Exploring Ca2+ Dynamics in Myelinating Oligodendrocytes through rAAV-Mediated jGCaMP8s Expression in Developing Spinal Cord Organ Cultures

Oligodendrocytes, the myelin-producing glial cells of the central nervous system (CNS), crucially contribute to myelination and circuit function. An increasing amount of evidence suggests that intracellular calcium (Ca2+) dynamics in oligodendrocytes mediates activity-dependent and activity-independent myelination. Unraveling how myelinating oligodendrocytes orchestrate and integrate Ca2+ signals, particularly in relation to axonal firing, is crucial for gaining insights into their role in the CNS development and function, both in health and disease. In this framework, we used the recombinant adeno-associated virus/Olig001 capsid variant to express the genetically encoded Ca2+ indicator jGCaMP8s, under the control of the myelin basic protein promoter. In our study, this tool exhibits excellent tropism and selectivity for myelinating and mature oligodendrocytes, and it allows monitoring Ca2+ activity in myelin-forming cells, both in isolated primary cultures and organotypic spinal cord explants. By live imaging of myelin Ca2+ events in oligodendrocytes within organ cultures, we observed a rapid decline in the amplitude and duration of Ca2+ events across different in vitro developmental stages. Active myelin sheath remodeling and growth are modulated at the level of myelin–axon interface through Ca2+ signaling, and, during early myelination in organ cultures, this phase is finely tuned by the firing of axon action potentials. In the later stages of myelination, Ca2+ events in mature oligodendrocytes no longer display such a modulation, underscoring the involvement of complex Ca2+ signaling in CNS myelination.


Introduction
Glial cells are key modulators of neural network formation and function (Allen and Lyons, 2018).Among these, oligodendrocytes, the CNS myelinating glia, extend segments of the lipid-rich myelin membrane in between nodes of Ranvier on numerous axons, facilitating rapid saltatory action potential conduction.Myelinating cells are adaptable and dynamically regulate the number, distribution, length, and thickness of myelin sheaths in response to neuronal activity (Paez and Lyons, 2020).Myelinating oligodendrocytes play a crucial function in providing metabolic support to axonal processes, and altered or dysfunctional axon-glia coupling can lead to neuronal/axonal degeneration (Monje, 2018).Dynamic interactions between myelinating oligodendrocytes and axons include ion homeostasis, regulated by channels localized in the myelin sheath at the axon-myelin interface.As a ubiquitous signaling molecule and second messenger, the dynamic of intracellular Ca 2+ concentration ([Ca 2+ ] i ) in oligodendrocytes can directly influence the formation and remodeling of myelin and may underlie other, yet unknown, cellular functions (Giorgi et al., 2018;Paez and Lyons, 2020).
During development, the modulation of [Ca 2+ ] i affects various aspects of oligodendrocyte progenitor cell (OPC) maturation (Wake et al., 2011), including their differentiation and the myelin sheath formation and growth (Baraban et al., 2018;Krasnow et al., 2018;Battefeld et al., 2019).On the other hand, demyelination occurs in the immune-mediated disease multiple sclerosis, and it is commonly seen in traumatic brain injury, spinal cord injury, stroke, and aging (Chen et al., 2020).Activation of Ca 2+ channels and receptors in OPCs and oligodendrocytes by neurotransmitters converges to regulate [Ca 2+ ] i , making Ca 2+ a signaling crossroad and a central candidate mediator tuning myelination (Paez and Lyons, 2020).Transgenic animal models, ad hoc modified to express genetically encoded Ca 2+ indicators (GECIs), are conventionally used for studying Ca 2+ signaling in mammalian oligodendrocytes (Lu et al., 2023).Although these models have provided valuable insights, they have substantial limitations, including costly breeding schemes and financial and animal welfare concerns, and are generally limited to one gene target at a time.Furthermore, relying on transgenic GECI-expressing lines restricts the flexibility to adopt new generations of GECIs with enhanced performance (Grødem et al., 2023).
This study introduces a recombinant adeno-associated virus (rAAV) to achieve robust expression of targeted GECIs in myelinating oligodendrocytes.By integrating the recently developed jGCaMP8s, with enhanced sensitivity and kinetics (Zhang et al., 2023), with the chimeric AAV/Olig001 capsid variant, which exhibits a preferential tropism for oligodendrocytes in vivo (Powell et al., 2016), our AAV-pMBP-jGCaMP8s-WPRE viral vector emerges as a potent and innovative tool.We show the vector efficiency in reporting live imaging of intracellular Ca 2+ dynamics specifically and selectively in myelinating oligodendrocytes, with sustained expression persisting for >2 weeks in spinal cord organotypic slices.Leveraging our knowledge of the molecular regulators of Ca 2+ in other cells, including neurons and astrocytes, we are now beginning to understand the factors converging on the regulation of Ca 2+ in oligodendrocytes during distinct stages of myelination.The ability to localize jGCaMP8s in myelinating oligodendrocytes provides the possibility to uncover the nature of Ca 2+ signaling within and between cells.

Materials and Methods
Mice husbandry.All animal studies were carried out with approval of the European Union (EU) and Italian Ministry of Health according to its regulations, under guidelines 2010/63/UE and Decree 26/14.The project was approved by SISSA Animal Care and Use Committee.Animal use was approved by the Italian Ministry of Health 22DABN1WO and 22DABNYQA, in agreement with the EU Recommendation 2007/526/CE.
Organotypic spinal cord cultures.Organotypic spinal cord and dorsal root ganglia slices were obtained from C57BL/6J mouse embryos at Embryonic Day 12.5.Pregnant mice were exposed to a CO 2 overdose and decapitated, and fetuses were delivered by cesarean section.Isolated fetuses were decapitated, and their backs were isolated from low-thoracic and high-lumbar regions and transversely sliced with a tissue chopper.Slices (275 μm) were cleaned by dissection, fixed on a glass coverslip with fibrin glue, that is, reconstituted chicken plasma (Rockland Immunochemicals) clotted with thrombin (Merck Millipore) and cultured within flat-bottom 15 ml screw cap tubes containing 1 ml standard Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific) supplemented with 25% fetal bovine serum (FBS; Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), 50 units/ml penicillin-streptomycin (Invitrogen), and mouse nerve growth factor (10 ng/ml, Alomone Labs).The tubes were placed in a roller drum, which rotated at a rate of 120 rph, at 37°C with 5% CO 2 .Experiments were performed on Day In Vitro 14 (DIV14) and DIV21.
Plasmid construct.A plasmid containing the jGCaMP8s and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) flanked by AAV2 inverted terminal repeats was used as a recipient plasmid (Addgene).It was digested by ApaI and BamHI-HF to replace the human synapsin promoter with the 1.9 kb MBP gene promoter, amplified by PCR from the plasmid (Addgene; FW, atgctctaggaagatctctgcagagggcccgagctccttcctgcttaggc; RV, tgatgatggtgatgatgcatggtggcgggtggatcctattcgagcttccggaagctg; Sigma-Aldrich; Table 1).To generate AAV-MBP-jGCaMP8s-WPRE, we performed a Gibson Assembly reaction by combining the digested backbone with the amplified MBP promoter (insert, backbone; ratio, 3:1 w/w).The resulting vector was validated by sequencing (detailed data reported in Extended Data Table 1-1).
Virus production.The AAV production protocol was adapted from Chakrabarti et al. (2020).Briefly, HEK293T cells, obtained from American type Culture Collection, were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 100 units/ml penicillin-streptomycin (Invitrogen) using a two-culture chambers CellSTACK (Corning).At each passage, cells were trypsinized for 1-3 min with 0.25% trypsin and 1 mM EDTA in PBS, pH 7.4 at 37°C, plated, and maintained for 2 d before transfection.Then, cells were triple transfected with the following plasmids: AAV-MBP-jGCaMP8s-WPRE (cargo) and helper and Rep/Cap Olig001 (Addgene) in a 1:2:1 (w/w) ratio using a linear polyethylenimine (PEI) reagent (Polysciences), at 0.33 mg/ml (1:11 plasmid:PEI w/v; Table 1).Three days after transfection, cells and media were harvested and treated with Triton X-100 and RNase A (Sigma-Aldrich) to a final concentration of 0.5% and 5 μg/ml, respectively, shaking for 1 h at 37°C.This mixture was centrifuged at 3,700 × g at RT for 30 min to remove debris and then concentrated by tangential flow filtration using the Vivaflow 200 cassette 100 kDa molecular weight cutoff (MWCO; Sartorius).
The concentrated medium containing rAAV particles was purified through an iodixanol column (OptiPrep density gradient medium; Serumwerk Bernburg) by ultracentrifugation (Optima XE-90 Ultracentrifuge; Beckman Coulter) at 44,400 rpm for 2 h at 18°C.After collecting the 40% layer, rAAV particles were further concentrated using a 50 kDa MWCO filter (Amicon, ultracentrifugal filters), followed by buffer exchange to 0.001% PBS-pluronic acid 200 mM NaCl solution.Viral titers (vg/ml) were measured by probing for inverted terminal repeats (FW, GGAACCCCTAGTGATGGAGTT; RV, CGGCCTCAGTGAGCGA) with quantitative polymerase chain reaction (qPCR) using a SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories), according to the manufacturer's instructions.The protocol was optimized to produce rAAVs at high titer [⋍5 × 10 14 vector genomes (vg/ml)].Due to the extensive virus's nomenclature, both the following text and images designate it as AAV-mbp:jGCaMP8s.
Quantification of AAV:mbp-jGCaMP8s transduction efficiency.The quantification of the viral expression efficiency was assessed using the "Analyze particles" function in the Fiji ImageJ software, employing Z-stacks of 20× or 40× images obtained from both the oligodendrocyte dissociated culture and the ventral horn of the spinal organ cultures at DIV14 and DIV21.This function identifies colocalization between eGFP+ cells and MBP+ cells, targeting transduced myelinating oligodendrocytes.
Live calcium imaging.For in vitro live calcium imaging, Oregon Green 488 BAPTA-1 AM (Invitrogen) and AAV-mbp: jGCaMP8s were employed.Primary cultures of oligodendrocytes were incubated with 4 µM Oregon Green 488 BAPTA-1 AM for 40 min (37°C; 5% CO 2 ; Rauti et al., 2020) or infected at DIV4 (at a final concentration of 2.3 × 10 8 vg in 2.2 ml) and recorded at DIV8.The samples were then placed in a coupled device camera and preincubated at 37°C in an extracellular solution for 15 min prior to live Ca 2+ imaging.Then, the samples were mounted on an inverted microscope (Nikon Eclipse Ti-U) and continuously perfused at RT with the same extracellular solution containing the following (in mM): 150 NaCl, 4 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 HEPES, 10 glucose; pH 7.4.Cells were visualized with a 40× objective (Plan Fluor 0.6 × NA), and recordings were performed from visual fields (680 × 680 µm 2 , binning 4 × 4).Samples were excited with a mercury lamp at 488 nm.We used a 395 nm dichroic mirror and DN filter (1/32); images were acquired by an ORCA-Flash 4.0 V2 sCMOS camera (Hamamatsu) and a setup controlled by the HCImage Live software (Hamamatsu).The exposure time ranged from 500 ms to 2 s to prevent bleaching.Live imaging recordings last 30 min for each sample, with movies captured every 10 min.
For ex vivo live Ca 2+ imaging, AAV-mbp:jGCaMP8s was utilized with the same technical settings employed for in vitro recording.Organotypic spinal cord cultures were infected at DIV7 or DIV14 and recorded after 7 d.The samples were continuously perfused at RT in an extracellular solution with the following composition (in mM): 152 NaCl, 4 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 HEPES, 10 glucose; pH 7.4.Samples were recorded for a total of 30 min, with movies collected every 10 min.Tetrodotoxin (TTX, HB1035, Hello Bio) was added into the extracellular solution, reaching a final concentration of 1 µM.Following a 20 min perfusion at RT, samples were recorded for a total of 30 min, with movies collected every 10 min.Additionally, we conducted an ex vivo longitudinal study, recording Ca 2+ signaling in oligodendrocytes at DIV14, DIV16, and DIV21 within the same field of view.Organotypic spinal cord slices were infected at DIV7 for 7 d.Live imaging recordings in extracellular solution last 20 min for each sample, with movies captured every 10 min.Postrecording at DIV14, half of the medium containing AAV-mbp:jGCaMP8s was replaced with fresh medium, while after the recording at DIV16, 25% of the medium was replaced with a fresh medium.Following the recording at DIV21, the samples were treated for 20 min with PI and fixed.In the context of this study, we define "Ca 2+ events" as Ca 2+ waves, specifically referring to events lasting longer than 1 s.
Electrophysiology and calcium imaging of oligodendrocytes.Live Ca 2+ imaging of oligodendrocytes and single-cell electrophysiology recordings from spinal neurons were simultaneously conducted in the ventral area of DIV14 organotypic cultures before and after TTX (1 μM) application.Briefly, whole-cell patch-clamp recordings were performed from ventral interneurons visually identified through a microscope (Nikon) on the basis of previously reported criteria (Avossa et al., 2003).During experiments, slices were perfused with extracellular solution as follows (in mM): 152 NaCl, 4 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 HEPES, 10 glucose, pH 7.4.
In voltage-clamp mode, we considered only recordings where uncompensated series resistance values were <20 MΩ, enabling recordings of synaptic currents without significant distortion.Spontaneous postsynaptic currents were monitored at a holding potential of −56 mV, not compensated for liquid junction potential (−14 mV).Ca 2+ events (defined as above) were recorded with an exposure time of 150 ms and 2 × 2 binning.
Ca 2+ live imaging analysis.Recorded movies were analyzed off-line using the Fiji ImageJ and Clampfit (pClamp suite, 10.7 version; Molecular Devices) software.[Ca 2+ ] i waves were expressed as a fractional amplitude increase ΔF / F 0 , where F 0 is the baseline fluorescence level and ΔF is the rise over the baseline.F 0 was determined by calculating the average fluorescence intensity during the period of cellular inactivity (for Oregon Green) or as the average of the lowest recorded fluorescence intensity within the same field of view and in the same movie (for AAV-mbp:jGCaMP8s).We defined Ca 2+ waves as increases in ΔF / F 0 exceeding 20% of the average maximum peak of amplitude recorded in each movie.We defined the frequency of Ca 2+ waves as the number of waves observed throughout each recording.We defined as duration the temporal difference between the endpoint and the starting point of the recorded wave; we defined amplitude as the fluorescence intensity at the peak of the detected wave.Data pooled from different cellular regions were integrated to generate a single data point presented as average frequency, average duration, and average amplitude.The collected data were derived from ≥5 experiments, each employing a different culture.
RNA extraction and RT-PCR.Total mRNA was isolated from the spinal cord organotypic cultures at DIV14 and DIV21 using a standard TRIzol RNA extraction protocol.The RNA obtained (50 ng) was reverse-transcribed and amplified using Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) with sequence-specific primers (1 μM) and probes (200 nM) in a 20 μl reaction, following the manufacturer's instructions.Primers and probes sequences are listed in Table 1.Quantification of Mbp gene expression was performed, and the expression of the housekeeping gene Gapdh was used for normalization.Amplifications were performed using a Bio-Rad Laboratories CFX96 machine.The real-time qPCR program was executed in accordance with the manufacturer's instructions (annealing 60°C for 30 s).
Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.Spinal cord organotypic culture lysates were prepared in the RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.2% (p/v) sodium deoxycholate) supplemented with a cocktail of protease inhibitors (Roche Diagnostics).For the quantification of the total protein in cellular lysates, the Bradford assay (Bio-Rad Laboratories) was used.All samples were diluted with the Laemmli buffer in the presence of 5% (v/v) β-mercaptoethanol (reduced SDS-PAGE) and heated at 95°C for 5 min.Proteins were resolved by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Sigma-Aldrich).Transferred protein bands were visualized by staining with 0.2% (p/v) Ponceau S in 1% (v/v) acetic acid.Subsequently, for MBP (1:1000, Abcam) and MAG (1:1000, D4G3 Cell Signaling Technology) detection, membranes were blocked for 1 h at RT with 5% (p/v) BSA in PBS containing 0.1% Tween 20 (PBS-T).For the detection of Gapdh (1:1000, 60004-1-lg, Proteintech), membranes were blocked with 5% (p/v) nonfat dry milk in PBS.Membranes were then incubated ON at 4°C with the primary antibody diluted in the appropriate blocking solution, washed three times with PBS-T or TBS-T, and incubated for 1 h at RT with the secondary antibody diluted in the blocking solution.Protein bands were visualized using the chemiluminescence detection system (Uvitec).Densitometric analysis of immunoblots was performed using the Fiji ImageJ software.

Statistics.
All experiments were performed as independent biological replicates.Statistical analyses and graphs were generated using GraphPad Prism 8.0.1.Testing was performed for normality by the D'Agostino-Pearson omnibus normality test.For normally distributed data, two-tailed unpaired parametric Student's t tests were utilized, with equal variance confirmed by the F test.Nonnormally distributed data were analyzed using a two-tailed Mann-Whitney test.Pearson's correlation test was used to assess correlation.Throughout all analyses, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Detailed statistical methods for each figure are provided in an attached table of statistics (Table 2).The number of cultures analyzed for each experiment and each condition was ≥3, unless otherwise stated.

Results
We engineered a rAAV to deliver the newly released GECI transgene, specifically jGCaMP8s, under the transcriptional control of the MBP promoter to drive its expression specifically in myelinating oligodendrocytes.Purified viral particles were used to infect both primary rat oligodendrocyte cultures and mouse organotypic spinal cord slices (Fig. 1).

AAV-mbp:jGCaMP8s expression in oligodendrocytes in vitro
It is well established that primary cultures of OPCs, in the absence of neurons, are capable of differentiating and synthesizing MBP, a protein produced solely by mature oligodendrocytes (Friess et al., 2016).Oligodendrocyte cultures were infected with AAV-mbp:jGCaMP8s and maintained in culture for 4 d prior to live Ca 2+ imaging at DIV8.We detected eGFP expression in >70% of MBP + oligodendrocytes (Fig. 2A; Extended Data Fig. 2-1).Our cultures, primarily consisting of enriched OPCs populations, were derived from mixed glial cultures; therefore, to assess the specificity of our construct, we excluded the expression of AAV-mbp:jGCaMP8s in glial fibrillary acidic protein (GFAP) + astrocytes.As illustrated in Figure 2B, eGFP + cells exhibit no colocalization with GFAP + cells.
We compared the efficiency of AAV-mbp:jGCaMP8s and Oregon Green 488 BAPTA-1 AM, a commonly used Ca 2+ imaging dye, to measure [Ca 2+ ] i signaling.In oligodendrocytes cultures, Ca 2+ events detected by AAV-mbp:jGCaMP8s displayed comparable dynamic features as those detected by Oregon Green, using the same acquisition time frame and exposure time (>1 s), but the rAAV strategy improved the oligodendrocyte processes spatial resolution (compare Fig. 2C,I,J; Movies 1 and 2).Overall, these results demonstrate the efficacy and specificity of the developed rAAV, allowing detection of Ca 2+ signals even from microdomains within the cell processes, thereby providing an experimental improvement in monitoring Ca 2+ dynamics in myelinating oligodendrocytes.Ca 2+ dynamics in oligodendrocytes during spinal slice culture development We exploited AAV-mbp:jGCaMP8s specificity in organotypic spinal cord cultures, where sensory, motor, and interneuron circuit development are accompanied by resident glia cell progressive maturation (Avossa et al., 2003).Spinal slices were administered with AAV-mbp:jGCaMP8s at DIV7 and imaged at DIV14.Immunostaining with MBP antibody confirmed that the rAAV expression was specific to myelinating oligodendrocytes; accordingly, eGFP + cells exhibited the unique oligodendrocyte localization and morphology (Fig. 3A,B), while neurons, marked by SMI-32, a neurofilament marker, were not eGFP + (Fig. 3C).This becomes notably clearer when examining confocal images at higher magnification of SMI-32 + axons ensheathed by eGFP + and MBP + processes (Fig. 3D; Extended Data Fig. 3-1).Quantification of MBP + /eGFP + cells further supported the specificity of rAAV expression in myelinating oligodendrocytes (Fig. 3E), without significantly affecting cell death (Extended Data Fig. 3-2).Staining with GFAP and Iba1, targeting astrocytes and microglia, respectively, confirmed the absence of colocalization with eGFP + cells (Fig. 3F,G).Collectively, this data indicates that AAV-mbp: jGCaMP8s in spinal cultures selectively targets myelinating oligodendrocytes while excluding neurons and other resident glial cell types.
Based on these findings, we performed live Ca 2+ imaging in spinal cord tissues at DIV14, detecting Ca 2+ events localized in oligodendrocytes processes (Fig. 3H,L) and measuring the average values of frequency, duration, and amplitude (Fig. 3I-K; Movies 3 and 4).Since neuronal activity might modulate [Ca 2+ ] i signaling in oligodendrocytes (Pitman and Young, 2016), we selectively blocked fast voltage-gated Na + channels by applying TTX (1 μM) to the spinal cultures (Panattoni et al., 2021).TTX treatment roughly halved the Ca 2+ events amplitude in oligodendrocytes processes while significantly decreasing their duration without affecting the average Ca 2+ frequency (Fig. 3I-L; Movies 3 and 4).Simultaneous recording of oligodendrocytes Ca 2+ signaling and electrophysiology in neurons allowed us to further confirm these findings (Extended Data Fig. 3-3).In both the control and TTX-treated slices, we did not observe a significant correlation between Ca 2+ amplitude and duration (Extended Data Fig. 3-4).
During differentiation, oligodendrocytes might undergo profound changes in cellular interactions, neurotransmitter receptor expression, and their overall response to neuronal activity (Clemente et al., 2013;Moura et al., 2022).We tracked Ca 2+ activity at a later stage of development, namely, at DIV21 to explore whether distinct Ca 2+ signals are generated by oligodendrocytes during spinal circuit maturation (Fig. 4A; Rosato-Siri et al., 2004).Immunohistochemical analysis showed that >80% of the eGFP+ cells colabeled with MBP, while no colocalization was detected with neurons and other glial cells (Extended Data Fig. 4-1).We investigated the effect of neuronal activity on oligodendrocyte Ca 2+ dynamics in spinal cord cultures at DIV21 (Fig. 4B-F).Our analysis showed no significant alterations in the average frequency, duration, and amplitude of Ca 2+ events when neuronal action potentials were inhibited by TTX (Fig. 4C-F; Movies 5 and 6).Differently from DIV14, we observed a significant positive correlation between amplitude and duration of Ca 2+ signals in both the control and TTX conditions (Fig. 4G).
Besides the changes in the amplitude/duration correlation and in TTX sensitivity over the period of oligodendrocyte in vitro maturation and myelin stabilization around axons, we directly compared the average values of frequency, amplitude, and duration of Ca 2+ signals in the spinal cultures between DIV14 and DIV21.We observed a remarkable decline in both amplitude and duration of Ca 2+ events as development progressed (Fig. 4I,J), while the frequency remained unaffected (Fig. 4H).Longitudinal live imaging highlighted dynamic changes in Ca 2+ duration and amplitude of oligodendrocytes throughout development without affecting long-term viability (Fig. 4K; Extended Data Fig. 4-2), along with increased Figure 1.Protocol overview.The procedure consists of two main steps: AAV production and evaluation of transgene expression through Ca 2+ imaging analysis in primary oligodendrocyte and in organotypic spinal cord cultures.The AAV-mbp:jGCaMP8s is obtained through a triple-plasmid transfection process using HEK293T cells, including the cloned vector containing a green fluorescent reporter (jGCaMP8s), the Rep/Cap plasmid Olig001, and the helper plasmid (Step 1).After collection, purification, and titration, AAV-mbp:jGCaMP8s viral particles were administered to primary oligodendrocyte cultures or organotypic spinal cord slices (Step 2).
oligodendrocyte branching complexity at DIV21 (Fig. 4L; Movies 7-9).Although sheath length remained consistent (Fig. 4M), the greater sheath number at DIV21 resulted in a higher total amount of myelin per oligodendrocyte (Fig. 4N).Accordingly, expression levels of MBP and MAG proteins were higher at DIV21 compared with those at DIV14 (Fig. 4O), accompanied by elevated Mbp RNA expression (Fig. 4P).These findings suggest an enhanced myelinating

Discussion
Our major result is the successful characterization of a recombinant viral vector expressing the jGCaMP8s under the control of the MBP promoter.We exploited AAV-mbp:jGCaMP8s to visualize and monitor Ca 2+ activity in myelinating oligodendrocytes in vitro and in ex vivo slice cultures.This model sheds light on the expression of MBP, a critical myelin protein (Chernoff, 1981), in concert with fluctuations in [Ca 2+ ] i during myelination.jGCaMP8s expression was histologically and functionally shown to be restricted to mature oligodendrocytes in both models, such a selectivity was particularly evident in spinal cord organotypic cultures, where numerous resident glial cells are present and eGFP + oligodendrocytes tightly ensheath SMI32 + axons.
In recent studies, myelin Ca 2+ activity was investigated in transgenic zebrafish and rodent animal models (Krasnow et al., 2018;Battefeld et al., 2019).While rAAVs are an easier and cost-effective approach, their utility is limited by the lack of natural AAV capsids with oligodendrocyte affinity.Our study successfully exploited the viral capsid Olig001 which displayed exceptional in vivo tropism for oligodendrocytes (Powell et al., 2016).The specificity of AAV-mbp:jGCaMP8s in myelinating oligodendrocytes was further reinforced by the absence of colocalization with microglia and neurons in organotypic cultures, as well as with astrocytes in both cultured models.
Comparing its kinetics to Oregon Green BAPTA-1, the current AAV-mbp:jGCaMP8s displayed a similar response, due to our imaging settings (Sun et al., 2013).However, the chemical probe showed a lower spatial resolution required for optimal measuring of Ca 2+ signals in oligodendrocytes processes.The robust expression of AAV-mbp:jGCaMP8s in mature  oligodendrocytes not only enabled precise imaging of Ca 2+ events in myelin processes but also demonstrated its suitability for chronic and high-resolution Ca 2+ signaling recordings without affecting total cell death.One methodological consideration is the sampling rate (∼0.03Hz) employed for detecting Ca 2+ events, determined by our imaging setup.This design limited our observations to Ca 2+ events longer than the subsecond duration Ca 2+ transients.
We focused on the development of our versatile tool to record the spatiotemporal pattern of Ca 2+ changes in myelinating spinal organ cultures.We observed a substantial number of traced axons ensheathed by MBP protein, together with a highly active period of spontaneous Ca 2+ events during the early postnatal week.At this age, individual oligodendrocytes make short myelin sheaths on multiple axons, retracting from some axons while maintaining sheaths on others (Czopka et al., 2013); this dynamic process is directly correlated with Ca 2+ signals as demonstrated by Baraban et al. (2018).Up to now, Ca 2+ changes in OPCs processes have largely been suggested as dependent on neuronal action potentials (Wake et al., 2011).Unexpectedly, our results indicated that the Ca 2+ frequency was independent of action potential generation both at DIV14 and DIV21.Conversely, only at DIV14, Ca 2+ amplitude and duration significantly decreased following the pharmacological blockade of neuronal activity, whereas no significant modulation was observed at DIV21.These data suggest that Ca 2+ events in oligodendrocytes can be sustained by both neuronal activity and intrinsic mechanisms (Hamilton et al., 2017;Rui et al., 2020;Fiore et al., 2022).In particular, myelination regulates the influx of extracellular Ca 2+ and its uptake from endoplasmic reticulum and mitochondria depending on the developmental stage (Krasnow et al., 2018;Paez and Lyons, 2020;Meyer and Rinholm, 2021).The morphology, density, and spatial distribution of mitochondria reflect the modulation of energy demand and Ca 2+ buffering capacity in oligodendrocytes during development.Mature oligodendrocytes are sustained by glycolysis and characterized by smaller and fewer mitochondria.In contrast, OPCs and developing oligodendrocytes have a higher metabolic demand, exhibiting a dense population of elongated mitochondria.These cells use both glycolysis and oxidative phosphorylation to produce energy for lipid biosynthesis and active myelination (Kirischuk et al., 1995;Rinholm et al., 2016;Rone et al., 2016;Meyer and Rinholm, 2021;Narine and Colognato, 2022).Our results showed a stage-dependent decline in the Ca 2+ amplitude and duration during myelination.Specifically, spinal slices at DIV14 display heightened amplitude and prolonged duration compared with those at DIV21, without alterations in the frequency of Ca 2+ events.Interestingly, large-amplitude of Ca 2+ waves occurred primarily between DIV13 and DIV15 in brain slices, coinciding with a phase of rapid longitudinal remodeling of myelin (Battefeld et al., 2019).
Our results suggest a decreased energy demand from oligodendrocytes with age, indicating possible modifications in mitochondrial morphology, density, and Ca 2+ buffering capacity, all issues needing further investigations.Interestingly, the absence of correlation between Ca 2+ events amplitude and duration at DIV14 in spinal organ slices, observed in both the control and TTX conditions, could be attributed to the fact that among MBP-expressing cells some are wrapping axons, while others are maintaining myelin.These simultaneous activities reveal a heterogeneous energy demand and Ca 2+ buffering capacity among oligodendrocytes, accompanied by a combined glycolysis-OXPHOS ATP production.In this phase, neuronal activity partially contributes to Ca 2+ buffering capacity, underscoring that myelination may also be regulated by additional Ca 2+ -mediated pathways.However, at DIV21, the correlation between Ca 2+ amplitude and duration was recovered, potentially due to the majority of axons being already myelinated and oligodendrocytes being mainly involved in maintaining myelin sheaths with a low ATP production through the glycolytic pathway.This reliance on glycolysis could result in a similar Ca 2+ buffering capacity among mitochondria.Thus, Ca 2+ dynamics in organ cultures between DIV14 and DIV21 align with significant morphological changes in oligodendrocytes, evidenced by enhanced myelin production per oligodendrocyte, increased number of branches, elevated expression of MBP and MAG proteins, and augmented levels showing AAV-mbp:jGCaMP8s expression (green) and mature oligodendrocytes (MBP, red).Nuclei (white) were labeled with DAPI dye.Note the ventral region highlighted by the ventral fissure (arrow).Scale bar, 300 μm.B, Representative higher magnification image of the spinal ventral region (same as in A) showing AAV-mbp:jGCaMP8s expression (green) and mature oligodendrocytes (MBP, red).Scale bar, 100 μm.See Extended Data Figure 3-1 for more details.C, Representative confocal micrograph of spinal organotypic ventral region showing neurons (SMI-32, cyan) and AAV-mbp:jGCaMP8s expression (green).Scale bar, 100 μm.D, Representative higher magnification images of spinal cord organotypic cultures showing AAV-mbp: jGCaMP8s expression (green), mature oligodendrocytes (MBP, red), and neurons (SMI-32, cyan).Scale bar, 30 μm.E, Quantification of the AAV-mbp: jGCaMP8s efficiency in spinal cord organotypic cultures at DIV14.Data are represented as percentages.The analysis was performed using maximum intensity projections of 3D z-stack images, and quantification was obtained by using the "Analyze particles" function of the Fiji ImageJ software; N = 3 spinal cord organotypic cultures.F, Representative image of ventral region of spinal cord organotypic cultures showing AAV-mbp:jGCaMP8s expression (green) and GFAP + astrocytes (magenta).Scale bar, 100 μm.G, Representative image of ventral region (same as in F) showing AAV-mbp:jGCaMP8s expression (green) and microglia (IbaI, white).Scale bar: 100 μm.H, Maximum intensity projection of a 3D z-stack, illustrating an extended-duration movie highlighting jGCaMP8s-expressing myelinating oligodendrocytes.Scale bar, 100 μm.Frames from the time-lapse imaging are indicated with a white box corresponding to panel h', h''; scale bar, 10 μm.I, Average distribution of Ca 2+ wave frequencies (Hz) per oligodendrocyte process before and after application of TTX.J, Average distribution of Ca 2+ wave duration per oligodendrocyte process before and after application of TTX.K, Average distribution of Ca 2+ amplitude per oligodendrocyte process before and after application of TTX.L, Representative traces of ΔF / F 0 over time before and after application of TTX.Scale bar, 40 s, and 5% ΔF / F 0 .Two hundred twenty (control) and one hundred thirty-five (TTX) oligodendrocyte processes, analyzed from N = 16 (control) and N = 10 (TTX) movies.Two-tailed statistical tests used: unpaired parametric t test in I and Mann-Whitney in J and K. *p < 0.05; ***p ≤ 0.001; ns, not significant.See Extended Data Figures 3-2  of MBP mRNA expression levels as development progress.Taken together, our study provides new evidence regarding the role of Ca 2+ signaling in mediating different stages of myelination.
Ca 2+ cellular sources and signaling pathways in mature oligodendrocytes remain poorly understood (Thornton and Hughes, 2020) and need further explorations.Nonetheless, the employment of AAV-mbp:jGCaMP8s with high-resolution Ca 2+ imaging, coupled with the manipulation of neuronal activity, not only enhances the spatiotemporal resolution of the recordings but also holds the potential to accelerate our understanding of how Ca 2+ intrinsically regulates oligodendrocyte myelination.
Figure Parameter Type of test Sample size Statistical data Figure 2H Correlation amplitude versus duration of Ca 2+ events Pearson's correlation t test, two-tailed

Figure 3 .
Figure3.Ex vivo AAV-mbp:jGCaMP8s screening in spinal cord organ cultures at DIV14.A, Representative image of the whole spinal organotypic culture showing AAV-mbp:jGCaMP8s expression (green) and mature oligodendrocytes(MBP, red).Nuclei (white) were labeled with DAPI dye.Note the ventral region highlighted by the ventral fissure (arrow).Scale bar, 300 μm.B, Representative higher magnification image of the spinal ventral region (same as in A) showing AAV-mbp:jGCaMP8s expression (green) and mature oligodendrocytes (MBP, red).Scale bar, 100 μm.See Extended Data Figure3-1 for more details.C, Representative confocal micrograph of spinal organotypic ventral region showing neurons (SMI-32, cyan) and AAV-mbp:jGCaMP8s expression (green).Scale bar, 100 μm.D, Representative higher magnification images of spinal cord organotypic cultures showing AAV-mbp: jGCaMP8s expression (green), mature oligodendrocytes (MBP, red), and neurons (SMI-32, cyan).Scale bar, 30 μm.E, Quantification of the AAV-mbp: jGCaMP8s efficiency in spinal cord organotypic cultures at DIV14.Data are represented as percentages.The analysis was performed using maximum intensity projections of 3D z-stack images, and quantification was obtained by using the "Analyze particles" function of the Fiji ImageJ software; N = 3 spinal cord organotypic cultures.F, Representative image of ventral region of spinal cord organotypic cultures showing AAV-mbp:jGCaMP8s expression (green) and GFAP + astrocytes (magenta).Scale bar, 100 μm.G, Representative image of ventral region (same as in F) showing AAV-mbp:jGCaMP8s expression (green) and microglia (IbaI, white).Scale bar: 100 μm.H, Maximum intensity projection of a 3D z-stack, illustrating an extended-duration movie highlighting jGCaMP8s-expressing myelinating oligodendrocytes.Scale bar, 100 μm.Frames from the time-lapse imaging are indicated with a white box corresponding to panel h', h''; scale bar, 10 μm.I, Average distribution of Ca 2+ wave frequencies (Hz) per oligodendrocyte process before and after application of TTX.J, Average distribution of Ca 2+ wave duration per oligodendrocyte process before and after application of TTX.K, Average distribution of Ca 2+ amplitude per oligodendrocyte process before and after application of TTX.L, Representative traces of ΔF / F 0 over time before and after application of TTX.Scale bar, 40 s, and 5% ΔF / F 0 .Two hundred twenty (control) and one hundred thirty-five (TTX) oligodendrocyte processes, analyzed from N = 16 (control) and N = 10 (TTX) movies.Two-tailed statistical tests used: unpaired parametric t test in I and Mann-Whitney in J and K. *p < 0.05; ***p ≤ 0.001; ns, not significant.See Extended Data Figures 3-2-3-4 and Movies 3 and 4 for more details.
Figure3.Ex vivo AAV-mbp:jGCaMP8s screening in spinal cord organ cultures at DIV14.A, Representative image of the whole spinal organotypic culture showing AAV-mbp:jGCaMP8s expression (green) and mature oligodendrocytes(MBP, red).Nuclei (white) were labeled with DAPI dye.Note the ventral region highlighted by the ventral fissure (arrow).Scale bar, 300 μm.B, Representative higher magnification image of the spinal ventral region (same as in A) showing AAV-mbp:jGCaMP8s expression (green) and mature oligodendrocytes (MBP, red).Scale bar, 100 μm.See Extended Data Figure3-1 for more details.C, Representative confocal micrograph of spinal organotypic ventral region showing neurons (SMI-32, cyan) and AAV-mbp:jGCaMP8s expression (green).Scale bar, 100 μm.D, Representative higher magnification images of spinal cord organotypic cultures showing AAV-mbp: jGCaMP8s expression (green), mature oligodendrocytes (MBP, red), and neurons (SMI-32, cyan).Scale bar, 30 μm.E, Quantification of the AAV-mbp: jGCaMP8s efficiency in spinal cord organotypic cultures at DIV14.Data are represented as percentages.The analysis was performed using maximum intensity projections of 3D z-stack images, and quantification was obtained by using the "Analyze particles" function of the Fiji ImageJ software; N = 3 spinal cord organotypic cultures.F, Representative image of ventral region of spinal cord organotypic cultures showing AAV-mbp:jGCaMP8s expression (green) and GFAP + astrocytes (magenta).Scale bar, 100 μm.G, Representative image of ventral region (same as in F) showing AAV-mbp:jGCaMP8s expression (green) and microglia (IbaI, white).Scale bar: 100 μm.H, Maximum intensity projection of a 3D z-stack, illustrating an extended-duration movie highlighting jGCaMP8s-expressing myelinating oligodendrocytes.Scale bar, 100 μm.Frames from the time-lapse imaging are indicated with a white box corresponding to panel h', h''; scale bar, 10 μm.I, Average distribution of Ca 2+ wave frequencies (Hz) per oligodendrocyte process before and after application of TTX.J, Average distribution of Ca 2+ wave duration per oligodendrocyte process before and after application of TTX.K, Average distribution of Ca 2+ amplitude per oligodendrocyte process before and after application of TTX.L, Representative traces of ΔF / F 0 over time before and after application of TTX.Scale bar, 40 s, and 5% ΔF / F 0 .Two hundred twenty (control) and one hundred thirty-five (TTX) oligodendrocyte processes, analyzed from N = 16 (control) and N = 10 (TTX) movies.Two-tailed statistical tests used: unpaired parametric t test in I and Mann-Whitney in J and K. *p < 0.05; ***p ≤ 0.001; ns, not significant.See Extended Data Figures 3-2-3-4 and Movies 3 and 4 for more details.

Figure 4 .
Figure 4. Ex vivo AAV-mbp:jGCaMP8s screening in spinal cord organ cultures at DIV21.A, Representative image of the ventral region of spinal cord showing AAV-mbp:jGCaMP8s expression (green) and mature oligodendrocytes (MBP, red).Scale bar, 30 μm.See Extended Data Figure 4-1 for more details.B, Maximum intensity projection of a 3D z-stack, illustrating an extended-duration movie highlighting jGCaMP8s-expressing myelinating oligodendrocytes.Scale bar, 100 μm.Frames from the time-lapse imaging are indicated with a white box corresponding to panel b'; scale bar, 10 μm.C, Distribution of average Ca 2+ wave frequencies (Hz) of 183 (control) and 49 (TTX) oligodendrocyte processes, analyzed from N = 13 (control) and N = 5 (TTX) movies.A two-sided statistical unpaired parametric t test was used, ns; not significant.D, Distribution of average Ca 2+ wave duration of 183 (control) and 49 (TTX) oligodendrocyte processes, analyzed from N = 13 (control) and N = 5 (TTX) movies.A two-tailed statistical Mann-Whitney test was used; ns, not significant.

Table 1 .
A comprehensive list of plasmids, primers, and probes used in this work

Table 2 .
Summary statistics