Correlative light–electron microscopy reveals fundamentally different axonal architectures for fast and modulatory transmission
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eLife Assessment
This paper provides fundamental data on the ultrastructural signatures of neuromodulatory axons in mouse cortex obtained from exceptionally high-quality correlated light- and volume electron microscopy. They report unique vesicle and synapse configurations of cholinergic and serotonergic axons, providing compelling structural support for volume transmission.
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Abstract
We used correlative light and focussed ion beam–scanning electron microscopy (FIBSEM) to characterise the ultrastructural organisation of neuromodulatory axons in the upper layers of the adult mouse somatosensory cortex. Cholinergic projections from the basal forebrain and serotonergic axons from the raphe nucleus were reconstructed, enabling quantitative analysis of synapse and vesicle distribution. Consistent with prior work, these axons formed only small and morphologically indistinct synaptic specialisations. However, these sites contained only a small fraction of the total axonal vesicle population, which were instead distributed along their length and spatially uncoupled from identifiable synaptic contacts. In contrast to classical excitatory and inhibitory axons, where vesicles are tightly clustered at defined presynaptic release sites, this organisation indicates that synaptic release contributes only minimally to neuromodulator output. Instead, our data support a model in which acetylcholine and serotonin signalling is mediated predominantly through non-synaptic release, consistent with a spatially diffuse transmission across cortical microcircuits.
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eLife Assessment
This paper provides fundamental data on the ultrastructural signatures of neuromodulatory axons in mouse cortex obtained from exceptionally high-quality correlated light- and volume electron microscopy. They report unique vesicle and synapse configurations of cholinergic and serotonergic axons, providing compelling structural support for volume transmission.
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Reviewer #1 (Public review):
Summary:
This study investigates the structural basis of neuromodulatory transmission in the cerebral cortex. Using genetic labelling, correlative light and electron microscopy (CLEM), and focused ion beam scanning electron microscopy (FIBSEM), the authors reconstruct cholinergic axons originating from the basal forebrain and serotonergic axons from the raphe nuclei in the mouse somatosensory cortex, and compare their ultrastructural organisation with that of glutamatergic and GABAergic axons.
The study provides detailed quantitative evidence that cholinergic and serotonergic axons have an axonal architecture fundamentally different from that of classical fast-transmitting glutamatergic and GABAergic axons. In particular, vesicles in cholinergic and serotonergic axons are distributed more diffusely along the …
Reviewer #1 (Public review):
Summary:
This study investigates the structural basis of neuromodulatory transmission in the cerebral cortex. Using genetic labelling, correlative light and electron microscopy (CLEM), and focused ion beam scanning electron microscopy (FIBSEM), the authors reconstruct cholinergic axons originating from the basal forebrain and serotonergic axons from the raphe nuclei in the mouse somatosensory cortex, and compare their ultrastructural organisation with that of glutamatergic and GABAergic axons.
The study provides detailed quantitative evidence that cholinergic and serotonergic axons have an axonal architecture fundamentally different from that of classical fast-transmitting glutamatergic and GABAergic axons. In particular, vesicles in cholinergic and serotonergic axons are distributed more diffusely along the axon and are only weakly associated with morphologically identifiable synaptic specialisations. These findings provide strong structural support for the idea that volume transmission represents a major mode of signalling by these neuromodulatory systems in the cortex.
Although the paucity of conventional synaptic contacts made by cortical neuromodulatory axons has been reported previously, including in classical work by Descarries and colleagues, the present study provides substantially more detailed and quantitative three-dimensional evidence. The study also reveals characteristic differences among neurotransmitter systems in vesicle density, vesicle distribution relative to synaptic membranes, and vesicle morphology.
Strengths:
A major strength of this study is the combination of genetic labelling, CLEM, and high-resolution FIBSEM, which allows molecularly identified cholinergic and serotonergic axons to be reconstructed without the electron-dense reaction products used in conventional immunoelectron microscopy. This enables detailed analysis of vesicles and membrane specialisations along extended axonal segments.
Another important strength is that the analysis is not restricted to individual boutons or varicosities. Instead, the authors analyse vesicle distributions continuously along reconstructed axons and compare them quantitatively with glutamatergic and GABAergic axons. This provides a more comprehensive assessment of the relationship between vesicle distribution and morphologically identifiable synaptic sites.
Overall, the data are convincing and support the authors' main conclusions. In particular, the finding that most vesicles in cholinergic and serotonergic axons are spatially dissociated from identifiable synaptic specialisations provides strong structural evidence for predominantly non-synaptic or volume transmission. The work should therefore be of broad interest to researchers studying neuromodulation, cortical circuitry, and connectomics.
Weaknesses:
I do not identify any major methodological weakness. The manuscript is well prepared, and the experimental results generally support the authors' conclusions. I have only a few points that could further improve the interpretation and discussion.
First, in the Introduction, ionotropic receptors are emphasised in the context of rapid actions of acetylcholine and serotonin. However, these neuromodulators can also produce relatively rapid effects on cortical neurons through G-protein-coupled receptors. This could be acknowledged, for example, with reference to Gulledge et al. (2009) and Tanaka & North (1993).
Second, the implications of the findings for volume transmission could be discussed somewhat more explicitly. If acetylcholine and serotonin are released extrasynaptically and are not immediately degraded or removed, they may diffuse through the extracellular space and activate receptors located at some distance from the release site. Thus, their actions need not be restricted to cellular elements immediately adjacent to vesicle-rich regions of the axon. A brief discussion of the sequence from action-potential-dependent Ca²⁺ entry and transmitter release to extracellular diffusion and activation of spatially distributed receptors would strengthen the interpretation.
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Reviewer #2 (Public review):
Summary:
This is an important, timely and extremely well-conducted study, providing 3D EM data of the highest quality for identification of modulatory axons in the neocortex. The field of connectomics, in which ever larger 3D EM volumes are being acquired, has so far focused on glutamatergic and GABAergic synaptic circuits in the cerebral cortex. These obviously constitute the major fraction of synapses and are known to form high-specificity networks. However, it has been clear that modulatory systems originating in subcortical structures are an important additional defining property of neocortical circuits. Without a clear identification of these specific axon types, their identification in 3D EM data has so far not been possible at scale.
Strength:
The present study addresses this issue and resolves the …
Reviewer #2 (Public review):
Summary:
This is an important, timely and extremely well-conducted study, providing 3D EM data of the highest quality for identification of modulatory axons in the neocortex. The field of connectomics, in which ever larger 3D EM volumes are being acquired, has so far focused on glutamatergic and GABAergic synaptic circuits in the cerebral cortex. These obviously constitute the major fraction of synapses and are known to form high-specificity networks. However, it has been clear that modulatory systems originating in subcortical structures are an important additional defining property of neocortical circuits. Without a clear identification of these specific axon types, their identification in 3D EM data has so far not been possible at scale.
Strength:
The present study addresses this issue and resolves the problem of identifying modulatory axons in 3D EM data of the cerebral cortex. Given the fast pace of development in the field of connectomics, such high-quality information is extremely relevant and of substantial impact for the field. The study is conducted beautifully, and the figures are very clear.
Slight weakness:
For the quantitative analyses, it would be highly appreciated to plot the raw data distributions as scatter plots. This is particularly important because, for many readers, these data will serve as a benchmark for classifying and identifying such synapses. Thus, any parameter distributions that are (close to) disjunct will be extremely informative. Along those lines, even at the level of the already extracted parameters, it would be very interesting to see whether the authors can already train a simple classifier that would allow the identification of cholinergic and serotonergic synapses or axons in the low-dimensional parameter space that has already been sampled.
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Reviewer #3 (Public review):
Summary:
This paper uses correlative FIB-SEM to reconstruct cholinergic and serotonergic axons in mouse somatosensory cortex. The authors wanted to examine how these neuromodulatory axons are organized at the ultrastructural level, specifically whether their vesicles are concentrated at synaptic structures like fast transmitters (glutamate and GABA) or distributed differently. They compare the two types against glutamate and GABA axons in the same datasets, and found that the synaptic vesicles are distributed more homogeneously in the neuromodulatory axons, supporting volume transmission.
Strengths:
The main strength is the dataset. FIB-SEM gives isotropic resolution, so vesicles can be counted and positioned accurately in 3D. The correlative light-EM approach lets them identify genetically labeled axons …
Reviewer #3 (Public review):
Summary:
This paper uses correlative FIB-SEM to reconstruct cholinergic and serotonergic axons in mouse somatosensory cortex. The authors wanted to examine how these neuromodulatory axons are organized at the ultrastructural level, specifically whether their vesicles are concentrated at synaptic structures like fast transmitters (glutamate and GABA) or distributed differently. They compare the two types against glutamate and GABA axons in the same datasets, and found that the synaptic vesicles are distributed more homogeneously in the neuromodulatory axons, supporting volume transmission.
Strengths:
The main strength is the dataset. FIB-SEM gives isotropic resolution, so vesicles can be counted and positioned accurately in 3D. The correlative light-EM approach lets them identify genetically labeled axons without using electron-dense stains that would obscure internal structures. This is a clear improvement over previous EM studies that relied on immunolabeling and only looked at varicosities.
The analysis is quantitative and systematic. They don't rely on identifying boutons or varicosities as the unit of analysis. Instead, they sample along the axon centerline at regular intervals, which avoids the bias of only looking at swellings. This is a cleaner way to ask where vesicles actually sit relative to synapses and other structures.
The comparison with glutamate and GABA axons is important. It shows that the differences are not just about neuromodulatory axons being "less synaptic" in a graded way, but qualitatively different in how vesicles are organized. Glutamate and GABA axons concentrate vesicles at synapses; ACh and 5-HT axons don't.
Weaknesses:
An intrinsic weakness of the EM approach is the missing of molecular information. The paper finds vesicles to be distributed homogeneously regardless of potential "post-synaptic" contact elements. However, protein machinaries for vesicle release might still statistically bind to certain postsynaptic elements.
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Correlative light-electron microscopy reveals fundamentally different axonal architectures for fast and modulatory transmission https://www.biorxiv.org/content/10.64898/2026.08.11.744243v1
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Correlative light-electron microscopy reveals fundamentally different axonal architectures for fast and modulatory transmission https://www.biorxiv.org/content/10.64898/2026.08.11.744243v1
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