Fluorescence microscopy constitutes nowadays an essential method for cell biologists to monitor the localization and function of most proteins. The discovery of the green fluorescent protein (GFP) and its application as a gene-fused reporter, together with the progress that followed with the isolation and evolution of variants that span the close-UV to near-IR spectrum with various photo-physical and -chemical properties, have largely contributed to the wide spreading of this approach (Rodriguez et al., 2017). Alternative labeling methods such as those relying on engineered enzymes have further expanded the possibilities of imaging approaches by allowing the direct coupling of high-performance organic dyes (Lavis, 2017; Xue et al., 2015). In parallel, technical breakthroughs in imaging methods have allowed to overcome the diffraction limit and are now enabling optical imaging of biological samples at the nanoscale (Liu et al., 2015; Sahl et al., 2017; Schermelleh et al., 2019). However, while these advances have expanded the scope of application of fluorescence imaging techniques, they have also generated a pressing need for improved labeling strategies (Choquet et al., 2021).

Indeed, the capacity to accurately investigate by fluorescence imaging the dynamics of endogenous proteins still constitutes a technical challenge. Antibodies, when available, can only be used against ectodomain-presenting proteins and still suffer from their large size and divalency. In parallel, the main drawbacks of most alternative labeling strategies for proteins (fluorescent protein, enzyme, or tag genetic fusions) are associated with non-physiological regulation of the modified gene expression level and the potential impact of the fusion on the protein of interest function. Recent developments in gene-editing methods (Bukhari and Müller, 2019) provide efficient means to circumvent the issue of expression level by directly modifying the endogenous gene, but their implementation is still not straightforward and furthermore intrinsically involves modifying the target protein with a fluorescent tag that can alter its function.

In this context, with the recent progress in directed evolution techniques, recombinant small domain binders capable of specifically recognizing endogenous proteins without impairing their function constitute a promising avenue for the development of minimally invasive and interfering labeling probes (Bedford et al., 2017; Dong et al., 2019; Helma et al., 2015). The increasing diversity in terms of validated molecular scaffolds, such as antibody fragments (scFv or V_HH) (Muyldermans, 2021) or other domains (DARPins [Boersma and Plückthun, 2011], monobodies [Sha et al., 2017], affimers [Tiede et al., 2017], etc.), provides a large variety of randomized surfaces that can recognize and bind virtually any protein of interest. In addition to their recombinant nature, which facilitates their characterization and allows further engineering – notably to convert them into fluorescent probes – these tools importantly alleviate the need to directly alter the gene of interest. Additionally, their small size allows to bring fluorophores coupled to the engineered evolved domain in close proximity of the targeted protein for advanced imaging techniques.

Two recent studies (Fukata et al., 2013; Gross et al., 2013) have applied such a strategy to PSD-95, the major postsynaptic scaffold protein at excitatory synapses (Chen et al., 2005; Cheng et al., 2006), by evolving recombinant binders as key recognition modules for developing imaging probes. PSD-95 plays a key role in organizing receptors, ion channels, adhesion proteins, enzymes, and cytoskeletal proteins at excitatory synapses (Won et al., 2017; Zhu et al., 2016). As a consequence, up- or downregulation of PSD-95 results in critical alterations in synapse morphology and function (Won et al., 2017). In particular, overexpression of fluorescent protein-fused PSD-95 for imaging purposes is phenotypically marked and leads to an increase in dendritic spine number and size, as well as frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) and affects synaptic plasticity (El-Husseini et al., 2000; Nikonenko et al., 2008; Zhang and Lisman, 2012). PSD-95 constitutes therefore an ideal candidate for developing labeling strategies that do not affect the protein expression levels. By exploiting evolved binders, a single-chain variable fragment (PF11) (Fukata et al., 2013) and a ¹⁰FN3-derived domain/monobody (PSD95.FingR) (Gross et al., 2013), the two groups have been able to directly label endogenous PSD-95. However, in both cases, the precise epitopes remain non-characterized, and, furthermore, one of the binders, PSD95.FingR, can also recognize SAP97 and SAP102, two closely related proteins (Gross et al., 2013; Li et al., 2018). The latter point may constitute a clear limitation, and, additionally, the lack of defined epitopes questions the possibility of PSD-95 function perturbation.

Using a phage display selection approach with a ¹⁰FN3-derived library, we have recently isolated and characterized three monobodies targeting PSD-95 (Rimbault et al., 2019). The clones were targeted against PSD-95 tandem PDZ domains and showed remarkable specificity for PSD-95, in particular when considering the high-sequence conservation of paralogs (SAP97, SAP102, and PSD-93). Importantly, all the clones recognized epitopes situated outside of the PDZ domain-binding groove in regions not subjected to post-translational modifications. These properties represent a prerequisite to identify binders having a minimal impact on the tandem domain function and consequently on the full-length protein. As such, they constitute ideal candidates to engineer and develop minimally interfering imaging probes to monitor endogenous PSD-95.

We describe here the exploitation of specific PSD-95 binders as a platform to develop a series of labeling tools for the endogenous synaptic scaffold protein as well as excitatory synapses targeting modules. We first evaluated the potential impact of each evolved ¹⁰FN3 domain binding on PSD-95 function as well as their capacity to be exploited as intrabody-type imaging tools. The selected binders were further engineered to allow their use in various super-resolution imaging (SRI) modalities (stimulated emission depletion [STED], photoactivation localization microscopy [PALM], and DNA-PAINT). Finally, beyond their direct exploitation as PSD-95 reporters, we validated the strategy to use their binding properties to enrich and address protein-based sensors to the postsynaptic density with the genetically encoded calcium reporter GCaMP6/7f (Chen et al., 2013; Dana et al., 2019). We termed the approach ReMoRA (Recombinant binding Modules for minimally interfering Recognition and Addressing of endogenous protein targets) as a sub-class of the intrabody general use with applications in fluorescence imaging where emphasis is set on the absence of interference with the targeted protein function.

With the objective to develop imaging probes to monitor endogenous PSD-95, we have exploited a series of evolved recombinant binders of PSD-95 tandem PDZ domains derived from the ¹⁰FN3 domain. Taking advantage of both their unique paralog-specific recognition properties and their respective epitopes all situated on regions distant from the PDZ domain-binding groove, we have first confirmed that binding to their target was not detectably affecting the PDZ domains nor the full-length protein function. Their use as ReMoRA endogenous PSD-95 probes in the form of fusions to fluorescent proteins was then evaluated in comparison to both antibodies and a similar – but not specific – monobody. The tools were next further engineered to adapt them for SRI applications. We demonstrated that the resulting probes could be exploited with STED, PALM, and DNA-PAINT techniques to provide nanoscale mapping as well as dynamics information on endogenous PSD-95. Finally, we also showed that the binders can be employed to enrich active protein-based modules, such as calcium fluorescent reporters, at the excitatory postsynapse.

The three monobodies we considered in this study were all selected primarily based on their capacity to discriminate PSD-95 from other strongly homologous paralogs (PSD-93, SAP97, and SAP102). As shown before, this remarkable specificity results from their ability to recognize epitopes situated in regions distant from the targeted PDZ domain-binding groove. Indeed, while the main site of interaction of the PDZ domains with their native protein partners is conserved across paralogs, their sequences are not strictly identical outside of these regions. Consequentially, binding of the PSD-95-specific monobodies does not obstruct the PDZ domain-binding grooves. We show here, however, that while Xph15 and Xph20, which bind exclusively to the first PDZ domain, do not detectably affect the domain nor the full-length protein function, the situation is slightly different for Xph18. Indeed, this evolved ¹⁰FN3 domain presents an epitope that encompasses regions on both PDZ domains 1 and 2. As a result, binding of Xph18 locks the two domains, otherwise free to rotate around a short linker, in a specific conformation. This conformational constraint was only detectably detrimental to the interactions of synthetic divalent PDZ domain ligands. We therefore excluded this binder from imaging applications to avoid potential impact on PSD-95 activity, even if its expression does not seem to affect AMPAR stabilization at synapses nor synaptic currents.

As previously reported, the Xph15 and Xph20 share very similar epitopes that are not known to engage in any reported PPI with neuronal partners. Importantly, these epitopes are conserved in a number of species (e.g., rodents, human, etc.) and are not subject to post-translational modifications. These features therefore guarantee a large spectrum of applications. Furthermore, we note that in the context of intrabody-like approaches, the ¹⁰FN3 scaffold, from which the binders are derived, is devoid of internal disulfide bonds, typically found, for instance, in antibody fragments, and thereby alleviating the risk of susceptibility to the intracellular reducing environment. Despite the differences in affinities and binding kinetics of Xph15 and Xph20, both allowed an efficient and specific labeling of PSD-95 independently of the associated reporter group (eGFP, mNeonGreen, SNAP-tag, mEos3.2, GCaMP). Xph20, as the tightest binder, should therefore be preferred for most applications. However, the faster binding kinetics of Xph15 can also be exploited to favor rapid renewal of the probes in live conditions, a decisive advantage when photobleaching prevents time-based experiments.

With the growing access and interest for intrabodies or their synthetic recombinant equivalents, there is a need to develop strategies to adapt the expression level of the probe to its target, in particular in the case of imaging applications. We have opted here for a regulation system developed by the group of Don Arnold for a similar application (Gross et al., 2013). It relies on the use of the excess (unbound) pool of probes to turn off further recombinant gene expression. In other words, the system is efficient if, on the one hand, the targeted protein is not nuclear and, on the other hand, the affinity of the evolved binder for the target is superior to the one of the appended zinc finger for its binding motif incorporated into the expression plasmid. Neuronal proteins that are located on cellular processes (dendrites or axons) are perfectly adapted for this strategy as the inevitable accumulation of fluorescent probes in the nucleus is not problematic for imaging purposes. We have observed here that the regulation system was functional for evolved binding modules with affinities in the 1–0.1 µM range in combination with a highly expressed target such as PSD-95. Indeed, for all probes and imaging techniques the expression profile was consistent with what would be expected from a directly labeled PSD-95 as confirmed by the strong colocalizations observed for the intrabodies and anti-PSD-95 antibody staining, the strong correlation obtained with FP-fused PSD-95 as well as by an estimation of synaptic copy number by PALM consistent with the literature. Furthermore, spt-PALM analysis on the millisecond timescale revealed a major population of probes in a mildly diffusive state, as would be expected from PSD-95-bound reporters.

We note, however, that while most of the reporter cargos we tried were compatible with this approach, the specific use of mScarlet-I and HaloTag resulted in the failure of the regulation system for reasons that are still unclear. The group that developed the regulation system has demonstrated that two orthogonal zinc finger systems could be used in concert (Gross et al., 2013). Alternative methods to regulate the effective expression levels of the probe in tune with the one of its molecular targets would also be highly valuable for multiplexing applications as well as for systems (target, binder or cargo) outside of the optimal conditions mentioned above. Developing probes that undergo fast degradation unless bound to their target constitutes an interesting alternative that has been successfully used for the nanobody scaffold (Gerdes et al., 2020; Keller et al., 2018; Tang et al., 2016). Another strategy for imaging applications would consist in conditioning the resulting fluorescence rather than the probe stability to its target binding by the development of fluorogenic probes (Wongso et al., 2017).

We have demonstrated here that the probes could be adapted to comply with a number of fluorescence-based imaging techniques. Besides the advantages of the system to monitor endogenous PSD-95 in live or fixed conditions with standard imaging procedures, SRI approaches can also be readily accessed with adequate probe engineering. Live imaging and protein dynamics investigations can be performed by exploiting STED or spt-PALM techniques. In the case of live STED, hour timescale studies will benefit from the straightforward use of most fluorescent protein fusions, whereas for studies that require a faster temporal resolution (minute timescale), coupling of brighter and more photorobust organic dyes can be achieved by the use of the SNAP-Tag. Precise nanoscale mapping of the protein target is accessed in fixed conditions either by STED with most probes, by PALM with photoswitchable fluorescent proteins such as mEos3.2, or by DNA-PAINT with a SNAP-Tag fusion as an anchoring point for the docking DNA strand. This large variety of imaging techniques applied to endogenous proteins highlights the potential of the labeling strategy compared to more conventional labeling schemes using either antibodies or direct genetic fusions. Importantly, we note that the PSD-95 labeling observed with the different microscopy modalities here was reproducible and largely independent of the method used despite the slight differences associated with technical aspects (signal to noise, acquisition procedure, nature of the dye and its associated effective labeling efficiency, etc.). The strategy can be easily implemented to other imaging techniques, and, for instance, STORM imaging could be achieved using either the SNAP-tag or the eGFP-based probes with respectively a BG or anti-GFP nanobody functionalized with dyes possessing photoswitching properties such as those from the Cy5 cyanine family. Importantly, given the central role of PSD-95 in synaptic function, we anticipate that the probes will open up numerous possibilities for investigations against various neuronal targets by providing straightforward solutions for the monitoring of PSD-95 in the context of multi-proteins studies.

As mentioned above, two other small recombinant PSD-95 binders have been recently reported by other groups in the context of imaging applications. One is a single-chain variable fragment (scFv), PF11, that was isolated against the palmitoylated form of PSD-95 and used as an intrabody (Fukata et al., 2013). While the epitope was not clearly identified, the study showed recognition by the scFv of a conformational variant of PSD-95 that implied both N-terminal palmitoylation and the C-terminal part of PSD-95. Specificity was confirmed against PSD-93, one of the closest PSD-95 paralogs that also possesses a palmitoylation site in its main isoform. The other binder is a monobody, therefore, in the same synthetic recombinant binder scaffold class as Xph15 and Xph20, isolated from a selection performed against the C-terminal domains of PSD-95 (SH3 and guanylate kinase domains) (Gross et al., 2013). The epitope was also not determined in this study, and the isolated monobody, PSD95.FingR, was shown to also recognize SAP97 and SAP102 paralogs but not PSD-93, a property that was exploited to investigate the role of SAP97/Dlg1 in cell polarity (Li et al., 2018). In both cases, affinities were not determined but the binders performed efficiently as intrabody-type probes for endogenous PSD-95. However, the absence of defined epitopes for both PF11 and PSD95.FingR does not allow to convincingly rule out possible perturbations of some of PSD-95 functions when any of the two probes is bound. PSD-95 is indeed a multidomain scaffold protein with a long list of identified partners (Dosemeci et al., 2007; Won et al., 2017; Zhu et al., 2020) as well as numerous post-translational regulation sites (Vallejo et al., 2017), which complicate evaluation of the impact resulting from a recombinant binder interaction. In addition, recent studies support the idea that the protein should not be viewed as a passive scaffolding element of the synapse but rather as an active actor with a capacity to respond to partners binding (Rademacher et al., 2019; Zeng et al., 2018). In this context, we note that the results we obtained with Xph18 illustrate the difficulty to establish with certainty whether a recombinant binder may impact the activity of its target even when the epitope is known. Indeed, while we could demonstrate that this particular monobody had a clear impact on PSD-95 conformation suggesting a plausible modification of its behavior in its native environment, we did not observe detectable perturbation of PSD-95 basic functions in basal conditions.

In comparison to PF11 and FingR.PSD-95, our study shows that Xph15 and Xph20 constitute valuable complementary molecular tools for standard imaging applications based on their unique specificity profile. They recognize both palmitoylated and non-palmitoylated PSD-95 and can discriminate PSD-95 vs its paralogs. Importantly, they present the net advantage of being characterized with respect to the identity of their respective epitope. While this was critical to clarify the molecular origin of the binders specificity for PSD-95, it also allowed us to relevantly adapt their evaluation in order to confirm the absence of impact of the probes on the target protein function. Critically, Xph15 and Xph20 remarkable specificity, as well as their binding properties, has allowed us to engineer the binders as SRI probes to investigate endogenous PSD-95.

Besides the use of evolved recombinant binders as a strategy to label endogenous PSD-95 in live conditions, a number of genetic approaches have been reported. They all rely on gene-editing methods and are typically used to generate PSD-95 fluorescent protein (Broadhead et al., 2016; Fortin et al., 2014; Willems et al., 2020; Zhu et al., 2020) or engineered self-labeling enzyme fusions (Masch et al., 2018). Comparatively, their main advantages over expressed exogenous probes are the ideal stoichiometric labeling (one fluorophore per target protein, to be tempered by the notion of effective labeling efficiency of the fluorophore; Thevathasan et al., 2019) together, for the knock-in approaches, with the possibility to achieve global labeling.

In contrast, the ReMoRA or intrabody-based approaches benefit from their ease of implementation by relying on standard cell biology techniques for the genetically encoded probe delivery (transfection, electroporation, or virus-mediated delivery). Indeed, gene-editing methods are still not accessible in routine use to most laboratories and are also not amenable to downstream adaptation to various imaging techniques, the possibilities being imposed by the initial choice of the fluorescent module. The modular design of the recombinant binder-based probes provides in turn a system more adapted to engineering and optimization (binding module, fluorescent system, promoter, etc.). Furthermore, we note that the rapid renewal of the probe obtained with the fast kinetics binders can be advantageous for imaging purposes over genetic modification of PSD-95 as its turnover is slow in basal conditions.

Finally, if we consider altogether the properties of the tools presented here and compare them to existing probes based on other recombinant PSD-95 binders (PF11 and PSD95.FingR) or to other labeling approaches (e.g., gene editing), we can identify a number of biological applications that cannot be currently achieved and that would now become accessible. Indeed, Xph15- and Xph20-derived probes currently constitute the only genetically encoded systems that allow the strict specific detection of endogenous PSD-95 for live and advanced imaging applications with, in addition, a comprehensive description of their target binding properties (epitopes, affinities and kinetics, absence detectable interference, etc.). The latter aspect is critical for ensuring that the tools can be exploited without modifying PSD-95 regulation and function and in turn not lead to artifactual results regardless of whether PSD-95 is the prime object of the investigation or just used as an excitatory postsynapse marker. This also constitutes unvaluable information for further engineering and molecular tool development. Specificity is the other salient feature of our tools, and along these lines, their full potential will be unlocked with their exploitation for comparative studies of the PSD-95 paralogs. As mentioned above, our tools provide an unprecedented access to endogenous PSD-95-specific labeling strategies that can be used in combination with complementary strategies to label PSD-93, SAP102, or SAP97, either existing ones with their respective limitations (antibodies, FP-fusion, etc.) or, ideally, with new sets of intrabodies that would here be specific for each of the other paralogs. A multiplexed ReMoRA approach to label the PSD-95 paralogs would open numerous possibilities for investigating the respective role of each of these major neuronal actors notably by providing access to advance imaging approaches with minimal interference on the targeted endogenous proteins.

In conclusion, we provide here a set of powerful probes for targeting PSD-95 with main applications for endogenous protein imaging as well as synaptic enrichment of active protein modules such as activity reporters. In comparison to other similar existing tools, the evolved monobodies described here constitute to this day the only binding modules displaying a strict specificity for PSD-95 regardless of its post-translational modification state. The molecular understanding of their mode of binding comforts our results, indicating undetectable perturbation of PSD-95 function. The probes presented here, which benefit from the simplicity of use of the ReMoRA design, provide direct access to different SRI techniques. We anticipate that beyond the direct benefit for nanoscale mapping and (single molecule) dynamics investigations of endogenous PSD-95, these probes will turn invaluable for investigations that require the implementation of multiplexing imaging strategies.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Charlotte Rimbault

   University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

   Contribution

   Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing, C.R. performed the biophysical experiments and generated the constructs, with the help of C.B., C.G., I.G

   Contributed equally with

   Christelle Breillat, Benjamin Compans and Estelle Toulmé

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4760-8430

2. Christelle Breillat

   University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

   Contribution

   Investigation, Methodology, Writing – review and editing, C.B. conducted the initial imaging experiments and performed the immunostaining, knock-down and correlation experiments together with the interactomics sample preparation

   Contributed equally with

   Charlotte Rimbault, Benjamin Compans and Estelle Toulmé

   Competing interests

   No competing interests declared

3. Benjamin Compans

   University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing, B.C. performed the uPAINT and PALM/sptPALM experiments

   Contributed equally with

   Charlotte Rimbault, Christelle Breillat and Estelle Toulmé

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7823-1499

4. Estelle Toulmé

   University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing, E.T. performed the electrophysiology experiments. E.T. and I.C. performed the GCaMP experiments

   Contributed equally with

   Charlotte Rimbault, Christelle Breillat and Benjamin Compans

   Competing interests

   No competing interests declared

5. Filipe Nunes Vicente

   University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

   Contribution

   Investigation, Methodology, Writing – review and editing, J.F.N.V. performed the DNA-PAINT experiments with the supervision of G.G

   Competing interests

   No competing interests declared

6. Monica Fernandez-Monreal

   University of Bordeaux, CNRS, INSERM, Bordeaux Imaging Center, BIC, UMS 3420, US 4, Bordeaux, France

   Contribution

   Investigation, Methodology, Writing – review and editing, M.F.M. performed the FRAP experiments

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7278-448X

7. Patrice Mascalchi

   University of Bordeaux, CNRS, INSERM, Bordeaux Imaging Center, BIC, UMS 3420, US 4, Bordeaux, France

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

8. Camille Genuer

   University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Virginia Puente-Muñoz

   University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

   Contribution

   Investigation

   Competing interests

   No competing interests declared

10. Isabel Gauthereau

    University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

    Contribution

    Investigation

    Competing interests

    No competing interests declared

11. Eric Hosy

    University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

    Contribution

    Investigation

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2479-5915

12. Stéphane Claverol

    University of Bordeaux, Bordeaux Proteome, Bordeaux, France

    Contribution

    Formal analysis, Investigation, Methodology, Writing – review and editing, S.C. performed the proteomics analysis

    Competing interests

    No competing interests declared

13. Gregory Giannone

    University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

    Contribution

    Supervision, Investigation, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

14. Ingrid Chamma

    University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

    Contribution

    Formal analysis, Investigation, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

15. Cameron D Mackereth

    University of Bordeaux, Inserm U1212, CNRS UMR 5320, IECB, Pessac, France

    Contribution

    Formal analysis, Funding acquisition, Investigation, Methodology, Writing – review and editing, C.D.M. designed NMR experiments, performed all NMR experiments and analyzed data

    Competing interests

    No competing interests declared

16. Christel Poujol

    University of Bordeaux, CNRS, INSERM, Bordeaux Imaging Center, BIC, UMS 3420, US 4, Bordeaux, France

    Contribution

    Funding acquisition, Investigation, Methodology, Writing – review and editing, C.P., M.F.M. and P.M. performed the STED experiments. C.B., V.P. and C.P. performed the FRET/FLIM experiments

    Competing interests

    No competing interests declared

17. Daniel Choquet

    University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Methodology, Writing – original draft, Project administration, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4726-9763

18. Matthieu Sainlos

    University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    sainlos@u-bordeaux.fr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-5465-5641

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