David Benacom

Camille Chataing

Jessica Apulei

Isabelle Queguiner

Alain Prochiantz

Di Nardo

Plasticity state-dependent changes in visual cortex parvalbumin interneuron mRNA translation and chromatin

Keywords: Critical period, perineuronal net, chondroitinase, double strand break, transposable elements

The juvenile brain is shaped by critical periods of heightened plasticity whose timing is regulated by the maturation of parvalbumin interneurons. Gene expression during brain developmental is regulated by neuronal activity, which can trigger the formation of DNA doublestrand breaks on early response genes regulatory elements. To determine if critical period plasticity induces double-strand breaks (γH2AX foci) and alters chromatin structure (DAPI foci) of parvalbumin cells, we analyzed the primary visual cortex of mice at peak ocular dominance plasticity (postnatal day P30) and in the non-plastic adult (at P90-P100). We also measured the expression of LINE-1 retrotransposons, known to induce double-strand breaks. Foci numbers and LINE-1 Orf1p expression were increased at P30 specifically in parvalbumin cells. TRAP-based RNA sequencing confirmed that LINE-1 translation in parvalbumin cells is activated at P30 compared to P100. ChABC injections in primary visual cortex were performed to determine whether reopened adult plasticity recapitulates the P30 translatome of parvalbumin cells. While the P30 TRAP was enriched for ion channel, postsynaptic membrane, and extracellular matrix components, the ChABC P100 TRAP was enriched for cytoskeletal structure and receptor binding proteins. Furthermore, ChABC treatment did not alter foci distribution or LINE-1 translation.

These results suggest that juvenile critical periods promote genomic instability in parvalbumin cells and that adult plasticity reopened by ChABC is distinct from critical period plasticity.

Introduction

During early postnatal brain development, sensory experience allows for the activity-dependent refinement of neuronal circuits. The seminal work of [START_REF] Hubel | Shape and arrangement of columns in cat's striate cortex[END_REF] involving monocular deprivation and analysis of ocular dominance (OD) columns in primary visual cortex (V1) revealed heightened plasticity was restricted to a transient critical period (CP) in the juvenile animal. It has since been shown that CP timing is controlled by the maturation of cortical layer IV fast-spiking parvalbumin (PV) inhibitory interneurons affecting the inhibitory/excitatory (I/E) balance in layers III/IV [START_REF] Hensch | Critical period plasticity in local cortical circuits[END_REF][START_REF] Reh | Critical period regulation across multiple timescales[END_REF].

While changes in PV cell activity and connectivity are highly experience-dependent during the CP, the molecular changes that occur have not been thoroughly characterized at the gene expression and epigenetic levels. Histone deacetylation (HDAC) activity and DNA methylation are involved in the maturation and consolidation of V1 cortex, although specific cell types remain unknown [START_REF] Baroncelli | Experience Affects Critical Period Plasticity in the Visual Cortex through an Epigenetic Regulation of Histone Post-Translational Modifications[END_REF][START_REF] Putignano | Developmental Downregulation of Histone Posttranslational Modifications Regulates Visual Cortical Plasticity[END_REF]Tognini et al., 2015). Regarding PV cells, changes in their nuclear distribution of the methyl-DNA binding MeCP2 and their expression of Gadd45b/g, which can regulate DNA methylation, have been reported during V1 CP and induced adult plasticity [START_REF] Apulei | Non-cell Autonomous OTX2 Homeoprotein Regulates Visual Cortex Plasticity Through Gadd45b/g[END_REF]. Taken together, these findings suggest that epigenetic changes occur within PV cells during CP maturation.

The triggering of CP onset induces the expression of immediate-early genes (IEGs), a process demonstrated in neuronal cultures to involve the histone mark γH2Ax [START_REF] Madabhushi | Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes[END_REF]. γH2Ax is a classic DNA double-stranded break (DSB) marker extensively studied in the context of irradiation, cancer, and oxidative stress, but appears to have an interesting physiological role in neurons, where it may be linked to cellular activity and the expression of long genes [START_REF] Madabhushi | Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes[END_REF][START_REF] Shanbhag | Early neuronal accumulation of DNA double strand breaks in Alzheimer's disease[END_REF]. DSBs can be produced by retrotransposons, in particular LINE-1, that encodes for ORF1p and ORF2p [START_REF] Gasior | The Human LINE-1 Retrotransposon Creates DNA Double-strand Breaks[END_REF]. Interestingly, retrotransposon elements can be remodeled in V1 by HDAC inhibitors that induce adult plasticity [START_REF] Lennartsson | Remodeling of retrotransposon elements during epigenetic induction of adult visual cortical plasticity by HDAC inhibitors[END_REF].

We therefore set out to investigate PV cell changes in gene expression sand DSB formation during CPs and induced adult plasticity in V1.

Results

Parvalbumin cell epigenetic marks are altered during the critical period

Given that DSBs are important for the expression of IEGs [START_REF] Madabhushi | Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes[END_REF], we assessed whether DSBs are associated with postnatal juvenile CP plasticity and/or induced adult plasticity. We quantified the number of DSBs in PV cells in juvenile (peak CP at P30) and adult (P90) V1 by using the nuclear protein γH2AX, a marker for activity-dependent DSBs classically mediated by the type II topoisomerase (Fig. 1A). To more clearly quantify changes in γH2AX distribution, we developed a new tool based on a deep-learning strategy to identify PV cells and analyze the entire cellular and extracellular volume (StarDist, Schmidt et al., 2018) for staining of PV, perineuronal nets (PNNs), and nuclear foci after limited deep neuronal network training (see Material & Methods). PNNs are formed by the condensation of proteoglycans, hyaluronic acid and linking proteins and are key actors in the maturation of PV cells and consolidation of circuitry [START_REF] Fawcett | The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function[END_REF][START_REF] Testa | Perineuronal nets in brain physiology and disease[END_REF]. We validated the classic changes observed during PV cell maturation in V1, such as a higher PV cell mean intensity (Fig. 1B) at P90 compared to P30 and a marked accumulation of PNNs stained by WFA at P90 (Fig. 1C). We also found a sharp decrease in γH2AX foci number at P90 compared to P30 (Fig. 1D) accompanied by a switch between a high number of low-intensity foci at P30, to a low number of high-intensity foci at P90 (Fig. 1E). These changes suggest global rearrangement of chromatin occurs between P30 and P90 in PV cells.

Interestingly we also found that DAPI foci, a marker of heterochromatin, also change with age similarly to γH2AX foci in PV cells (Fig. 1D,E).

L1 translation is activated during the critical period

We investigated whether LINE-1 expression varies within PV cells during plasticity periods by using an antibody raised against ORF1p (a protein encoded by the LINE-1 bicistronic mRNA) [START_REF] De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF]. Quantification of expression before (P17), during (P30) and after the plasticity period (P100), reveals that ORF1p staining intensity is significantly higher at P30 within WFA+ cells (Fig. 2A). Our laboratory reported that EN1 homeoprotein transcription factor directly represses LINE-1 expression at the level of the ventral midbrain dopaminergic (mDA) neurons, thus limiting DSB formation in oxidative stress conditions or during ageing [START_REF] De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF]. Given that LINE-1 ORF2p, the second product of the bicistronic LINE-1 mRNA, has both endonuclease and reverse-transcriptase activities, we used stavudine (2',3'-didehydro-2',3'-dideoxythymidine, d4T), a nucleoside analogue and strong LINE-1 reverse transcriptase inhibitor [START_REF] Jones | Nucleoside Analogue Reverse Transcriptase Inhibitors Differentially Inhibit Human LINE-1 Retrotransposition[END_REF], to determine whether LINE-1 expression is implicated in DSB formation.

Mice were infused in V1 for 3 days starting at P17 with either stavudine or vehicle and the changes in the average number of DSBs identified by γH2AX staining was analyzed at P20 (Fig. 2B,C).

The number of DSBs was shown to be lower in WFA+ cells at P17 and to increase significantly at P20 (Fig. 2C, left panel) to levels similar to those of WFA-cells (Fig. 2C, right panel), which include neurons and glia cells. This increase was blocked by stavudine infusion, suggesting involvement of ORF2p activity in PV cells during CP onset (Fig. 2C, left panel). No changes were observed in WFA-cells (Fig. 2C, right panel).

Given that LINE-1 retro-transposition in non-mitotic cells remains controversial, and that ORF1p itself does not possess endonuclease activity, we sought to more precisely quantify LINE-1 expression during the CP. We crossed the NuTrap-flox mouse line, which allows for separate isolation of ribosomal-associated RNA in translation (TRAP) and nuclei [START_REF] Roh | Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo[END_REF], with the PV::Cre mouse line in order to express the NuTrap construct specifically in PV cells (Fig. 2D).

This specificity was confirmed by staining for PV, WFA and GFP in V1 sections at P30, which showed that nearly 100% of PV-expressing cells also expressed GFP (Fig. 2E). Interestingly, while most GFP-expressing cells also expressed PV, some mice were found to have GFP in cells lacking PV, suggesting heterogeneous and subthreshold expression of PV in V1 at this age [START_REF] Beurdeley | Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex[END_REF][START_REF] Lee | Genetic Otx2 mis-localization delays critical period plasticity across brain regions[END_REF]. Using this PV::Cre x NuTrap-flox line (PVNT), we isolated and sequenced RNA from both nuclei and TRAP to determine differentially expressed genes (DEGs) and differentially translated genes (DTGs), respectively, at P30, the peak of V1 OD CP, and at P90, an adult non-plastic stage. We found a high expression of LINE-1 subfamilies in PV cell nuclei, although it was not significantly different between P30 and P90 at the nuclear level (Fig. 2F). In the TRAP fraction, we found lower levels of LINE-1 RNA (by approximately 100-fold, depending on the LINE-1 subfamily), but we confirmed a specific and significant upregulation of transcripts in translation at P30 compared to P90 in 10 LINE-1 subfamilies (Fig. 2G).

Parvalbumin cell translation is activated during the critical period

Based on the same dataset generated for the LINE-1 analysis, we compared overall DEG and DTG changes between P30 and P90. We sequenced the input fraction corresponding to bulk sequencing, the PV purified nuclei fraction and the PV purified ribosomal fraction at both ages.

First, we analyzed RNA from V1 tissue at P30 and P90 namely P30_input and P90_input, and identified 323 up-regulated genes and 186 down-regulated genes (Fig. 3A). We identified classic activity genes involved in CP such as Egfr2, Arc, Fos and Nr4a1, and found GO analysis of Cellular Component and Molecular Function to be highly enriched in synapse and junction formation (Fig. 3B), confirming the endogenous CP plasticity status of the tissue used for precipitation of PV nuclei and TRAP.

We found a large disparity between the PV-specific transcripts identified by nuclear and TRAP fractions. In our nuclear fraction, P30_nuc versus P90_nuc, we only report 40 DEGs between both time points, suggesting that transcription changes remain limited during and after the CP (Fig. 3C).

In the TRAP fraction, we report more than 1000 DTGs, suggesting substantial translation regulation processes between both time points (Fig. 3D). GO Cellular components reveal an enrichment in post-synaptic, extracellular PNN, and perisynaptic components known to be implicated in PV cell maturation during the CP (Fig. 3E). GO Molecular Function analysis of DTGs shows strong enrichment in channel activity and transmembrane signaling components amongst other markers of neuronal activity (Fig. 3E). These results confirm that the changes observed in PV cell connectivity between P30 and P90 depend primarily on post-transcriptional regulation at the cell body and/or synaptic level.

Adult visual cortex plasticity with ChABC treatment involves alternate mechanisms compared to critical period plasticity

In order to determine whether an adult plasticity re-opening strategy also modifies the number or structure of γH2AX foci, we injected P90 mice unilaterally with ChABC, which is a wellestablished strategy for adult plasticity reopening that degrades proteoglycans and removes the PNNs of PV cells [START_REF] Pizzorusso | Reactivation of Ocular Dominance Plasticity in the Adult Visual Cortex[END_REF]. We validated the efficiency of ChABC injection, as it resulted in a large reduction of the proportion of PV+WFA+ cells compared to PV+WFA-cells in the enzyme-injected hemisphere (Fig. 4A). ChABC treatment also decreased the number of observable PV+ cells (Fig. 4B). A similar decrease has been previously reported by our laboratory [START_REF] Beurdeley | Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex[END_REF] to be accompanied by restored PV cell numbers two weeks after ChABC treatment, whereby the decrease is not due to cell death but to PV staining transiently falling below detection levels. Here, we did not observe changes in PV intensity (Fig. 4C), as reported in the literature [START_REF] Carceller | Perineuronal Nets Regulate the Inhibitory Perisomatic Input onto Parvalbumin Interneurons and γ Activity in the Prefrontal Cortex[END_REF]. Thus, the discrepancy between the number of PV cells and PV intensity might reflect a switch in PV intensity from mean basal expression to an undetectable level.

We also analyzed the effect of ChABC on various nuclear foci parameters, and found no change in either γH2AX foci number (Fig. 4D) or intensity (Fig. 4E). DAPI co-staining of chromocenters also shows no change in foci number or intensity (Fig. 4D,E). These results suggest that different mechanisms of plasticity are at play in the adult compared to those during physiological CP in the juvenile mouse.

In order to build upon our findings, we analyzed TRAP RNAseq to identify PV-specific ribosome-associated DTGs between ChABC treated and P90 control PVNT mice. We identify 696 upregulated genes and 126 downregulated genes (Fig. 4F). Strikingly, the GO Cellular Component analysis revealed that transcripts undergoing modulation in PV-specific TRAP were highly associated with cytoskeletal structure, in particular actin (Fig. 4G). Post-synaptic density components were also enriched that are known to be highly dependent on cytoskeleton structure for synaptic receptor anchorage and dendritic organization. The GO Molecular Function analysis revealed that upregulated proteins were enriched with actin binding and glutamate receptor binding function. (FDR < 10 -5 ). These results suggest that ChABC engages the translational regulation of core synaptic proteins and their associated cytoskeletal proteins that might take place in local synaptic compartments. Given the implication of the LINE-1 family translation in CP plasticity at P30, we also evaluated whether ChABC-induced adult plasticity similarly involves LINE-1 activation. We found no change in polysome-associated LINE-1 mRNA (Fig. 4H), suggesting that these LINE-1 subfamilies undergo specific translation only during the V1 CP.

Discussion

We demonstrated that some aspects of the chromatin status and genetic stability of PV cells, as suggested by γH2AX and DAPI foci, are modified during the physiological V1 OD CP. The increase in γH2AX foci number during the CP is antagonized by stavudine treatment, a potent inhibitor of LINE-1 retro-transposition activity. LINE-1 transcripts are highly represented in PV cell nuclei, and are found in the TRAP ribosomal fraction, suggesting that they are transcribed and translated in PV cells. During the CP peak, we observe a significant increase in LINE-1 transcripts in translation that is not recapitulated by inducing plasticity in the adult. This increase is reflected at the protein level with an increase in ORF1p staining at P30. Similarly, the comparison between nuclear DEGs and polysome-associated DTGs at P30 and P90 reveals that a vast majority of the regulated transcripts are only observed in the polysome mRNA fraction. This suggests that regulation is generally not at the transcription level but, rather, at the initiation of translation. After ChABC treatment to reopen adult plasticity, γH2AX marks do not reverse to the increased levels observed during CP while DTGs relate to cytoskeletal changes and synaptic activity, suggesting that this treatment modifies morphological and physiological traits with little influence on chromatin structure. To confirm this hypothesis, a more direct analysis of the PV cell chromatin structure using methods such as Hi-C will need to be performed before, during and after CP and after induction of adult plasticity with ChABC.

Physiological significance of γH2AX staining and LINE-1 expression

We observed a transient increase in the number, but not intensity, of γH2AX foci in PV cells during V1 CP, followed by an almost twofold increase in mean γH2AX intensity at P90. This transient increase in foci numbers could be explained by the expression at P30 of IEGs, a class of genes which requires topo-isomerase related DSB formation for transcription [START_REF] Madabhushi | Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes[END_REF]. The observation that stavudine, an inhibitor of the retro-transcriptase activity of ORF2p, prevents the increase of γH2AX foci number during CP is intriguing. Stavudine may cause allosteric changes in ORF2p conformation upon binding, thus impairing endonuclease activity or DNA binding. Accordingly, ORF2p endonuclease may act as a "topo-isomerase" during endogenous CP, but this assertion remains to be demonstrated, as the retro-transposase domain may also possess different functions, such as the generation of RNA-DNA hybrids [START_REF] Schwertz | Endogenous LINE-1 (Long Interspersed Nuclear Element-1) Reverse Transcriptase Activity in Platelets Controls Translational Events Through RNA-DNA Hybrids[END_REF]. Another hypothesis is the increase in DSBs during the CP is a byproduct of a specific activity of ORF1p. ORF1p has an RNA binding activity and has been suggested to regulate RNA processing in p-bodies [START_REF] Briggs | RIP-seq reveals LINE-1 ORF1p association with p-body enriched mRNAs[END_REF]. Specific functions of ORF1p might be required during CP, while associated unregulated ORF2p expression might result in unwanted endonuclease activity. In order to test this hypothesis and determine if ORF2p-dependent γH2AX foci formation is required for endogenous CP plasticity, we could infuse stavudine over the course of the CP coupled with MD. An absence of OD shift would then demonstrate the role of ORF2p for CP plasticity through the formation of breaks revealed by γH2AX foci. It would also be of great interest to evaluate the distribution of H3K27me3 marks, which have been shown to be remodeled by LINEs for the fine-tuning of gene expression during brain development [START_REF] Mangoni | LINE-1 regulates cortical development by acting as long non-coding RNAs[END_REF].

Re-opening strategies do not recapitulate endogenous CP mechanisms

The development of tools to assess chromatin foci and genetic instability in PV cells has enabled us to determine important new parameters for plasticity. The nature of chromatin changes differs between physiological CP and plasticity reopening, as shown by the comparison of γH2AX staining between P30, P90 and in ChABC-injected adult mice. Interestingly, while we first analyzed DAPI as a control, we reported a significant decrease in chromocenter number between P30 and P90, once again not recapitulated with ChABC treatment. However, we cannot totally preclude that there exist several independent pathways to reopen plasticity in the adult and that they differ according to the strategy. Strategies include ChABC, employed in this study, as well as fluoxetine, HDAC inhibitors, and single chain antibodies against OTX2 [START_REF] Bernard | A Mouse Model for Conditional Secretion of Specific Single-Chain Antibodies Provides Genetic Evidence for Regulation of Cortical Plasticity by a Non-cell Autonomous Homeoprotein Transcription Factor[END_REF][START_REF] Casarotto | Antidepressant drugs act by directly binding to TRKB neurotrophin receptors[END_REF][START_REF] Silingardi | Epigenetic treatments of adult rats promote recovery from visual acuity deficits induced by long-term monocular deprivation[END_REF].

Taken together, these observations lead us to propose that juvenile CP plasticity and induced adult plasticity are distinct phenomena. Analyses of the molecular actors identified by TRAP either for physiological CP or re-opened plasticity reveal the high diversity of mechanisms underlying plasticity, and provide an extensive resource on pathways which require validation in future studies. Some re-opening strategies might provide useful adaptive pathways for an altered functional network, but could come at the expense of other physiological functions, suggesting that further characterization of the safety of these methods is needed. Further studies comparing the transcriptomic signature of PV cells subjected to a plasticity reopening strategy with or without physical challenge could reveal different genes involved in a plasticity "permissive" state, and genes actually responsible for plastic changes, or "effectors" genes. Identification of plasticity permissive genes in comparison to plasticity effector genes in future studies might provide an important framework to consider when assessing plasticity re-opening strategies.

Material and methods

Mice and surgery

The C57Bl/6 J mice were purchased from Janvier Laboratories, while PV::Cre mice and NuTrap mice were obtained from Jackson Labs, USA. The PVNT mice were generated by crossing PV-Cre females and NuTrap males to avoid spurious recombination in the testis, which have been shown to transiently express PV. Stereotaxic infusions into V1(lambda: x = 1.7 mm, y = 0 mm, z = 0.5 mm) with 3-day osmotic mini pump (Alzet 1003D, Charles River Laboratories) were used to deliver stavudine (d4T, 10 μM, Sigma Y0000408) diluted in 0.9% NaCl. Recombinant ChABC (Bio-Techne) was injected in V1 (lambda: x = ±1.7 mm, y = 0 mm, z = 0.5 mm) with a single dose of 0.5 μl (50 U/mL) at a rate of 0.1 μl/min using a Hamilton 1701 syringe mounted with a 33G needle. All animal procedures, including housing, were carried out in accordance with the recommendations of the European Economic Community (2010/63/UE) and the French National Committee (2013/118). For surgical procedures, animals were anesthetized with xylazine (Rompun 2%, 5 mg/kg) and ketamine (Imalgene 500, 80 mg/kg). For biochemical analysis, mice either underwent transcardial perfusion or were sacrificed by cervical elongation.

Immunohistochemistry

Mice were perfused transcardially with PBS followed by 4% paraformaldehyde prepared in PBS. Brains were postfixed for 24 h at 4 °C and immunohistochemistry was performed on cryosections (20 μm) encompassing the entire visual cortex. Heat-induced antigen retrieval in 10 mM sodium citrate was performed prior to overnight primary antibody incubation at 4 °C. Primary antibodies included anti PV (rabbit, Swant 27) anti-γH2AX (mouse, 1/200, millipore clone JBW301), anti-ORF1p (guinea pig, 1/ 200, in house), and biotinylated WFA (1/100, Sigma L1516).

Secondary antibodies were Alexa Fluor-conjugated (Molecular Probes). Images were acquired with a Leica SP5 or LSM 800(Zeiss) confocal microscope with 20 µm of the slide acquired with a 1 µm step.

Image Analysis

Microscopy analysis was conducted using using ImageJ. For manual foci quantification, a Z projection was applied before manual foci counting. For intensity quantification, a mask was traced following inner WFA staining, and measured in the Orf1p channel. An in-house script used a custom analysis macro in Java for image analysis, using a trained model for automatic PV and PNN detection. Briefly, we trained the model to detect PNN and PV staining based on a subsampling of random images in the datasets. DAPI signal within PV masks was used for nuclear delimitation. Foci detection was performed using an in-house script with user-defined parameters, and validated on a subset of 10 images.

TRAP protocol

Brain V1 cortices of PVNT mice were manually dissected with a particular emphasis on myelin removal, and placed into a pre-chilled 2 ml dounce containing 1 ml cold lysis buffer (50mM TrisHCl 7,5 + 100mM KCl + 12mM MgCl2 + 1% NP40 + 1mM DTT + 100 µg/ml cycloheximide (sigma) + 0,4 U/µl RNAse OUT + Protease inhibitor (Pierce) 1X). Dounce with a loose pestle was performed 10 times, followed by a tight pestle for 10 strokes, 5' incubation, and 10 supplementary strokes. Homogenates were filtered on 40 µm strainer and transferred to a pre-chilled 2 ml LoBind tube. Homogenates were subsequently centrifuged at 4 °C, 10,000 rcf for 10 min. A 50 µl sample of supernatant was kept as input, and the remaining supernatant was transferred to a pre-chilled 1.5 ml LoBind tube. A 10' pre-clear step was performed, using 10µl un-conjugated DynaBeads Protein G (Thermofisher, 1003D) per sample at 4°. Beads were removed with a Dynamag magnet (Thermofisher) and samples were incubated with 1 µg anti-GFP antibody (ab290, abcam) for 1h at 4°. DynaBeads Protein G (Thermofisher, 1003D) were blocked in 1 mL lysis buffer supplemented with 2% BSA and 0.2 mg/ml yeast tRNA for 3x 1' incubation, and resuspended in their original volume. 30µL Dynabeads Protein G were added in each sample, for a supplementary 2h incubation at 4 °C. Samples were placed on a DynaMag magnet for 1 min until complete clearing of the beads in solution. Beads were washed 3 times with high salt lysis buffer (50mM TrisHCl 7,5 + 300mM KCl + 12mM MgCl2 + 1% NP40 + 2mM DTT + 100 µg/ml cycloheximide + 0,4 U/µl RNAse OUT + IP 1X). After the third wash, samples were removed from the magnet and beads were resuspended in 500 µL QIAzol and kept at -80° until RNA extraction using QIAgen Micro Kit with standard procedure. Double DNAse treatment was performed. Purified RNA were quantified on a Qubit 4 device and analyzed on a BioAnalyzer for quality control.

Nuclear isolation protocol

Brain V1 cortices of PVNT mice were manually dissected and placed into a pre-chilled 2 ml dounce containing 1 ml cold homogenization buffer (HB) 1X (sucrose 0.25M, inhibitor protease 1X, DTT 1mM, spermidine 0.5 mM, spermine 0.15 mM, NP40 0.3%, H2O). HB was supplemented with 10 µl RiboLock per ml of HB. Dounce with a loose pestle was performed 10 times, followed by a tight pestle for 10 strokes, 5' incubation, and 10 supplementary strokes.

Homogenates were filtered on 40 µm strainer and transferred to a pre-chilled 2 ml LoBind tube.

Homgenate were subsequently centrifugated at 4 °C, 350 rcf, soft settings for 5 min in swinging buckets. 50 µl of supernatant was removed as input (containing cytoplasmic RNAs) and transferred to a pre-chilled 1.5 ml LoBind tube. Nuclei were gently resuspended in a total volume of 400 µl 1x HB and mixed with 50% optiprep solution to a final 25% optiprep. 600 µl of 30% optiprep solution was laid under the 25% mixture. The gradient was centrifugated in a pre-chilled swinging bucket centrifuge, for 10 min at 4 °C, 3000 rcf with soft settings. The top layer was slowly removed down to within 50 µl of the nuclei pellet. The nuclei pellet was then resuspended in FACS Buffer (BSA 0.1%, RNAse-IN 0.01%, 0.1% triton, PBS) to a volume of 500 µL. FACS gating were performed using nuclei extracted from WT mice using the same procedure. The GFP population was sorted directly into QIAzol and kept at -80 °C until RNA extraction using QIAgen Micro Kit with standard procedure. Double DNAse treatment was performed. Purified RNA were quantified on a Qubit 4 device and analyzed on a BioAnalyzer for quality control.

Sequencing & Bioinformatic analysis

Samples were sequenced by the Curie Institute Genomics facility with the SMARTer Stranded Total RNA-Seq Kit Pico Input Mammalian. Bioinformatic analysis was conducted using OneStopRNAseq [START_REF] Li | OneStopRNAseq: A Web Application for Comprehensive and Efficient Analyses of RNA-Seq Data[END_REF] with standard parameters for DEG analysis, and SalmonTE for transponable element analysis [START_REF] Jeong | An ultra-fast and scalable quantification pipeline for transposable elements from next generation sequencing dataBiocomputing[END_REF]. Enrichr was used for GO analysis (Xie et al., 2021).

Statistical Analysis

Statistical analysis was performed on Prism 9 (GraphPad). Pairwise comparison was performed with Student t-test, while multiple group analyses were done with ANOVA (one-or two-way) followed by Bonferroni correction. Changes in frequency distribution were assessed by Kolmogorov-Smirnov test.

Figure 1 .

 1 Figure 1. Parvalbumin cell epigenetic marks are altered during the critical period. A. Representative immunostainings for γH2AX and WFA in V1 layer IV of WT mice at P30 and P90. Scale bar, 40 µm. B-C. Quantification of cells double-stained or not for PV and WFA (B) and of PV staining intensity (C). D-E. Quantification of foci number (E) and staining intensity (F) for γH2AX and DAPI in PV cells. Values mean ± SEM; unpaired t-test in C-E; ANOVA in B; n = 4 mice; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3 .

 3 Figure 3. Parvalbumin cell translation is activated during the critical period A. MA plot with Log2(fold change) as a function of Log2(mean expression) showing differential expression between P30 and P90 obtained from RNAseq of the input fractions. B. Input fraction RNAseq GO analysis (Enrichr software) of Cellular Component, for genes involved in the composition of a specific cellular compartment, and of Molecular Function, for protein functional classes. C-D. MA plots showing differential expression between P30 and P90 obtained from RNAseq of PV cell nuclear fractions (C) and PV cell TRAP fractions (D). E. TRAP RNAseq GO analysis of Cellular Component and Molecular Function. Red dots and blue dots indicate significantly (FDR < 0.1) upregulated and downregulated genes, respectively (N = 3-4 samples). *FDR enrichment value.

Figure 4 .

 4 Figure 4. Adult visual cortex plasticity with ChABC treatment involves alternate mechanisms compared to critical period plasticity A-C. Quantification of cells double-stained or not for PV and WFA (A), PV cell numbers (B), and PV staining intensity (C). D-E. Quantification of foci number (D) and staining intensity (E) for γH2AX and DAPI in PV cells. F. MA plots with Log2(fold change) as a function of Log2(mean expression) showing differentially translated genes between vehicle and ChABC treated mice obtained from RNAseq of PV cell TRAP fractions. Red dots and blue dots indicate significantly (FDR < 0.1) upregulated and downregulated genes, respectively (N = 3 samples). G. TRAP RNAseq GO analysis (Enrichr software) of Cellular Component, for genes involved in the composition of a specific cellular compartment, and of Molecular Function, for protein functional classes. *FDR enrichment value. H. The expression in TPM of L1 subfamilies in PV cell TRAP from V1 of P90 WT and ChABCinjected mice. Values in A-E, mean ± SEM; unpaired t-test; n = 3 mice; **p < 0.01, ****p < 0.0001.

Acknowledgements

We would like to thank Damien Testa and Uma Mani for technical assistance. Funding was provided by the European Research Council Advanced Grant (ERC-2013-ADG-339379) and by the NeuroGlia Foundation for supporting research costs and salaries.

Experience-dependent DNA methylation regulates plasticity in the developing visual cortex.

Nat


Aimeric Bruno

Jean-Marc Aury

Stefan Engelen
email: sengelen@genoscope.cns.fr

BoardION: real-time monitoring of Oxford Nanopore sequencing instruments

Keywords: Nanopore sequencing, Real-time monitoring, Web application

Background

Since 2014, Oxford Nanopore Technologies (ONT) has launched a range of nanopore sequencing devices, from the portable MinION device to the flexible, high-throughput PromethION sequencer. The ONT technology allows the sequencing of long DNA or RNA molecules with a read accuracy of currently 96% [1] and already has a wide range of applications [2-7]. Unlike traditional next-generation sequencing platforms, which provide data at the end of the sequencing experiment, ONT sequencing data is generated in real time. Users can access time-critical information [8] and thus be able to stop an analysis once the result is obtained. This eventually allows washing and reusing of the flowcells [9]. However, the ONT interface (MinKnow) does not provide enough interactivity and reporting to thoroughly explore the quality of the sequencing data. In addition, almost all quality control tools such as MinIONQC [10], pycoQC [11], Nano-Pack [12] and ToulligQC [13] do not take full advantage of the real-time capability of

the ONT instrument as they analyze the sequencing data at the end of the run. Herein, we introduce BoardION, an interactive web application for real-time evaluation of ONT sequencing runs. BoardION offers the possibility for sequencing platforms to remotely and simultaneously monitor all their ONT devices (MinION, Mk1C, GridION and Pro-methION). It also allows the comparison of several sequencing experiments to assess the library's preparations or the quality of the input samples. In addition, we compare BoardION and Minotour [START_REF] Loose | minoTour: Real time data analysis tools for the MinION sequencing platform[END_REF] which is currently the only software able to process the data in real-time.

Implementation

BoardION is organized in two components, the first one is dedicated to the preprocessing of the files generated by the ONT basecaller (guppy) and the second one is a dynamic web application, the central part of BoardION, which allows the visualization and comparison of sequencing metrics. BoardION can be installed and used directly from the source code or via a docker image containing all the necessary dependencies. Documentation, github and dockerhub repositories, as well as an interactive demo version of BoardION are available at http:// www. genos cope. cns. fr/ board ion/.

Data preprocessing

Assessment of an in-progress run is performed by using information contained in the sequencing summary file generated by the guppy basecaller, but this file is not directly read and load by the web application. Indeed, the sequencing summary can be quite large (more than 10 GB) and will become larger in the future with the increase of the throughput. Instead, a preprocessing tool analyzes it periodically to generate lightweight datasets that contain only the metrics of interest. On each launch, the software searches for the sequencing summary files in the input folder and uses the presence of the final_ summary.txt file to detect completed experiments. This preprocessing step, developed in C++, uses the seekg function of the iostream library to directly access and process only newlines. With these optimizations, statistics can be computed and updated in the web application every minute. For each flowcell, the preprocessing script generates five small files (< 3 MB): four containing statistics calculated every 10 min and one containing the read length distribution. The C++ software also creates a file listing all the flowcells, their status (completed or not) and the latest metrics. The cpu time and memory usage of the preprocessing script are correlated with the number of reads of a run. It takes one minute and 60 Mo on average to process a single run and less than 5 min and 100 Mo on our largest PromethION run (83 M reads).

BoardION web application

We developed a web application because it has the advantage of being easily accessible regardless of the user's configuration. Thus, users can remotely and simultaneously monitor all the sequencing runs of their ONT devices. The BoardION web application is based on the R shiny package [START_REF] Chang | Shiny: web application framework for R. R package version 1[END_REF] that allows us to describe the content of the interface and the relationships between the elements of the application. It also uses ggplot2 [START_REF] Wickham | ggplot2: elegant graphics for data analysis[END_REF] and plotly R library [START_REF] Sievert | plotly for R[END_REF] to create dynamic JavaScript graphs. The memory usage of the web application is dependent on the use of the interface, but generally requires less than 400 Mo. The BoardION application computes widely used metrics such as yield, translocation speed, read length and quality. All graphs, dynamically generated, can be exported and saved as images. The interface is divided into three tabs: «Run in progress» that emphasizes the sequencing experiments currently in-progress, «Run archive» that allows users to accurately evaluate a given experiment across all stored experiments and «Sequencing overview» that compares metrics from all sequencing experiments (in progress or finished).

The «Run in progress» tab displays an overview of the main sequencing metrics (read length, yield, quality, translocation speed …) in real time for all runs being sequenced (Fig. 1). Metrics are plotted cumulatively from the start of the experiment to assess the overall efficiency or every ten minutes to quickly detect a drop in quality. This dashboard allows users to follow the in-progress runs and to investigate more precisely a given experiment using the « Run archive» tab.

The «Run archive» tab contains the results of in-progress and finished runs (Fig. 2). Metrics are displayed through dynamic and customizable graphs as users can select the metrics to plot. These metrics can be observed temporally to analyze the run quality according to experiment time or spatially on the channel grid of the flowcell, to detect the presence of bubbles or contaminants, which can reduce the efficiency of the sequencing experiment.

The « Sequencing overview» tab is dedicated to the comparison of sequencing metrics from multiple flowcells (Fig. 3). This tab is flexible and the user can interactively choose between stored flowcells and proposed metrics and add them to current plots. The first panel gives an overview of the sequencing metrics for all stored sequencing runs and allows users to follow the evolution of the ONT technology according to the releases of new protocols, pores, sequencers or basecallers. Next panels are dedicated

Results and discussion

Real-time monitoring

Generally, «Run in progress» and «Run archive» tabs are used to monitor ongoing sequencing experiments. During the sequencing, the consumption of reagents leads to a decrease in the translocation speed that can affect the read quality or the yield [START_REF] Tyler | Evaluation of Oxford Nanopore's minion sequencing device for microbial whole genome sequencing applications[END_REF]. Indeed, ONT recommends keeping the translocation rate above 300 bases per second Here, the panel shows the monthly variation of the run yields. In run comparison panel users can select several runs to compare read length distributions and all other metrics over run time [START_REF]Refuelling a sequencing run[END_REF]. Thanks to its real-time capability, BoardION allows the users to detect a decrease of the translocation rate, and then add reagents (refueling) at the right time to avoid an irreversible drop in effectiveness. Figure 4 shows an example of a PromethION run, refueled after 20 h, following the observation of a decrease in translocation speed as well as read length and base quality. After refueling, the sequencing experiment recovers an optimal translocation rate, generates longer and higher-quality reads and finally achieves a yield of 83 Gb. In addition, BoardION allows sequencing platforms to define metric thresholds (speed, quality or yield) below which a flowcell must be refueled or washed to sequence a new sample.

Comparison of flowcells

We compare four flowcells, where two strains of the same species were sequenced using different library sizes. We observe that the yield is not the same between the two strains (Fig. 5a). In contrast, the yield is the same for a given strain even if the size of the library is different (Fig. 5c). Library preparation like flowcell F2 seems to be a better option for generating longer reads. One main advantage of the ONT technology is the ability to sequence very long DNA fragments (> 30 kb). As an illustration, we also used Board-ION to compare long-reads library preparation protocols of ONT and Circulomics by sequencing the same genotype of a plant. With the Circulomics protocol the peak at a read size of 5 kb is almost removed (Fig. 5d). This protocol generates a higher proportion of bases in reads longer than 10 kb. Nevertheless, the ONT protocol generates almost twice more bases than the Circulomics protocol (Fig. 5b). Another usage of the run comparison panel is the possibility to compare in real time the yield and the read size distribution of simultaneous runs being sequenced (Fig. 5e).

Discussion and future developments

Like BoardION, the ONT sequencing interface Minknow2 displays real-time information on translocation speed, yield, read length and quality but adds graphs on temperature, voltage, pores states, barcodes distribution and reference coverage. Nevertheless, the graphs lack interactivity to fully explore the quality of the run. In contrast, BoardION provides interactivity to plot all the metrics cumulatively from the start of the run, every ten minutes, according to the channel grid of the flowcells or to correlate the metrics using customizable panels. Furthermore, BoardION adds some functionalities such as storing statistics of all sequenced runs, the comparison of the metrics of these runs and the possibility to remotely and simultaneously monitor the runs of many ONT devices. Minknow is a great application to monitor a single run, but BoardION is dedicated to the monitoring and the comparison of all the runs performed in a given facility.

MinoTour is currently the only platform able to process the ONT sequencing data in real-time. The platform is composed of a database to store sequencing metrics and a web user interface. The interface displays metrics describing run efficiency (yield, read length and quality, rate, pore activity) and alignment to a reference genome (coverage). Users can also setup alerts to be informed when a threshold is reached. We tested mino-Tour to compare its functionalities with BoardION. Installation of minoTour on unix is quite hard for biologists without strong computer skills. MinoTour developers propose to upload data on their server but due to web transfer this solution does not allow a real time analysis of data and is problematic in term of confidentiality. In contrast, the docker image of BoardION is easy to install and platform independent. We tested minoTour using data provided by the minoTour platform. Unlike BoardION, minoTour offers the possibility to explore the signal of the runs and the coverage obtained on the sequenced genome. However, BoardION has some additional features such as comparison of runs, overview of all runs and customizable plots that allow users to thoroughly explore their sequencing experiments. Furthermore, we did not succeed to upload our own sequencing data on the minoTour server. It seems that minoTour has not been updated since 2017, so, as ONT technology evolves quickly, minoTour seems to be currently unusable on recent ONT sequencing data. Several applications can take advantages of all ONT generated data (assembly, structural variation) but others need only high quality data (identification, SNV, epigenetics). Guppy basecalling report distinguishes pass and fail reads (quality threshold of 7). It will be useful to report metrics for pass reads in order to help sequencing platforms to optimize the pass yield. It will also be useful to report the yield of each sample of barcoding runs to assess barcoding efficiency and evaluate the generated data obtained for each sample. We aim to add an alert feature that sends emails to users when a threshold is reached for a metric. With this feature, the sequencing platforms will be able to configure their own alert thresholds in order to react faster in case of sequencing problems or to stop a run once a yield is obtained. We also want to add a report generator to export readable documents containing all metrics and graphs computed by BoardION.

Conclusion

BoardION is an interactive web application for real-time monitoring of ONT sequencing runs. The application can be installed via the Docker distribution. BoardION's dynamic and interactive interface allows users to explore sequencing metrics easily and to optimize in real time the quantity and the quality of the generated data. It also offers the possibility of comparing several sequencing experiments, which can be useful to analyze sequencing conditions and follow the evolution of the ONT technology. We believe BoardION will help sequencing platforms to establish the best sequencing guidelines for different types of samples.

Fig. 1

 1 Fig. 1 BoardION «Run in progress» tab. A first panel lists sequencing metrics of all in-progress experiments. Below, each panel is dedicated to one of these experiments.Users can open the panels to display the throughput, the read length distribution and important metrics of each experiment. The throughput can be plotted cumulatively from the start of the run or every ten minutes. All graphs are interactive as users can also zoom or display popup above the mouse to highlight particular values. In the example, the panel of the first experiment is opened while the second is minimized

Fig. 2 Fig. 3

 23 Fig. 2 BoardION «Run archive» tab. Channel view panel: distribution on the flowcell channel grid of a metric chosen by the user. Customizable plot panel: the variation of the chosen metric is represented as a function of the elapsed time and colored according to the quality. In this example, yield and read length distribution panels are minimized

Fig. 4 a

 4 Fig. 4 a, c, d Examples of customizable graphs for a refueled run: the metrics (yield, speed and median size) are calculated (sum or average) in ten minutes slices and displayed in function of the elapsed time. b Number of reads (color scale) of a given quality (y axis) generated during the sequencing experiment (x axis). Dotted black lines have been added manually and highlight the refueling time (1200, 1650 and 2450 mn). The plots show a drop of the median read size (d) and quality (b) after 500 mn of sequencing at a speed behind 260 b/s (c). The first refuel at 1200 mn restores initial speed (c) and median size (d) but also an almost initial yield per ten minutes (400 Mb vs. 460 Mb, plot a). The last two refuels were made when reaching a translocation rate around 300 b/s as recommended by ONT support

Fig. 5

 5 Fig. 5 Examples of metrics and plots depicted by the run comparison panel of the sequencing overview tab. The panel allows us to compare runs according to yield and read length. a Ten minutes cumulative yield of four runs. b Cumulative yield from the sequencing start of two runs. c Number of bases contained in reads of a particular length of four runs. d Percent of bases contained in reads of a particular length of two runs. e Cumulative yield from the experiment start of three runs being sequenced

Acknowledgements

The authors are grateful to the Oxford Nanopore staff for technical help and for granting Genoscope access to the Pro-methION Early Access Programme (PEAP). We thank the France Génomique Infrastructure (www. france-genom ique. org) for its support. We also thank Frédérick Gavory, Paul Mielle and Thomas Mejean for their insight and support and Corinne Cruaud for testing the tool in real conditions.

Funding

This work was supported by the Genoscope, the Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA) and France Génomique (ANR-10-INBS-09-08).

Availability and requirements

Project name: BoardION.

Project home page: https:// github. com/ insti tut-de-genom ique/ Board ION. Operating system(s): Platform independent. Programming language: c++, R. Other requirements: R library shiny 1.4.0.2, plotly 4.9.2.1 and ggplot2 3.3.1. License: CeCILL. Any restrictions to use by non-academics: no.

Abbreviations

ONT: Oxford Nanopore Technology; DNA: Deoxyribo nucleic acid; GB: Giga bytes; MB: Mega bytes; SNV: Single nucleotid variation.

Authors' contributions

AB designed and implemented the application, and wrote the manuscript. SE designed the application, tested it on datasets, wrote the manuscript and supervised the project. JMA wrote the manuscript and supervised the project. All authors read and approved the final manuscript.

Availability of data and materials

Not applicable.

Declarations Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests. J.-M.A. received travel and accommodation expenses to speak at ONT conferences.

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