Chandelier cell anatomy and function reveal a variably distributed but common signal
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Evaluation Summary:
This paper will be of high interest to a broad audience of neuroscientists, as it provides a major advancement of our understanding of cortical circuits. The quality and quantitative nature of the neuroanatomical reconstructions at synaptic resolution are remarkable. Complementing the reconstructions with computational modeling and activity measurements, the study proposes a likely circuit function for a specific inhibitory cell type during behavior.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
The activity and connectivity of inhibitory cells has a profound impact on the operation of neuronal networks. While the average connectivity of many inhibitory cell types has been characterized, we still lack an understanding of how individual interneurons distribute their synapses onto their targets and how heterogeneous the inhibition is onto different individual excitatory neurons. Here, we use large-scale volumetric electron microscopy (EM) and functional imaging to address this question for chandelier cells in layer 2/3 of mouse visual cortex. Using dense morphological reconstructions from EM, we mapped the complete chandelier input onto 153 pyramidal neurons. We find that the number of input synapses is highly variable across the population, but the variability is correlated with structural features of the target neuron: soma depth, soma size, and the number of perisomatic synapses received. Functionally, we found that chandelier cell activity in vivo was highly correlated and tracks pupil diameter, a proxy for arousal state. We propose that chandelier cells provide a global signal whose strength is individually adjusted for each target neuron. This approach, combining comprehensive structural analysis with functional recordings of identified cell types, will be a powerful tool to uncover the wiring rules across the diversity of cortical cell types.
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Evaluation Summary:
This paper will be of high interest to a broad audience of neuroscientists, as it provides a major advancement of our understanding of cortical circuits. The quality and quantitative nature of the neuroanatomical reconstructions at synaptic resolution are remarkable. Complementing the reconstructions with computational modeling and activity measurements, the study proposes a likely circuit function for a specific inhibitory cell type during behavior.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
All of the experiments and modeling described are beautifully executed and the data and analyses are clearly presented. All conclusions are well supported by the data presented. The introduction and discussion provide a good overview of the prior literature and put the new observations in context. Some aspects of the introduction could be improved by providing a more nuanced view of the prior work on functional interactions between different inhibitory cell types in the cortex, but this does not impact the quality or novelty of the results and analyses that are presented.
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Reviewer #2 (Public Review):
Summary:
The authors used large volume complete EM reconstruction to analyze and quantify the synapses around AIS of 153 pyramidal cells from mouse visual cortex. Totally 1127 putative axon-axon synapses from 122 axon fragments were revealed. They found that the number of axon-axon synapses per AIS is highly variable and correlates with target cell properties. Monitoring the activity of ChCs by genetic expression of calcium indicator in ChCs revealed increased and collective activity during spontaneous walking bout in mice. This is an important work which provide the ultimate resolution and ground truth data on chandelier cell innervation patterns. However, there are several important issues that the author should address and clarify in Results and Discussion. In particular, it is unfortunate and surprising …
Reviewer #2 (Public Review):
Summary:
The authors used large volume complete EM reconstruction to analyze and quantify the synapses around AIS of 153 pyramidal cells from mouse visual cortex. Totally 1127 putative axon-axon synapses from 122 axon fragments were revealed. They found that the number of axon-axon synapses per AIS is highly variable and correlates with target cell properties. Monitoring the activity of ChCs by genetic expression of calcium indicator in ChCs revealed increased and collective activity during spontaneous walking bout in mice. This is an important work which provide the ultimate resolution and ground truth data on chandelier cell innervation patterns. However, there are several important issues that the author should address and clarify in Results and Discussion. In particular, it is unfortunate and surprising that the work used P36 rather than P56 or older mice. There is no good evidence that P36 visual cortex in mice is fully mature; most likely it is not. Therefore, some of the observations may not be describing the mature state of ChC-PyN connectivity, but rather a developing intermediate state. This point or possibility should be highlighted in the Results and Discussion, otherwise it will mislead the field.
Review:
The authors used large volume complete EM reconstruction to analyze and quantify the synapses around AIS of 153 pyramidal cells from mouse visual cortex. Totally 1127 putative axon-axon synapses from 122 axon fragments were revealed. They found that the number of axon-axon synapses per AIS is highly variable and correlates with target cell properties. Monitoring the activity of ChCs by genetic expression of calcium indicator in ChCs revealed increased and collective activity during spontaneous walking bout in mice. This is an important work which provide the ultimate resolution and ground truth data on chandelier cell innervation patterns. However, there are several important issues that the author should address and clarify in Results and Discussion. In particular, it is unfortunate and surprising that the work used P36 rather than P56 or older mice. There is no good evidence that P36 visual cortex in mice is fully mature; most likely it is not. Therefore, some of the observations may not be describing the mature state of ChC-PyN connectivity, but rather a developing intermediate state. This point or possibility should be highlighted in the Results and Discussion, otherwise it will mislead the field.
Main concerns:
It is puzzling why the authors used a P36 mouse. For such a demanding and effort intensive work, they should have made sure that they would be studying the "end product" of ChC-PyN connectivity. They cited Inan et al., 2013 for evidence of "mature ChCs" at P36, but Inan et al did not study older mice. We have unpublished evidence from high resolution complete single cell reconstruction that ChC axons and synapse "cartridges" continue to increase in complexity even at 2 months of age in the frontal cortex in mice; this timeline may also apply to visual cortex. If this were the case, then some of the results may pertain to a developing rather mature state of ChC -PyN connectivity. For example, the sparse non-ChC synapses at AIS may be further reduced or eliminated. I am not suggesting the authors to do another mouse at P60 for this paper, but this should be done at some point. For now, the authors should discuss the possibility that their results may not be describing the mature ChC circuit, and certain result could be pertaining to postnatal maturation.
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Reviewer #3 (Public Review):
Schneider-Mizell et al., investigate the structure and function of chandelier cells, a class of inhibitory neurons in the superficial layers of the cerebral cortex that is characterized by specially forming synaptic connections with the axon initial segments (AISs) of excitatory pyramidal neurons (PNs). For this purpose, the authors employ dense electron-microscopy to reconstruct all inhibitory synapses along the AISs of PNs within a relatively large volume of mouse primary visual cortex. These data provide an unprecedented quantitative account of the number of axo-axonic synapses per PN, their distributions along the AISs, whether they originate from chandelier or other inhibitory cells, and how these connectivity parameters depend on morphological properties of the targeted PNs. Simulations of activity …
Reviewer #3 (Public Review):
Schneider-Mizell et al., investigate the structure and function of chandelier cells, a class of inhibitory neurons in the superficial layers of the cerebral cortex that is characterized by specially forming synaptic connections with the axon initial segments (AISs) of excitatory pyramidal neurons (PNs). For this purpose, the authors employ dense electron-microscopy to reconstruct all inhibitory synapses along the AISs of PNs within a relatively large volume of mouse primary visual cortex. These data provide an unprecedented quantitative account of the number of axo-axonic synapses per PN, their distributions along the AISs, whether they originate from chandelier or other inhibitory cells, and how these connectivity parameters depend on morphological properties of the targeted PNs. Simulations of activity patterns in PNs with anatomically-inspired synaptic input from chandelier cells appear to be more effective in suppressing excitatory output compared to alternative scenarios for axo-axonic connectivity. Moreover, functional imaging in behaving animals revealed highly correlated activity across genetically identified chandelier cells, which correlated with pupil dilation. Taken together, the authors propose that arousal drives collective activation of chandelier cells, which is then passed onto PNs such that cell-specific structural differences of the PNs are compensated by heterogeneous axo-axonic connectivity patterns.
The major strength of this study are the electron-microscopic reconstructions of axo-axonic connections for a representative sample of 153 PNs. The authors show convincingly that they have solved many of the challenges for reconstructing such a complex dataset by combining automated image segmentation with manual proof-reading. The reconstructions provide an unprecedented quantitative account for the number and distributions axo-axonic connections, and their cell-cell variability. Moreover, the simulations provide first insight for how these empirically observed structural configurations of axo-axonic connections could impact the function of PNs. However, the simulation predictions remain to be tested empirically. While the functional imaging experiments performed here indicate how such testing might be achieved in future studies, direct evidence for how chandelier cells impact PN activity is not provided. In sum, the key claims of the paper are well supported by the data, and the approaches used are thoughtful and rigorous.
This study will have a strong impact on neuroscience research for many reasons. For example, the quantitative reconstruction data is available online, which will allow others to constrain more comprehensive simulations of cortex function. Moreover, the study reports a novel approach that allows imaging specifically the activity of chandelier cells, setting the stage for future studies to explore how these cells are embedded into local and long-range circuits, and how they are engaged during different behavioral states. Finally, the methods used here will facilitate dense reconstructions of larger cortical volumes, as emphasized by the authors in the last paragraph of the discussion section.
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