ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the lamprey central canal
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Evaluation Summary:
This manuscript is of broad interest for the neuroscience and imaging community. The authors employ an array of advanced imaging techniques to bridge the understanding of neuronal function in whole organisms to the sub-cellular physiology of specific neuronal types. The microscopical observations, combined with system perturbations strongly support the claims.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Cerebrospinal fluid-contacting (CSF-c) neurons line the central canal of the spinal cord and a subtype of CSF-c neurons expressing somatostatin, forms a homeostatic pH regulating system. Despite their importance, their intricate spatial organization is poorly understood. The function of another subtype of CSF-c neurons expressing dopamine is also investigated. Imaging methods with a high spatial resolution (5–10 nm) are used to resolve the synaptic and ciliary compartments of each individual cell in the spinal cord of the lamprey to elucidate their signalling pathways and to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine- and somatostatin-expressing CSF-c neuronal subtypes. The density of somatostatin-containing dense-core vesicles, resolved by stimulated emission depletion microscopy, was shown to be markedly reduced upon each exposure to either alkaline or acidic pH and being part of a homeostatic response inhibiting movements. Their cilia symmetry was unravelled by stimulated emission depletion microscopy in expanded tissues as sensory with 9 + 0 microtubule duplets. The dopaminergic CSF-c neurons on the other hand have a motile cilium with the characteristic 9 + 2 duplets and are insensitive to pH changes. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell-type classification.
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Author Response:
Reviewer #2 (Public Review):
In all vertebrate species investigated, cerebrospinal fluid contacting the cerebrospinal fluid express the channel PKD2L1 (in macaques, mice and zebrafish: Djenoune et al., Frontiers in Neuroanatomy 2014; in lamprey: Jalalvand et al., Current Biology 2016b; Jalalvand et al J Neurosci 2018). However, in all species investigated these cells fall into two functional types based on their axial sensitivity to detect spinal curvature (in vivo for zebrafish: Bohm et al., Nature Communications 2016; Hubbard et al., Current Biology 2016), expression of neuropeptides and neuromodulators (in lamprey;: Christenson et al., Neurosci Letter 1991; Schotland et al., JCN 1996; in zebrafish: Djenoune et al., Scientific Reports 2017) or their firing patterns (in mouse: Petracca et al J Neurosci 2016; Di …
Author Response:
Reviewer #2 (Public Review):
In all vertebrate species investigated, cerebrospinal fluid contacting the cerebrospinal fluid express the channel PKD2L1 (in macaques, mice and zebrafish: Djenoune et al., Frontiers in Neuroanatomy 2014; in lamprey: Jalalvand et al., Current Biology 2016b; Jalalvand et al J Neurosci 2018). However, in all species investigated these cells fall into two functional types based on their axial sensitivity to detect spinal curvature (in vivo for zebrafish: Bohm et al., Nature Communications 2016; Hubbard et al., Current Biology 2016), expression of neuropeptides and neuromodulators (in lamprey;: Christenson et al., Neurosci Letter 1991; Schotland et al., JCN 1996; in zebrafish: Djenoune et al., Scientific Reports 2017) or their firing patterns (in mouse: Petracca et al J Neurosci 2016; Di Bella et al., Cell Reports 2019).
While the microscopy techniques used here are outstanding and bring without a doubt important evidence on the location and density of DSVs, there are concerns to address regarding the consolidation and interpretation of the physiological recordings of the ciliated neurons and pharmacology based on evidence that only ASIC1 channel is expressed in lamprey (see phylogenic analysis: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047259/), and that the lamprey ASIC1a is proton insensitive (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464184/).
In the article of Coric et al 2005 they had identified one clone of cDNA that corresponded to ASIC1 and when expressed in oocytes, no pH sensitivity was found under these conditions. They do not comment regarding the possible presence of ASIC3. The review of Grunder and Chen (2010) has a focus on ASIC1a and base their comment in lamprey on Coric et al 2005. Our evidence for the presence of ASIC3 in lamprey is that both the mechanical and pH response are blocked by APETx2, a selective antagonist of ASIC3, (Jalalvand et al 2016, Nature. com), strongly suggesting the presence of ASIC3 in the lamprey. ASIC3 is present in both the peripheral and central nervous system in mammals.
Reviewer #3 (Public Review):
This manuscript uses a variety of optical superresolution techniques to explore the structure and function of different cerebrospinal fluid contacting (CSF-c) neurons. First, Expansion Microscopy and Lightsheet Microscopy are combined to image large volumes of tissue from lamprey and to demonstrate the known organization of somatostatin-expressing and dopaminergic CSF-c neurons (Fig. 1). The authors then used STED to explore the subcellular location of somatostatin and dopamine in CSF-c neurons and demonstrate their presences in vesicle-like structures ranging between 60 and 200 nm (Fig. 2). Subsequently, the relation between GABA and somatostatin is examined in somatostatin CSF-c neurons. The authors show that there is no obvious colocalization between these molecules and that only somatostatin levels are altered in response to changes in the extracellular pH (Fig. 3). The authors furthermore demonstrate that dopamine levels with dopaminergic CSF-c neurons are also insensitive to pH changes (Fig. 4).
The authors have previously shown that somatostatin CSF-c neurons are mechanosensitive and now also demonstrate this for dopaminergic CSF-c neurons (Ext Data Fig. 1). They also show that their mechanosensitivity is mediated differently (ie. Not through ASIC3). To further explore this difference in mechanosensitivity, the authors set out to explore ciliary structure of these ciliated neurons. By combining expansion and STED, the authors succeed in resolving ciliary ultrastructure and demonstrate that they can distinguish between between motile (9+2) and primary cilia (9+0) (Fig. 5). They find that the majority of cilia on somatostatin-expressing CSF-c neurons is primary, whereas all cilia on dopaminergic CSF-c neurons were motile (Fig. 6).
Overall, this is an interesting imaging study that reports a number of technical steps that enable tissue-imaging with exquisite detail, such as discriminating between motile and primary cilia. It also nicely demonstrates what sort of new data can now be obtained in tissue, e.g. changes in vesicle numbers upon certain stimuli. However, as explained below, both the composition of the main text and the reproduction quality of the figures make it hard to judge the biological significance of this work.
Comments:
1/ Overall, I feel the paper needs a thorough rewrite. The introduction should give more insights into the underlying biology and also clarify which questions are being asked and why these are important. Currently the introduction is mostly a long summary of all results, but it doesn't help to understand the biology that underlies this work. Because of that the different experimental pieces currently feel a bit random and disconnected.
We have rewritten part of the Introduction to better expose the underlying biological questions.
2/ The reproduction quality of Figures 1, 2, 3 and 6 in the merged PDF is not great. In many cases, I cannot read the annotations or appreciate the content of the images. This make it rather impossible to judge the quality of the work. The data shown in Figure 5 is very impressive and I am sure the raw data for the other figures is equally great, but I can only judge what I see for myself.
We provide Figures at high resolution.
3/ Page 8: the conclusion that PKD2L1 is the mechanosensitive receptor for dopaminergic CSF-c neurons is only based on its presence in this cells. To really demonstrate this loss-of-function experiments would be needed.
PKD2L1 has been found to be responsible for mechanosensitivity in Zebrafish (Böhm et al. 2016).
We agree that to demonstrate a loss of function in lamprey a knock-out experiment would be needed. Transgenic techniques unfortunately cannot be used in lamprey since each generation lasts around 7 years, and there is no specific blocker for PKD2L1 to apply either. Therefore, we have modified the sentences.
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Evaluation Summary:
This manuscript is of broad interest for the neuroscience and imaging community. The authors employ an array of advanced imaging techniques to bridge the understanding of neuronal function in whole organisms to the sub-cellular physiology of specific neuronal types. The microscopical observations, combined with system perturbations strongly support the claims.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this paper Jalalvand et al. employ various imaging techniques to investigate the function and physiology of cerebrospinal fluid contacting (CSF-c) neurons in lamprey. By combining expansion microscopy techniques with lattice light sheet microscopy, they can reveal the spatial organization of somatostatin and dopaminergic neurons along the spinal cord. Zooming into these neurons with STED super-resolution microscopy they can show that somatostatin and dopamine are stored into dense core vesicles. Combining STED with alteration of extracellular pH and electrophysiology the authors could show that somatostatin, but not dopaminergic neurons are responsible for pH regulation. Further, they employ expansion microscopy combined with STED to investigate the architecture of cilia in the two neuronal types and …
Reviewer #1 (Public Review):
In this paper Jalalvand et al. employ various imaging techniques to investigate the function and physiology of cerebrospinal fluid contacting (CSF-c) neurons in lamprey. By combining expansion microscopy techniques with lattice light sheet microscopy, they can reveal the spatial organization of somatostatin and dopaminergic neurons along the spinal cord. Zooming into these neurons with STED super-resolution microscopy they can show that somatostatin and dopamine are stored into dense core vesicles. Combining STED with alteration of extracellular pH and electrophysiology the authors could show that somatostatin, but not dopaminergic neurons are responsible for pH regulation. Further, they employ expansion microscopy combined with STED to investigate the architecture of cilia in the two neuronal types and discover that somatostatin neurons have mainly sensory cilia while dopaminergic neurons have motile cilia.
Based on these observations, the authors suggest that pH/mechanosensitive somatostatin-expressing CSF-c neurons are sensory neurons. On the other hand, non-pH- but mechanosensitive dopaminergic CSF-c neurons may act as cerebrospinal fluid flow generator. I believe the data presented nicely support these claims.
Data is of excellent quality, considering the technical challenges or working with expansion microscopy and STED super-resolution in tissues. Expansion microscopy STED is extremely challenging in cell monolayers and the data in expanded tissues is of extremely good quality. Additionally, application of the same work pipeline to other tissues/cell types promises to help us gain insights into whole organism physiology down at the single cell level.
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Reviewer #2 (Public Review):
In all vertebrate species investigated, cerebrospinal fluid contacting the cerebrospinal fluid express the channel PKD2L1 (in macaques, mice and zebrafish: Djenoune et al., Frontiers in Neuroanatomy 2014; in lamprey: Jalalvand et al., Current Biology 2016b; Jalalvand et al J Neurosci 2018). However, in all species investigated these cells fall into two functional types based on their axial sensitivity to detect spinal curvature (in vivo for zebrafish: Bohm et al., Nature Communications 2016; Hubbard et al., Current Biology 2016), expression of neuropeptides and neuromodulators (in lamprey;: Christenson et al., Neurosci Letter 1991; Schotland et al., JCN 1996; in zebrafish: Djenoune et al., Scientific Reports 2017) or their firing patterns (in mouse: Petracca et al J Neurosci 2016; Di Bella et al., Cell …
Reviewer #2 (Public Review):
In all vertebrate species investigated, cerebrospinal fluid contacting the cerebrospinal fluid express the channel PKD2L1 (in macaques, mice and zebrafish: Djenoune et al., Frontiers in Neuroanatomy 2014; in lamprey: Jalalvand et al., Current Biology 2016b; Jalalvand et al J Neurosci 2018). However, in all species investigated these cells fall into two functional types based on their axial sensitivity to detect spinal curvature (in vivo for zebrafish: Bohm et al., Nature Communications 2016; Hubbard et al., Current Biology 2016), expression of neuropeptides and neuromodulators (in lamprey;: Christenson et al., Neurosci Letter 1991; Schotland et al., JCN 1996; in zebrafish: Djenoune et al., Scientific Reports 2017) or their firing patterns (in mouse: Petracca et al J Neurosci 2016; Di Bella et al., Cell Reports 2019).
While the microscopy techniques used here are outstanding and bring without a doubt important evidence on the location and density of DSVs, there are concerns to address regarding the consolidation and interpretation of the physiological recordings of the ciliated neurons and pharmacology based on evidence that only ASIC1 channel is expressed in lamprey (see phylogenic analysis: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047259/), and that the lamprey ASIC1a is proton insensitive (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464184/).
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Reviewer #3 (Public Review):
This manuscript uses a variety of optical superresolution techniques to explore the structure and function of different cerebrospinal fluid contacting (CSF-c) neurons. First, Expansion Microscopy and Lightsheet Microscopy are combined to image large volumes of tissue from lamprey and to demonstrate the known organization of somatostatin-expressing and dopaminergic CSF-c neurons (Fig. 1). The authors then used STED to explore the subcellular location of somatostatin and dopamine in CSF-c neurons and demonstrate their presences in vesicle-like structures ranging between 60 and 200 nm (Fig. 2). Subsequently, the relation between GABA and somatostatin is examined in somatostatin CSF-c neurons. The authors show that there is no obvious colocalization between these molecules and that only somatostatin levels are …
Reviewer #3 (Public Review):
This manuscript uses a variety of optical superresolution techniques to explore the structure and function of different cerebrospinal fluid contacting (CSF-c) neurons. First, Expansion Microscopy and Lightsheet Microscopy are combined to image large volumes of tissue from lamprey and to demonstrate the known organization of somatostatin-expressing and dopaminergic CSF-c neurons (Fig. 1). The authors then used STED to explore the subcellular location of somatostatin and dopamine in CSF-c neurons and demonstrate their presences in vesicle-like structures ranging between 60 and 200 nm (Fig. 2). Subsequently, the relation between GABA and somatostatin is examined in somatostatin CSF-c neurons. The authors show that there is no obvious colocalization between these molecules and that only somatostatin levels are altered in response to changes in the extracellular pH (Fig. 3). The authors furthermore demonstrate that dopamine levels with dopaminergic CSF-c neurons are also insensitive to pH changes (Fig. 4).
The authors have previously shown that somatostatin CSF-c neurons are mechanosensitive and now also demonstrate this for dopaminergic CSF-c neurons (Ext Data Fig. 1). They also show that their mechanosensitivity is mediated differently (ie. Not through ASIC3). To further explore this difference in mechanosensitivity, the authors set out to explore ciliary structure of these ciliated neurons. By combining expansion and STED, the authors succeed in resolving ciliary ultrastructure and demonstrate that they can distinguish between between motile (9+2) and primary cilia (9+0) (Fig. 5). They find that the majority of cilia on somatostatin-expressing CSF-c neurons is primary, whereas all cilia on dopaminergic CSF-c neurons were motile (Fig. 6).
Overall, this is an interesting imaging study that reports a number of technical steps that enable tissue-imaging with exquisite detail, such as discriminating between motile and primary cilia. It also nicely demonstrates what sort of new data can now be obtained in tissue, e.g. changes in vesicle numbers upon certain stimuli. However, as explained below, both the composition of the main text and the reproduction quality of the figures make it hard to judge the biological significance of this work.
Comments:
1/ Overall, I feel the paper needs a thorough rewrite. The introduction should give more insights into the underlying biology and also clarify which questions are being asked and why these are important. Currently the introduction is mostly a long summary of all results, but it doesn't help to understand the biology that underlies this work. Because of that the different experimental pieces currently feel a bit random and disconnected.2/ The reproduction quality of Figures 1, 2, 3 and 6 in the merged PDF is not great. In many cases, I cannot read the annotations or appreciate the content of the images. This make it rather impossible to judge the quality of the work. The data shown in Figure 5 is very impressive and I am sure the raw data for the other figures is equally great, but I can only judge what I see for myself.
3/ Page 8: the conclusion that PKD2L1 is the mechanosensitive receptor for dopaminergic CSF-c neurons is only based on its presence in this cells. To really demonstrate this loss-of-function experiments would be needed.
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