Axo-vascular coupling mediated by oligodendrocytes
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eLife assessment
This manuscript provides the first cellular analysis of how neuronal activity in axons (in this case the optic nerve) regulates the diameter of nearby blood vessels and hence the energy supply to neuronal axons and their associated cells. This is an important subject because, in a variety of neurological disorders, there is damage to the white matter that may result from a lack of sufficient energy supply. This paper will stimulate work on this important subject.
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Abstract
The high energy requirements of the cortical gray matter are met by the precise cooperation of neurons, glia, and vascular cells in a process known as neurovascular coupling (NVC). In contrast, the existence and significance of NVC in white matter (WM) are still debated and basic regulatory mechanisms are unknown. We recently discovered that oligodendrocytes sense the spiking axons’ activity via NMDA receptors and regulate their cell surface expression of glucose transporter GLUT1 allowing an increase in glycolytic metabolism that enables lactate release to metabolically support the axons. Here, we show for the mouse optic nerve (ON), a model WM tract, that the vascular support is also dynamically controlled. Axonal spiking activity induces small vessel dilations which are sustained for more than 20 minutes upon the ending of electrical stimulation. Pharmacological inhibition shows that the electrically evoked dilation is mediated by the prostaglandin E 2 receptor EP 4 and can be modulated by the oxygen concentration, as has been shown in the grey matter. Importantly, we found in ONs from conditional mouse mutants that oligodendroglial NMDA receptors are required for this type of neurovascular response, demonstrating a critical role of oligodendrocytes in coupling axonal activity to pericyte function. Reminiscent of NVC in cortical slices, the “axo-vascular” response is slower and may represent a more rudimentary form of neurovascular coupling.
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Author Response
Reviewer #1 (Public Review):
This manuscript provides the first cellular analysis of how neuronal activity in axons (in this case the optic nerve) regulates the diameter of nearby blood vessels and hence the energy supply to neuronal axons and their associated cells. This is an important subject because, in a variety of neurological disorders, there is damage to the white matter that may result from a lack of sufficient energy supply, and this paper will stimulate work on this important subject.
Axonal spiking is suggested to release glutamate which activates NMDA receptors on myelin-making oligodendrocytes wrapped around the axons: the oligodendrocytes - either directly or indirectly via astrocytes - then generate prostaglandin E2 which relaxes pericytes on capillaries, thus decreasing the resistance of the …
Author Response
Reviewer #1 (Public Review):
This manuscript provides the first cellular analysis of how neuronal activity in axons (in this case the optic nerve) regulates the diameter of nearby blood vessels and hence the energy supply to neuronal axons and their associated cells. This is an important subject because, in a variety of neurological disorders, there is damage to the white matter that may result from a lack of sufficient energy supply, and this paper will stimulate work on this important subject.
Axonal spiking is suggested to release glutamate which activates NMDA receptors on myelin-making oligodendrocytes wrapped around the axons: the oligodendrocytes - either directly or indirectly via astrocytes - then generate prostaglandin E2 which relaxes pericytes on capillaries, thus decreasing the resistance of the vascular bed and (presumably) increasing blood flow in the nerve.
Strengths of the paper
The paper identifies some important characteristics of axon-vascular coupling, notably its slow temporal development and long-lasting nature, the involvement of PgE2 in an oxygen-dependent manner, and a role for NMDARs. Rigorous criteria (constriction and dilation of capillaries by pharmacological agents) are used to select functioning pericytes for analysis.
Weaknesses of the paper
The study focuses exclusively on pericytes. It would have been interesting to assess whether arteriolar SMCs also contribute to regulating blood flow
We thank reviewer #1 for his/her positive comment on our manuscript. We also share the future interest in the optic nerve’s arteriole (there is only one main arteriole covered by SMC). However, it is not always visible in the preparation due to the orientation of the nerve - if not on the surface and directly under the microscope it is not possible to image it.
Reviewer #2 (Public Review):
This paper describes a new concept of "axo-vascular coupling" whereby action potential traffic along white matter axons induces vasodilation in the mouse optic nerve. This is an initial report dissecting some of the mechanisms that are undoubtedly complex as in gray matter NVC. I like the novel AVC concept.
We really appreciate the reviewer’s positive comments.
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eLife assessment
This manuscript provides the first cellular analysis of how neuronal activity in axons (in this case the optic nerve) regulates the diameter of nearby blood vessels and hence the energy supply to neuronal axons and their associated cells. This is an important subject because, in a variety of neurological disorders, there is damage to the white matter that may result from a lack of sufficient energy supply. This paper will stimulate work on this important subject.
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Reviewer #1 (Public Review):
This manuscript provides the first cellular analysis of how neuronal activity in axons (in this case the optic nerve) regulates the diameter of nearby blood vessels and hence the energy supply to neuronal axons and their associated cells. This is an important subject because, in a variety of neurological disorders, there is damage to the white matter that may result from a lack of sufficient energy supply, and this paper will stimulate work on this important subject.
Axonal spiking is suggested to release glutamate which activates NMDA receptors on myelin-making oligodendrocytes wrapped around the axons: the oligodendrocytes - either directly or indirectly via astrocytes - then generate prostaglandin E2 which relaxes pericytes on capillaries, thus decreasing the resistance of the vascular bed and …
Reviewer #1 (Public Review):
This manuscript provides the first cellular analysis of how neuronal activity in axons (in this case the optic nerve) regulates the diameter of nearby blood vessels and hence the energy supply to neuronal axons and their associated cells. This is an important subject because, in a variety of neurological disorders, there is damage to the white matter that may result from a lack of sufficient energy supply, and this paper will stimulate work on this important subject.
Axonal spiking is suggested to release glutamate which activates NMDA receptors on myelin-making oligodendrocytes wrapped around the axons: the oligodendrocytes - either directly or indirectly via astrocytes - then generate prostaglandin E2 which relaxes pericytes on capillaries, thus decreasing the resistance of the vascular bed and (presumably) increasing blood flow in the nerve.
Strengths of the paper
The paper identifies some important characteristics of axon-vascular coupling, notably its slow temporal development and long-lasting nature, the involvement of PgE2 in an oxygen-dependent manner, and a role for NMDARs. Rigorous criteria (constriction and dilation of capillaries by pharmacological agents) are used to select functioning pericytes for analysis.
Weaknesses of the paper
The study focuses exclusively on pericytes. It would have been interesting to assess whether arteriolar SMCs also contribute to regulating blood flow.
The slow (~10 minutes) time scale of the responses seen is remarkable when compared to grey matter neurovascular coupling which occurs in seconds. The authors suggest that this reflects movement of messengers along the nerve, but the action potential moves so rapidly down the nerve that it will activate the release of vasodilators essentially at the same time everywhere along the nerve, and there will be no concentration (or voltage) gradient to drive such a movement of messengers (unless there is a spatially localized unique site in the vascular bed where propagating responses are generated). Thus it remains unclear why the responses are so slow.
The activity-evoked dilation is thought to reflect PgE2 release at what is probably a physiological O2 concentration, but not at the hyperoxic 95% O2 used in most of the experiments. The involvement of NMDA receptors is apparently only shown (using OL-specific receptor subunit deletion) at the unphysiological high O2 level. This raises two questions: are NMDA receptors also involved in the response at low O2 (as schematized in Fig 6), and what is the messenger downstream of NMDARs at high O2 (NO would be an obvious candidate, previously shown to contribute to NVC in the grey matter).
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Reviewer #2 (Public Review):
This paper describes a new concept of "axe-vascular coupling" whereby action potential traffic along white matter axons induces vasodilation in the mouse optic nerve. This is an initial report dissecting some of the mechanisms that are undoubtedly complex as in gray matter NVC. I like the novel AVC concept.
Some minor corrections and suggestions:
p3: "The cerebral white matter (WM) in the adult brain is particularly vulnerable to cerebrovascular diseases such as ischemia":this may be misleading since WM is actually far less vulnerable to ischemia than gray matter
p4-5: "The ON exhibited a median of 175.8 pc/mm2 {plus minus} 35.7 pc/mm2, more than twice the number of pericytes observed in the corpus callosum [...] and lower than cortex ": this seems incorrect, the density in cortex is not significantly …
Reviewer #2 (Public Review):
This paper describes a new concept of "axe-vascular coupling" whereby action potential traffic along white matter axons induces vasodilation in the mouse optic nerve. This is an initial report dissecting some of the mechanisms that are undoubtedly complex as in gray matter NVC. I like the novel AVC concept.
Some minor corrections and suggestions:
p3: "The cerebral white matter (WM) in the adult brain is particularly vulnerable to cerebrovascular diseases such as ischemia":this may be misleading since WM is actually far less vulnerable to ischemia than gray matter
p4-5: "The ON exhibited a median of 175.8 pc/mm2 {plus minus} 35.7 pc/mm2, more than twice the number of pericytes observed in the corpus callosum [...] and lower than cortex ": this seems incorrect, the density in cortex is not significantly different than ON
p5: what is the unit 'pc'? (A cellI I presume but please define at first use)
p7 : "To evaluate if pericytes have and retain their contractile properties, we applied the vasoconstrictor U46619 (100 nM) for 15 min followed by acetylcholine (ACh - 100 μM) as a vasodilator": if they saw an effect, how would the authors know these were mediated my pericytes and not smooth muscle cells?
in Fig. 3i there is a sharp step after U466... application: is this an artifact or evidence of a delayed constriction? Could a clearer trace be shown that does not confuse?
Fig. 4I: what does "20% CAP (norm)" mean? Why not just mV for the y-axis? Also what pulse width was used for stimulation?
Fig.5: it would be good to show both the CAPs (at various frequencies) and the vasorespones at 95% vs 20% O2. In particular, are the ONs able to sustain conduction at the higher frequencies (showing overlays as in 4I), and if not, could this at least partially account for the different responses at the two O2 levels?
Fig. 6G,H is somewhat misleading as it implies no change in AVC, at odds with 6E. Suggest some clearer labeling to reduce confusion surrounding this very important point.
P18 authors state radius of a MON is 150um but on p4 they say "150 μm - 200 μm thickness", pls clarify.
p19-20: as part of their second messenger speculation authors may also want to include NO that has been shown to induce important effects in WM. Indeed, testing the tat uncoupling peptides could be interesting to see of oligodendroglial NMDARs have a similar singling arrangement with NOS as do neurons. This may have important implications for WM neuroprotective strategies in stroke that have typically focused on gray matter mechanisms.
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Reviewer #3 (Public Review):
This study used the ex vivo optic nerve preparation from adult mice to examine the organization of blood vessels and the mechanisms or neurovascular coupling (NVC). Strengths of the study include the benefits of the isolated preparation, which allows visualization of vessels and pericytes with high resolution and control over axonal activity and the extracellular environment, and the elegant analyses performed. Imaging at high resolution is critical, because vessel diameter changes can be small and slow to develop. The authors leverage this preparation to define the organization of blood vessels and pericytes in the nerve. They then examine the extent of NVC, showing that some aspects appear to be distinct. In particular, dilation does not present rapidly (over minutes) during axon stimulation, but rather …
Reviewer #3 (Public Review):
This study used the ex vivo optic nerve preparation from adult mice to examine the organization of blood vessels and the mechanisms or neurovascular coupling (NVC). Strengths of the study include the benefits of the isolated preparation, which allows visualization of vessels and pericytes with high resolution and control over axonal activity and the extracellular environment, and the elegant analyses performed. Imaging at high resolution is critical, because vessel diameter changes can be small and slow to develop. The authors leverage this preparation to define the organization of blood vessels and pericytes in the nerve. They then examine the extent of NVC, showing that some aspects appear to be distinct. In particular, dilation does not present rapidly (over minutes) during axon stimulation, but rather emerges after the stimulation, increasing progressively over tens of minutes. It is similarly dependent on oligodendrocyte NMDARs and prostaglandin E4 receptors, but the latter only appears to be engaged during low oxygen conditions. There are several notable limitations of these studies. Less is known about NVC in the intact optic nerve, so it is unclear how well this preparation mimics the in vivo environment. All studies of NVC were performed in the presence of U46619 (an agonist of prostaglandin H2 receptors) to pre-constrict the vessels, which may interfere with NVC. The degree of vessel change was small and slow to develop, and the magnitude and timecourse of the dilation were not closely linked to the stimulation frequency, raising concerns about tissue stability and cell viability. Finally, the studies examined the role of oligodendrocyte NMDARs in NVC using a conditional gene knockout strategy to inactive the NR1 subunit in these cells. To control for possible developmental effects, additional studies could be performed using acute application of NMDAR antagonists, as this preparation contains only neuronal axons, and a further analysis of vessel structure and pericyte organization should be performed using the methodologies developed to characterize their properties in control nerves. Importantly, extracellular stimulation of the nerve, which triggers near simultaneous activation of axons may not mimic activity patterns in these nerves that occur during vision.
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