The brain: a network of neurons… and supporting cells

Our brain is made up of neurons…but not only neurons! Neurons are specialized cells that transmit information to other cells, whether nerve or non-nerve. Information travels as electrical impulses along the axon, a sort of “highway” that carries the nerve signal.

At the end of each axon are synapses, small communication zones composed of:

• the axon terminal of the presynaptic neuron (which sends the signal),
• the membrane of the postsynaptic neuron (which receives it),
• and the synaptic cleft, the narrow gap where neurotransmitters are released to pass the message.

To keep neurons functioning properly, the brain also contains glial cells, which provide structural and metabolic support and regulate many physiological processes. Among them are astrocytes, star-shaped cells located close to synapses.

Since the 1990s and the discovery of the “tripartite synapse” concept, scientists have revealed the crucial role astrocytes play in information transmission.

However, astrocytes still hold some secrets, particularly concerning their finest extensions, called “leaflets”!

Cutting-edge imaging strategies for unprecedented results

To explore the structure and function of astrocytes in greater detail, the authors developed an innovative multimodal imaging workflow, combining two-photon and electron microscopy.

The electron microscopy experiments were led by several France-BioImaging units: CEA-Grenoble, Grenoble Institute of Neurosciences and the TIMC laboratory. The 2-photon imaging was performed at Lausanne University.

• FIB-SEM microscopy to reveal the 3D ultrastructure of astrocytes and, in particular, their leaflets. This technique was combined with 3D immunolabeling to detect the presence of specific proteins:

• IP₃R1, a marker of the endoplasmic reticulum involved in calcium signaling, 

• Connexin-43, a protein found at gap junctions connecting different structures.

• 2-photon calcium imaging to monitor real-time calcium variations in live leaflets in 3D, thereby assessing their role in cell-to-cell communication.

Architecture of the leaflets

Leaflets are extremely thin extensions (<250 nm), sometimes smaller than the wavelength of visible light, which explains why they long escaped direct observation.

They contain tiny segments of endoplasmic reticulum (i-ER) but no mitochondria, indicating a specialization for rapid and localized calcium signaling.

Several leaflets are interconnected via gap junctions, forming micro-networks within the astrocyte.

Figure 1. Leaflets are complex subdomains of the astrocyte interconnected by gap junctions. (B) Top: segmentation of a complete astrocyte fragment, including: bottom left: shafts with inner diameter >250 nm (gray); bottom right: leaflets, ≤250 nm (green). Bars: 3 μm.

Figure 3. i-ER: Minuscule organelles observed specifically in the leaflet domains of astrocytes. (C) Examples of i-ER saccules (magenta overlaid to EM images). Top: i-ERs surrounded by associated ribosomes (magenta arrowheads). Bottom: i-ERs contacting the astrocyte plasma membrane (yellow arrowheads). Bar: 500 nm.

Interaction with synapses

3D reconstructions revealed that the majority of synapses (around 90%) are shared by several leaflets of a single astrocyte.

Thus, one astrocyte can link together multiple synapses within the same neuronal network.

This finding challenges the “one synapse ↔ one astrocyte” model: in reality, astrocytes connect neurons to one another through their leaflets.

Figure 2. Leaflets are the astrocyte’s specialized subdomains that interact with synapses and preferentially embed multiple vs. individual synapses. (B) Arrangement of synaptic elements with leaflets and other astrocyte structures; 2D FIB-SEM images overlaid with segmentation map (leaflet: green; axonal bouton: yellow; spine: orange; synaptic cleft: red). Left: five synapses encompassed in a leaflet domain, contacted by leaflet units. Bar: 0.5 μm. Top right: synapse in direct contact with a shaft (S, gray); bar: 0.5 μm. Middle right: synapse in contact with both leaflets emerging from a shaft and the shaft itself; bar: 0.5 μm. Bottom right: synapse in contact with leaflets emerging from cell body (soma). Bar: 1 μm.

Calcium activity in leaflets

2-photon calcium imaging revealed very rapid, localized calcium micro-events, often triggered by neuronal activity.

These signals depend on IP₃R1 receptors in the i-ER and can propagate from one leaflet to another via gap junctions.

When they merge, these signals create broader calcium waves capable of synchronizing several neighboring synapses.

Figure 7. Leaflet domains host multiple synapses from independent circuits and display complex Ca²⁺ events that can integrate their ongoing activities in space and time. (B) Left: single-plane time-series capturing a representative multi-originated Ca²⁺ event in “Leaflets” (pseudocolor Ca²⁺ intensity scale, see “Two-photon 3D+t imaging data analysis” in STAR Methods). “Mitochondria” regions (azure pseudocolor) display an independent event. Right: oriented graph representation of the evolution of the event in “Leaflets,” visualizing spatial and time aspects, directionality, and intensity. (i), (ii), and (iii): timings of the images on the left. Arrowheads in (i) and (ii) identify distinct origination sites, and in (iii) their merging into a larger, long-lasting event. Bar: 1 μm. (C) Left: time-series as in (B), here capturing a Ca²⁺ event in “Leaflets” with three distinct originations (arrowheads). Two of them appear simultaneously in (i), the third, delayed, at another location in (ii), while in (iii) they all merge into a larger, long-lasting, high-intensity Ca²⁺ elevation. Noteworthy, the event expands around “Mitochondria” regions without touching them. Bar: 1 μm. Right: oriented graph representation of the Ca²⁺ event’s dynamics as in (B).

Conclusion

The results show that astrocytes are not mere support cells, but true biological computation units, where each leaflet acts as a functional “pixel” in a mosaic capable of interpreting and integrating multiple neuronal signals through the language of calcium.
This new astrocyte-oriented approach could pave the way for further studies exploring other brain regions to determine whether similar mechanisms exist elsewhere in the nervous system.

Read the scientific article here!

Benoit L, Hristovska I, Liaudet N, Jouneau PH, Fertin A, de Ceglia R, Litvin DG, Di Castro MA, Jevtic M, Zalachoras I, Kikuchi T, Telley L, Bergami M, Usson Y, Hisatsune C, Mikoshiba K, Pernet-Gallay K, Volterra A. Astrocytes functionally integrate multiple synapses via specialized leaflet domains. Cell. 2025 Sep 24:S0092-8674(25)01028-1. doi: 10.1016/j.cell.2025.08.036. Epub ahead of print. PMID: 40997814.

Last week, France-BioImaging participated in the Global BioImaging Exchange of Experience 2025. This year’s edition brought together scientists, facility managers, and infrastructure leaders from the international imaging community to explore “Imaging in 2035 – Sustaining Infrastructure Ecosystems & Advanced Technologies.”

Co-organized by BioImaging North America and Canada BioImaging, the event fostered rich exchanges among imaging infrastructure stakeholders on how to ensure the long-term sustainability of our global imaging ecosystem. France-BioImaging was represented by Caroline Thiriet and Jean Salamero who, as Mission Officer for Inter-Infrastructures Relationships at France-BioImaging and member of the Global BioImaging Working Group on Impact, moderated the session dedicated to “Micro-to-Macro: Measuring the Hidden Impacts of Imaging Scientists & Networks.” The session gathered a vibrant panel of experts — Susan Warner, Nick Souter, Johanna Bischof, Caron Jacobs, Leonel Malacrida, and Michelle Itano — who discussed how to communicate impact, reduce research’s carbon footprint, and recognize the essential contributions of imaging scientists worldwide.

The event also featured an inspiring keynote by Teng-Leong Chew (HHMI Janelia Research Campus) on “Microscopy Dissemination and Adoption Across the Globe.”

A big thank you to our global partners for an inspiring week of exchange and vision-building for the future of bioimaging!

This new edition, dedicated to the complete imaging workflow, from sample preparation to image analysis, will take place from November 5 to 7, 2025, in Versailles.

Program

The program includes numerous conferences and practical workshops on major topics:

• Fluorescence lifetime imaging (FLIM/multi-color)
• Live imaging – Biosensors
• 2D and 3D electron imaging
• Label-free imaging
• Supervised learning, AI, and image analysis
• Chemical imaging

Find the detailed program below:

This event is open to all! Whether you are a researcher, engineer, or technician, don’t miss this opportunity to network with other members of the community.

General information

When? November 5-7, 2025

Where? Centre INRAE Versaille-Saclay

The registration is free but mandatory – Deadline October 24

Find more information and registration link here: https://rmui-2025.journees.inrae.fr/

Bio-protocol is organizing a webinar focused on live-cell imaging on October 15, 2025.

On this occasion, Magali Bénard and Christophe Chamot from the PRIMACEN imaging platform (Normandie node) will present an optimized protocol for maintaining cell viability during live-cell imaging.

When? October 15, 2025 – 11:00 AM
How to join? https://bio-protocol.org/webinar/detail?number=577683940903485442

MIDOG is a challenge organized on the occasion of the MICCAI conference, dedicated to medical image computing and computer-assisted intervention. This bioinformatics competition focused on mitotic figures, which are a key biomarker in tumor grading. The variability of clinical samples makes the detection and classification of mitotic figures difficult for current AI models. The aim of MIDOG 2025 was to address this problem by testing algorithms in atypical conditions.

With their solutions, Thomas Walter’s team won:

• 2nd place in Task 1 focused on mitotic figures detection. They designed a robust and efficient YOLOv12 one-stage model, combined with a streamlined preprocessing pipeline. The approach enables fast detection of both mitotic figures and hard negatives, and benefits from a precise data augmentation process integrating multi-target Macenko stain normalization.
• 1st place in Task 2 dedicated to atypical mitotic figure classification. They leveraged the DINOV3-H+ foundation model, originally pretrained on natural images; and fine-tuned it with LoRA, requiring only ~1.3M trainable parameters. To tailor the model to histopathology, they incorporated extensive domain-specific data augmentations and implemented a domain-aware focal loss, allowing them to better handle both class and domain imbalance during training.

You can read the preprint of their solutions here:

• Task 1: https://www.arxiv.org/abs/2509.02593 (update soon)
• Task 2: https://arxiv.org/abs/2508.21041

The Plant Imaging Expert Group of Euro-BioImaging is organizing a new webinar to introduce the DREAM project, Wednesday, September 24, at 13:00!

What is the DREAM project?

DREAM stands for “Dynamic Regulation of Photosynthesis in Light-Acclimated Organisms”. This research initiative aims to develop a new generation of instrumentation and data acquisition protocols to better evaluate the dynamics of photosynthesis. A better understanding of this mechanism in plants could open promising pathways toward sustainable agriculture that meets global challenges.

A webinar with France-BioImaging members

Members of France-BioImaging and DREAM, Ludovic Jullien, Ian Coghill – from the Chemistry Department of ENS (Sorbonne University/CNRS) – and Aliénor Lahlou (Chemistry Department ex-member, now PhD research at Sony CSL) will present the overall objectives of the project as well as its latest outcomes.

How to join?

Meeting link here.

More info? https://www.eurobioimaging.eu/events/plant-imaging-expert-group-meeting-dynamic-regulation-of-photosynthesis-in-light-acclimated-organisms/

The France-BioImaging Preclinical Microscopy working group is organizing its next webinar on Thursday, September 25th from 14:00 to 16:00.

This session, open to all, will focus on advanced imaging solution with two keynotes:

• Label-free 3D imaging of thick samples by Kaer Labs – 14:00
• Quantitative blood vessel photoacousticmicroscopy coupled with ultrasonicmicroscopy by Bastien Arnal (LiPhy, Université Grenoble Alpes, CNRS) – 14:55

This webinar will conclude with an open discussion!

To join us, click here!

Meeting ID: 339 429 931 973 3
Passcode: PY6E2v4A

The 2nd edition of the Single Cell Conference of Nantes focused on “Deciphering single cell interactions in health and disease” will take place on October 7 and 8, 2025.

This event aims to highlight the use of single cell analysis technologies applied to the study of cellular mechanisms. Many international and local speakers will present the latest methods and applications in single-cell analysis.

In addition, participants will have the opportunity to present their research project after the submission and selection of an abstract.

A bioinformatics workshop dedicated to spatial data analysis will also be held on the afternoon of the second day (14:00-17:00).

General information

The participant have until September 19, 2025 at 17:00 to submit their abstract and/or apply for the bioinformatics workshop.

The registration is free for standard academic registration.
A fee of 50€ is asked for Senior Academic Registration (CR, DR, MCU, PU, Lecturer, goup leader…) and of 100€ for Industry Registration.

Find the program and the registration link here: https://nantescell.sciencesconf.org/

A research project* at the IBDM (Marseille Developmental Biology Institute, CNRS – AMU), led by Clément Rodier, has recently challenged long-standing scientific hypotheses about how muscles elongate during development, thanks to advanced microscopy. Let’s take a closer look!

Sarcomeres, the “bricks” of muscles

Striated muscles are made up of muscle fibers, themselves composed of myofibrils, chains of thousands of contractile “bricks” called sarcomeres. In mammals, a sarcomere measures between 2 and 3 micrometers.

As an organism grows, its muscles must expand to keep pace with skeletal development. Yet sarcomeres maintain roughly the same size. For decades, the prevailing hypothesis held that new bricks, sarcomeres, were added exclusively at the ends of myofibril chains. However, it had never been directly observed. Clément Rodier’s goal was to visualize, using both light and electron microscopy**, how new sarcomeres are inserted during muscle growth, with the fly Drosophila as a model system.

A scientific hypothesis challenged

The research team analyzed Drosophila flight muscles between 32 and 40 hours after puparium formation, the developmental stage following the larval phase. This model has a key advantage: its growth is extremely fast (around 100 new sarcomeres are added in just 4 hours!) making the mechanism easier to capture.

Contrary to the initial hypothesis, microscopy images showed that sarcomere insertion was not restricted to the ends of the myofibrillar chain. Each brick is capped by terminal structures called Z-discs, which should have shown changes if new sarcomeres were only added there.

(B) Schematic of a Drosophila hemithorax at pupal stages with the six dorsal longitudinal flight muscles (DLMs) in magenta and the tendon cell epithelium in green. DLM4 is highlighted and was used for quantifications in (E). The stable connection of the flight muscles to the tendon cell extensions (green lines) during the growth phase from 32 to 40 hours after puparium formation (APF). h, hours. (C and D) Confocal images of pupae of the indicated stages displaying the six flight muscles stained for actin (phalloidin in red) and Shot (anti-Shortstop in gray, also labeling the tendon extensions) in (C). High magnifications displaying the myofibrils stained for actin (red) and myosin [anti–Mhc (myosin heavy chain) in blue] in (D). Scale bars, 100 μm in (C) and 5 μm in (D). Note the marked muscle length increases after 32 hours APF. 

(B and C) About 34 hours APF, a pupa expressing Sls-GFP with a focus on the anterior flight muscle end. Stills from movie S1 are shown (B), and a kymograph of a selected region is shown (C). The myofibril ends, marked with yellow arrowheads, move to the left, while muscle fiber length increases and thus moves away from the reference point marked with the orange arrowhead. No obvious sarcomere addition is seen at the ends.

So how do muscles really grow?

To probe this mechanism further, the team developed a computational tool capable of analyzing the length of thousands of sarcomeres and identifying those with abnormal size.
Using this tool combined with high-resolution microscopy techniques (sush as in vivo bi-photon imaging), the researchers observed that some sarcomeres gradually elongated and then split into two daughter sarcomeres of normal length. The essential proteins needed for proper sarcomere function were also restored.

Unlike the long-accepted end hypothesis, this division mechanism allows new sarcomeres to be inserted at multiple points along the myofibril.

Fig. 7. Sarcomere division model. Working model of the tension-induced sarcomere division in Drosophila flight muscle sarcomeres. The growth of the skeleton, marked by integrin-based attachments, induces high tension, which triggers the division of the highlighted middle sarcomere. Thus, the sarcomere number increases from three to four. For details, see Discussion.

Does this mechanism also occur in mammals?

The researchers next turned to 3D electron microscopy and FRAP to test whether this mechanism also applied to other muscle types, namely Drosophila larval muscles, which are similar to mammalian skeletal muscles.

These imaging techniques revealed a zipper-like division process of sarcomeres during larval development. Further analyses confirmed the recruitment of new proteins that reestablish proper function in the daughter sarcomeres.

Looking ahead

Thanks to the combination of light and electron microscopy, both realized on France-BioImaging’s core facilities*, the team was able to put a long-standing hypothesis to the test… and overturn it! This work sheds new light on developmental mechanisms in insects, and possibly in mammals, including humans. Better understanding how muscles grow at the microscopic level could one day pave the way for novel therapies to fight muscle degenerative diseases.

* Clement Rodier et al., Muscle growth by sarcomere divisions. Sci. Adv.11, eadw9445 (2025). DOI:10.1126/sciadv.adw9445

** Electronic microscopy (3D EM) was performed on a PICsL core facility and advanced photonic microscopy (FRAP imaging, in vivo bi-photon imaging) on IBDM core facility.