These flies displayed normal STM, 3-h MTM, and LTM (Fig. 2A). The distinct temporal windows of Rab3 requirement in memory formation could potentially be attributed to the specific contributions of distinct subclasses of KCs. We thus specifically reduced Rab3 in the adult αβ neurons, known to be specifically involved in MTM and LTM (Bouzaiane et al. 2015). Indeed, those flies present normal STM, consistent with αβ neurons not playing an essential role for STM formation (Fig. 2B). At the same time, the αβ KC-specific driver reproduced results similar to the pan-KC driver: MTM at 1 h was normal, but MTM at 3 h and LTM were defective (Fig. 2B). Thus, Rab3 is likely needed within other KCs for STM formation, probably the γ-KCs as they are known to be required for STM (Blum et al. 2009; Qin et al. 2012), and later within αβ-KCs for MTM at 3 h and LTM.

It is tempting to speculate that Rab3's role in facilitating SV recruitment could be essential for its function in STM. This notion is supported by findings showing that the loss of Synapsin, typically considered critical for SV recruitment (Zhang and Augustine 2021), is crucial for STM formation in KCs as well (Michels et al. 2011). Similarly, we previously showed that Synaptotagmin, a key protein for efficient SV release, is required in adult MB lobes for STM (Turrel et al. 2022).

The observation that Rab3 is dispensable for 1-h MTM is intriguing, suggesting the involvement of partly independent plasticity mechanisms in supporting distinct memory phases such as STM versus MTM. However, the requirement of Rab3 for late memory, specifically at 3 h postconditioning, initially appears perplexing, indicating potential differences in the underlying plastic processes between MTM at 1 and 3 h, or possibly involvement of different sets of synapses. Regarding the molecular plasticity processes specific to 3-h MTM, it is noteworthy that Rab3 has been implicated in controlling the assembly of BRP presynaptic AZ scaffold during synapse formation at the larval NMJ (Graf et al. 2009). Thus, AZ scaffold remodeling steered by Rab3 might be particularly relevant for the plasticity processes involved in displaying the 3-h MTM (Turrel et al. 2022). Another intriguing possibility is the involvement of Rab3 in aspects of cAMP-mediated plasticity. Recent reports have suggested that Rab3 plays a crucial role as a mediator of cAMP-mediated plasticity, also involving rearrangements of the release factor Unc13A (Sachidanandan et al. 2023). It has also been shown that cAMP production is involved in the induction of long-term depression in mammals, meant to underlie aversive olfactory associative learning in flies (Hige et al. 2015; Handler et al. 2019; Yamada et al. 2024). Alternatively, several known effectors of Rab3, importantly Rabphilin (Li et al. 1994), have been involved in changes in the organization of the cytoskeleton via interaction and phosphorylation of α-actinin and F-actin at the NMJ (Giovedi et al. 2004a,b; Baldini et al. 2005), offering intriguing alternative mechanistic avenues to explain the 3-h MTM function of Rab3 and a possible role in the AZ protein remodeling processes underlying MTM (Turrel et al. 2022).

In contrast to Rab3, Neurexins serve as essential synaptic cell adhesion proteins, playing roles in synapse formation and plasticity via the interaction with postsynaptic cell adhesion protein partners, importantly Neuroligins (Gomez et al. 2021). We tested flies suffering a postdevelopmental decrease of Neurexin-1 (Nrx), the sole member of the Neurexin family in Drosophila, in the adult MB lobes (Fig. 1A). We observed that this genetic scenario provoked a memory defect for STM, 1-h MTM, and 3-h MTM (Fig. 2C). Importantly, the Nrx-knockdown flies kept at 18°C displayed normal memory scores (Fig. 2C). Surprisingly, LTM was normal in these animals (Fig. 2C).

Nrx contributes to successive phases of AZ formation and maturation. It interacts sequentially with two distinct postsynaptic Neuroligin (Nlg) binding partners: Nlg1 for AZ/synapse assembly initiation and Nlg2 for AZ/synapse assembly maturation. In this process, Nrx interacts via its carboxyl terminus with two antagonistic presynaptic AZ regulators, Syd-1 and Spinophilin (Spn), with Syd-1 promoting assembly initiation and Spn promoting maturation (Banovic et al. 2010; Owald et al. 2012; Muhammad et al. 2015; Ramesh et al. 2021). Interesting in this regard, it was recently found that Spn pan-adult KC knockdown in the adult MB provokes MTM 1- and 3-h defects, whereas Syd1 knockdown only leads to a MTM 3-h defect (Ramesh et al. 2023). Moreover, at the NMJ, spn mutants also fail to allow for AZ scaffold remodeling upon triggering homeostatic PHP (Ramesh et al. 2023). Taken together, it is tempting to speculate that plasticity processes supporting 1-h MTM are dominated by remodeling and maturation processes at AZs existing at the time of conditioning. In contrast, the processes mediating 3-h MTM might involve essential elements of new AZ formation and initiation. Considering the normal LTM phenotype, it is interesting to note that in mammals, even if the trans-synaptic interactions between Nrx–Nlg are needed for long-term facilitation, so are the interactions between two other diptychs of trans-synaptic proteins, indicating possible compensation mechanisms allowing for normal LTM even without the trans-synaptic communication by Nrx–Nlg (Bailey et al. 2015). Concerning STM, it could be argued that trans-synaptic communication and postsynaptic receptor incorporation would be altered after Nrx knockdown, similarly as at NMJs (Owald et al. 2012; Ramesh et al. 2021), leading to improper memory formation or expression.

Despite using a timed, postdevelopmental knockdown approach, it could still be argued that an irreversible damage to AZ or synaptic functional organization might be responsible for the spectrum of memory phenotypes observed, rather than an acute lack of AZ or synaptic regulation. However, such an interpretation is not easily reconciled with the observed defect in 3-h MTM but unchanged 1-h MTM seen in both pan-KCs and αβ KC-specific knockdown of Rab3 or the normal LTM observed in pan-KCs knockdown of Nrx. To further explore this question, we analyzed whether the memory deficits provoked via acute knockdown using the TARGET system could be reversed when shifting the animals from 30°C back to 18°C. After 10 d, flies resumed normal protein expression (staining data not shown) and present restored STM (Fig. 3), indicating that the acute knockdown is indeed temporary, and that the system permits the flies to return to a normal state after a time window plausible for a recovery of the targeted proteins. Thus, we are confident that our analysis pictures a real, on the spot, regulatory requirement of plasticity factors.

Figure 3.

Figure 3.

Open in a new tab

The TARGET system offers a reversal of the behavior after a return to the permissive temperature. (A) Gal80ts;OK107/RNAi-Rab3 flies show normal STM 10 d at 18°C after 5 d of induction at 30°C (F(2,14) = 2.972, P = 0.0894, n = 5). (B) Gal80ts;OK107/RNAi-Nrx flies show normal STM 10 d at 18°C after 5 d of induction at 30°C (F(2,14) = 1.577, P = 0.2466, n = 5). Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad Software). Differences were tested by one-way ANOVA with Tukey's post hoc test. Half scores are presented in Supplemental Table S4.

Supplementary Material

Supplement 1
Supplemental_Table_S1.xlsx (254.9KB, xlsx)
Supplement 2
Supplemental_Table_S2.xlsx (250.7KB, xlsx)
Supplement 3
Supplemental_Table_S3.xlsx (253.7KB, xlsx)
Supplement 4
Supplement 5
Supplemental_Fig_S1.pdf (423.4KB, pdf)

Acknowledgments

This work was supported by grants to S.J.S. (FOR 2705, NanoSYNDIV, and ERC SynProtect 101097053), O.T. (Fondation Fyssen and Bettencourt Schueller Foundation), and L.G. (China Scholarship Council).

Footnotes

[Supplemental material is available for this article.]

Freely available online through the Learning & Memory Open Access option.

References

  1. Bailey CH, Kandel ER, Harris KM. 2015. Structural components of synaptic plasticity and memory consolidation. Cold Spring Harb Perspect Biol 7: a021758. 10.1101/cshperspect.a021758 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldini G, Martelli AM, Tabellini G, Horn C, Machaca K, Narducci P, Baldini G. 2005. Rabphilin localizes with the cell actin cytoskeleton and stimulates association of granules with F-actin cross-linked by α-actinin. J Biol Chem 280: 34974–34984. 10.1074/jbc.M502695200 [DOI] [PubMed] [Google Scholar]
  3. Banovic D, Khorramshahi O, Owald D, Wichmann C, Riedt T, Fouquet W, Tian R, Sigrist SJ, Aberle H. 2010. Drosophila neuroligin 1 promotes growth and postsynaptic differentiation at glutamatergic neuromuscular junctions. Neuron 66: 724–738. 10.1016/j.neuron.2010.05.020 [DOI] [PubMed] [Google Scholar]
  4. Binotti B, Jahn R, Chua JJ. 2016. Functions of Rab proteins at presynaptic sites. Cells 5: 7. 10.3390/cells5010007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blum AL, Li W, Cressy M, Dubnau J. 2009. Short- and long-term memory in Drosophila require cAMP signaling in distinct neuron types. Curr Biol 19: 1341–1350. 10.1016/j.cub.2009.07.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Böhme MA, Beis C, Reddy-Alla S, Reynolds E, Mampell MM, Grasskamp AT, Lützkendorf J, Bergeron DD, Driller JH, Babikir H, et al. 2016. Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling. Nat Neurosci 19: 1311–1320. 10.1038/nn.4364 [DOI] [PubMed] [Google Scholar]
  7. Böhme MA, McCarthy AW, Grasskamp AT, Beuschel CB, Goel P, Jusyte M, Laber D, Huang S, Rey U, Petzoldt AG, et al. 2019. Rapid active zone remodeling consolidates presynaptic potentiation. Nat Commun 10: 1085. 10.1038/s41467-019-08977-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bouzaiane E, Trannoy S, Scheunemann L, Plaçais P-Y, Preat T. 2015. Two independent mushroom body output circuits retrieve the six discrete components of Drosophila aversive memory. Cell Rep 11: 1280–1292. 10.1016/j.celrep.2015.04.044 [DOI] [PubMed] [Google Scholar]
  9. Chen S, Gendelman HK, Roche JP, Alsharif P, Graf ER. 2015. Mutational analysis of Rab3 function for controlling active zone protein composition at the Drosophila neuromuscular junction. PLoS ONE 10: e0136938. 10.1371/journal.pone.0136938 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Citri A, Malenka RC. 2008. Synaptic plasticity: multiple forms, functions, and mechanisms. Neuropsychopharmacology 33: 18–41. 10.1038/sj.npp.1301559 [DOI] [PubMed] [Google Scholar]
  11. Crittenden JR, Skoulakis EM, Han KA, Kalderon D, Davis RL. 1998. Tripartite mushroom body architecture revealed by antigenic markers. Learn Mem 5: 38–51. 10.1101/lm.5.1.38 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dubnau J, Grady L, Kitamoto T, Tully T. 2001. Disruption of neurotransmission in Drosophila mushroom body blocks retrieval but not acquisition of memory. Nature 411: 476–480. 10.1038/35078077 [DOI] [PubMed] [Google Scholar]
  13. Gerber B, Scherer S, Neuser K, Michels B, Hendel T, Stocker RF, Heisenberg M. 2004. Visual learning in individually assayed Drosophila larvae. J Exp Biol 207: 179–188. 10.1242/jeb.00718 [DOI] [PubMed] [Google Scholar]
  14. Ghelani T, Escher M, Thomas U, Esch K, Lützkendorf J, Depner H, Maglione M, Parutto P, Gratz S, Matkovic-Rachid T, et al. 2023. Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca2+ channels drives sustained active zone potentiation. Sci Adv 9: eade7804. 10.1126/sciadv.ade7804 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giovedi S, Darchen F, Valtorta F, Greengard P, Benfenati F. 2004a. Synapsin is a novel Rab3 effector protein on small synaptic vesicles. II. Functional effects of the Rab3A–synapsin I interaction. J Biol Chem 279: 43769–43779. 10.1074/jbc.M404168200 [DOI] [PubMed] [Google Scholar]
  16. Giovedi S, Vaccaro P, Valtorta F, Darchen F, Greengard P, Cesareni G, Benfenati F. 2004b. Synapsin is a novel Rab3 effector protein on small synaptic vesicles. I. Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals. J Biol Chem 279: 43760–43768. 10.1074/jbc.M403293200 [DOI] [PubMed] [Google Scholar]
  17. Gomez AM, Traunmüller L, Scheiffele P. 2021. Neurexins: molecular codes for shaping neuronal synapses. Nat Rev Neurosci 22: 137–151. 10.1038/s41583-020-00415-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Graf ER, Daniels RW, Burgess RW, Schwarz TL, DiAntonio A. 2009. Rab3 dynamically controls protein composition at active zones. Neuron 64: 663–677. 10.1016/j.neuron.2009.11.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Handler A, Graham TGW, Cohn R, Morantte I, Siliciano AF, Zeng J, Li Y, Ruta V. 2019. Distinct dopamine receptor pathways underlie the temporal sensitivity of associative learning. Cell 178: 60–75.e19. 10.1016/j.cell.2019.05.040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hige T, Aso Y, Modi Mehrab N, Rubin Gerald M, Turner Glenn C. 2015. Heterosynaptic plasticity underlies aversive olfactory learning in Drosophila. Neuron 88: 985–998. 10.1016/j.neuron.2015.11.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huang S, Piao C, Beuschel CB, Götz T, Sigrist SJ. 2020. Presynaptic active zone plasticity encodes sleep need in Drosophila. Curr Biol 30: 1077–1091.e5. 10.1016/j.cub.2020.01.019 [DOI] [PubMed] [Google Scholar]
  22. Isabel G, Preat T. 2008. Molecular and system analysis of olfactory memory in Drosophila. In Molecular mechanisms of memory. Vol. 4 of learning and memory: a comprehensive reference (ed. Byrne JH), pp. 103–118. Academic Press, Cambridge, MA. [Google Scholar]
  23. Kittel RJ, Heckmann M. 2016. Synaptic vesicle proteins and active zone plasticity. Front Synaptic Neurosci 8: 8. 10.3389/fnsyn.2016.00008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krashes MJ, Keene AC, Leung B, Armstrong JD, Waddell S. 2007. Sequential use of mushroom body neuron subsets during Drosophila odor memory processing. Neuron 53: 103–115. 10.1016/j.neuron.2006.11.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li C, Takei K, Geppert M, Daniell L, Stenius K, Chapman ER, Jahn R, De Camilli P, Südhof TC. 1994. Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C. Neuron 13: 885–898. 10.1016/0896-6273(94)90254-2 [DOI] [PubMed] [Google Scholar]
  26. Liu KSY, Siebert M, Mertel S, Knoche E, Wegener S, Wichmann C, Matkovic T, Muhammad K, Depner H, Mettke C, et al. 2011. RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release. Science 334: 1565–1569. 10.1126/science.1212991 [DOI] [PubMed] [Google Scholar]
  27. McGuire SE, Le PT, Osborn AJ, Matsumoto K, Davis RL. 2003. Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302: 1765–1768. 10.1126/science.1089035 [DOI] [PubMed] [Google Scholar]
  28. Michels B, Chen YC, Saumweber T, Mishra D, Tanimoto H, Schmid B, Engmann O, Gerber B. 2011. Cellular site and molecular mode of synapsin action in associative learning. Learn Mem 18: 332–344. 10.1101/lm.2101411 [DOI] [PubMed] [Google Scholar]
  29. Muhammad K, Reddy-Alla S, Driller JH, Schreiner D, Rey U, Böhme MA, Hollmann C, Ramesh N, Depner H, Lützkendorf J, et al. 2015. Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function. Nat Commun 6: 8362. 10.1038/ncomms9362 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Owald D, Fouquet W, Schmidt M, Wichmann C, Mertel S, Depner H, Christiansen F, Zube C, Quentin C, Körner J, et al. 2010. A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila. J Cell Biol 188: 565–579. 10.1083/jcb.200908055 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Owald D, Khorramshahi O, Gupta VK, Banovic D, Depner H, Fouquet W, Wichmann C, Mertel S, Eimer S, Reynolds E, et al. 2012. Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly. Nat Neurosci 15: 1219–1226. 10.1038/nn.3183 [DOI] [PubMed] [Google Scholar]
  32. Qin H, Cressy M, Li W, Coravos Jonathan S, Izzi Stephanie A, Dubnau J. 2012. γ Neurons mediate dopaminergic input during aversive olfactory memory formation in Drosophila. Curr Biol 22: 608–614. 10.1016/j.cub.2012.02.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ramesh N, Escher MJF, Mampell MM, Böhme MA, Götz TWB, Goel P, Matkovic T, Petzoldt AG, Dickman D, Sigrist SJ. 2021. Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly. Curr Biol 31: 1711–1725.e5. 10.1016/j.cub.2021.01.093 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ramesh N, Escher M, Turrel O, Lützkendorf J, Matkovic T, Liu F, Sigrist SJ. 2023. An antagonism between Spinophilin and Syd-1 operates upstream of memory-promoting presynaptic long-term plasticity. Elife 12: e86084. 10.7554/eLife.86084.sa2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reddy-Alla S, Böhme MA, Reynolds E, Beis C, Grasskamp AT, Mampell MM, Maglione M, Jusyte M, Rey U, Babikir H, et al. 2017. Stable positioning of Unc13 restricts synaptic vesicle fusion to defined release sites to promote synchronous neurotransmission. Neuron 95: 1350–1364.e12. 10.1016/j.neuron.2017.08.016 [DOI] [PubMed] [Google Scholar]
  36. Rizo J, Rosenmund C. 2008. Synaptic vesicle fusion. Nat Struct Mol Biol 15: 665–674. 10.1038/nsmb.1450 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sachidanandan D, Aravamudhan A, Mrestani A, Nerlich J, Lamberty M, Hasenauer N, Ehmann N, Pauls D, Seubert T, Maiellaro I, et al. 2023. Rab3 mediates cyclic AMP-dependent presynaptic plasticity and olfactory learning. bioRxiv 10.1101/2023.12.21.572589 [DOI] [Google Scholar]
  38. Südhof TC. 2012. The presynaptic active zone. Neuron 75: 11–25. 10.1016/j.neuron.2012.06.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Takamori S, Holt M, Stenius K, Lemke EA, Grønborg M, Riedel D, Urlaub H, Schenck S, Brügger B, Ringler P, et al. 2006. Molecular anatomy of a trafficking organelle. Cell 127: 831–846. 10.1016/j.cell.2006.10.030 [DOI] [PubMed] [Google Scholar]
  40. Tully T, Quinn WG. 1985. Classical conditioning and retention in normal and mutant Drosophila melanogaster. J Comp Physiol A 157: 263–277. 10.1007/BF01350033 [DOI] [PubMed] [Google Scholar]
  41. Turrel O, Ramesh N, Escher MJF, Pooryasin A, Sigrist SJ. 2022. Transient active zone remodeling in the Drosophila mushroom body supports memory. Curr Biol 32: 4900–4913.e4. 10.1016/j.cub.2022.10.017 [DOI] [PubMed] [Google Scholar]
  42. Woitkuhn J, Ender A, Beuschel CB, Maglione M, Matkovic-Rachid T, Huang S, Lehmann M, Geiger JRP, Sigrist SJ. 2020. The Unc13A isoform is important for phasic release and olfactory memory formation at mushroom body synapses. J Neurogenet 34: 106–114. 10.1080/01677063.2019.1710146 [DOI] [PubMed] [Google Scholar]
  43. Yamada D, Davidson AM, Hige T. 2024. Cyclic nucleotide-induced bidirectional long-term synaptic plasticity in Drosophila mushroom body. bioRxiv 10.1101/2023.09.28.560058 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang M, Augustine GJ. 2021. Synapsins and the synaptic vesicle reserve pool: floats or anchors? Cells 10: 658. 10.3390/cells10030658 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1
Supplemental_Table_S1.xlsx (254.9KB, xlsx)
Supplement 2
Supplemental_Table_S2.xlsx (250.7KB, xlsx)
Supplement 3
Supplemental_Table_S3.xlsx (253.7KB, xlsx)
Supplement 4
Supplemental_Table_S4.xlsx (56.4KB, xlsx)
Supplement 5
Supplemental_Fig_S1.pdf (423.4KB, pdf)

Articles from Learning & Memory are provided here courtesy of Cold Spring Harbor Laboratory Press

RESOURCES