Detection of changes in membrane potential by magnetic resonance imaging

Reviewer #1 (Public review):

Summary:

This paper examines changes in relaxation time (T1 and T2) and magnetization transfer parameters that occur in a model system and in vivo when cells or tissue are depolarized using an equimolar extracellular solution with different concentrations of the depolarizing ion K+. The motivation is to explain T2 changes that have previously been observed by the authors in an in vivo model with neural stimulation (DIANA) and to try provide a mechanism to explain those changes.

Strengths:

The authors argue that the use of various concentrations of KCL in the extracellular fluid depolarize or hyperpolarize the cell pellets used and that this change in membrane potential is the driving force for the T2 (and T1-supplementary material) changes observed. In particular, they report an increase in T2 with increasing KCL concentration in the extracellular fluid (ECF) of pellets of SH-SY5Y cells. To offset the increasing osmolarity of the ECF due to the increase in KCL, the NaCL molarity of the ECF is proportionally reduced. The authors measure the intracellular voltage using patch clamp recordings, which is a gold standard. With 80 mM of KCL in the ECF, a change in T2 of the cell pellets of ~10 ms is observed with the intracellular potential recorded as about -6 mv. A very large T1 increase of ~90 ms is reported under the same conditions. The PSR (ratio of hydrogen protons on macromolecules to free water) decreases by about 10% at this 80 mM KCL concentration. Similar results are seen in a Jurkat cell line and similar, but far smaller changes are observed in vivo, for a variety of reasons discussed. As a final control, T1 and T2 values are measured in the various equimolar KCL solutions. As expected, no significant changes in T1 and T2 of the ECF were observed for these concentrations.

Weaknesses:

While the concepts presented are interesting, and the actual experimental methods seem to be nicely executed, the conclusions are not supported by the data for a number of reasons. This is not to say that the data isn't consistent with the conclusions, but there are other controls not included that would be necessary to draw the conclusion that it is membrane potential that is driving these T1 and T2 changes. Unfortunately for these authors, similar experiments conducted in 2008 (Stroman et al. Magn. Reson. in Med. 59:700-706) found similar results (increased T2 with KCL) but with a different mechanism, that they provide definite proof for. This study was not referenced in the current work.

It is well established that cells swell/shrink upon depolarization/hyperpolarization. Cell swelling is accompanied by increased light transmittance in vivo, and this should be true in the pellet system as well. In a beautiful series of experiments, Stroman et al. (2008) showed in perfused brain slices that the cells swell upon equimolar KCL depolarization and the light transmittance increases. The time course of these changes is quite slow, of the order of many minutes, both for the T2-weighted MRI signal and for the light transmittance. Stroman et al. also show that hypoosmotic changes produce the exact same timecourse as the KCL depolarization changes (and vice versa for the hyperosmotic changes - which cause cell shrinkage). Their conclusion, therefore, was that cell swelling (not membrane potential) was the cause of the T2-weighted changes observed, and that these were relatively slow (on the scale of many minutes).

What are the implications for the current study? Well, for one, the authors cannot exclude cell swelling as the mechanism for T2 changes, as they have not measured that. It is however well established that cell swelling occurs during depolarization, so this is not in question. Water in the pelletized cells is in slow/intermediate exchange with the ECF, and the solutions for the two compartment relaxation model for this are well established (see Menon and Allen, Magn. Reson. in Med. 20:214-227 (1991). The T2 relaxation times should be multiexponential (see point (3) further below). The current work cannot exclude cell swelling as the mechanism for T2 changes (it is mentioned in the paper, but not dealt with). Water entering cells dilutes the protein structures, changes rotational correlation times of the proteins in the cell and is known to increase T2. The PSR confirms that this is indeed happening, so the data in this work is completely consistent with the Stroman work and completely consistent with cell swelling associated with depolarization. The authors should have performed light scattering studies to demonstrate the presence or absence of cell swelling. Measuring intracellular potential is not enough to clarify the mechanism.

So why does it matter whether the mechanism is cell swelling or membrane potential? The reason is response time. Cell swelling due to depolarization is a slow process, slower than hemodynamic responses that characterize BOLD. In fact, cell swelling under normal homeostatic conditions in vivo is virtually non-existent. Only sustained depolarization events typically associated with non-naturalistic stimuli or brain dysfunction produce cell swelling. Membrane potential changes associated with neural activity, on the other hand, are very fast. In this manuscript, the authors have convincingly shown a signal change that is virtually the same as what was seen in the Stroman publication, but they have not shown that there is a response that can be detected with anything approaching the timescale of an action potential. So one cannot definitely say that the changes observed are due to membrane potential. One can only say they are consistent with cell swelling, regardless of what causes the cell swelling.

For this mechanism to be relevant to explaining DIANA, one needs to show that the cell swelling changes occur within a millisecond, which has never been reported. If one knows the populations of ECF and pellet, the T2s of the ECF and pellet and the volume change of the cells in the pellet, one can model any expected T2 changes due to neuronal activity. I think one would find that these are minuscule within the context of an action potential, or even bulk action potential.

There are a few smaller issues that should be addressed.
(1) Why were complicated imaging sequences used to measure T1 and T2? On a Bruker system it should be possible to do very simple acquisitions with hard pulses (which will not need dictionaries and such to get quantitative numbers). Of course, this can only be done sample by sample and would take longer, but it avoids a lot of complication to correct the RF pulses used for imaging, which leads me to the 2nd point.
(2) Figure S1 (H) is unlike any exponential T2 decay I have seen in almost 40 years of making T2 measurements. The strange plateau at the beginning and the bump around TE = 25 ms are odd. These could just be noise, but the fitted curve exactly reproduces these features. A monoexponential T2 decay cannot, by definition, produce a fit shaped like this.
(3) As noted earlier, layered samples produce biexponential T2 decays and monoexponential T1 decays. I don't quite see how this was accounted for in the fitting of the data from the pellet preparations. I realize that these are spatially resolved measurements, but the imaging slice shown seems to be at the boundary of the pellet and the extracellular media and there definitely should be a biexponential water proton decay curve. Only 5 echo times were used, so this is part of the problem, but it does mean that the T2 reported is a population fraction weighted average of the T2 in the two compartments.
(4) Delta T1 and T2 values are presented for the pellets in wells, but no absolute values are presented for either the pellets or the KCL solutions that I could find.

Reviewer #2 (Public review):

Summary:

Min et al. attempt to demonstrate that magnetic resonance imaging (MRI) can detect changes in neuronal membrane potentials. They approach this goal by studying how MRI contrast and cellular potentials together respond to treatment of cultured cells with ionic solutions. The authors specifically study two MRI-based measurements: (A) the transverse (T2) relaxation rate, which reflects microscopic magnetic fields caused by solutes and biological structures; and (B) the fraction or "pool size ratio" (PSR) of water molecules estimated to be bound to macromolecules, using an MRI technique called magnetization transfer (MT) imaging. They see that depolarizing K+ and Ba2+ concentrations lead to T2 increases and PSR decreases that vary approximately linearly with voltage in a neuroblastoma cell line and that change similarly in a second cell type. They also show that depolarizing potassium concentrations evoke reversible T2 increases in rat brains and that these changes are reversed when potassium is renormalized. Min et al. argue that this implies that membrane potential changes cause the MRI effects, providing a potential basis for detecting cellular voltages by noninvasive imaging. If this were true, it would help validate a recent paper published by some of the authors (Toi et al., Science 378:160-8, 2022), in which they claimed to be able to detect millisecond-scale neuronal responses by MRI.

Strengths:

The discovery of a mechanism for relating cellular membrane potential to MRI contrast could yield an important means for studying functions of the nervous system. Achieving this has been a longstanding goal in the MRI community, but previous strategies have proven too weak or insufficiently reproducible for neuroscientific or clinical applications. The current paper suggests remarkably that one of the simplest and most widely used MRI contrast mechanisms-T2 weighted imaging-may indicate membrane potentials if measured in the absence of the hemodynamic signals that most functional MRI (fMRI) experiments rely on. The authors make their case using a diverse set of quantitative tests that include controls for ion and cell type-specificity of their in vitro results and reversibility of MRI changes observed in vivo.

Weaknesses:

The major weakness of the paper is that it uses correlational data to conclude that there is a causational relationship between membrane potential and MRI contrast. Alternative explanations that could explain the authors' findings are not adequately considered. Most notably, depolarizing ionic solutions can also induce changes in cellular volume and tissue structure that in turn alter MRI contrast properties similarly to the results shown here. For example, a study by Stroman et al. (Magn Reson Med 59:700-6, 2008) reported reversible potassium-dependent T2 increases in neural tissue that correlate closely with light scattering-based indications of cell swelling. Phi Van et al. (Sci Adv 10:eadl2034, 2024) showed that potassium addition to one of the cell lines used here likewise leads to cell size increases and T2 increases. Such effects could in principle account for Min et al.'s results, and indeed it is difficult to see how they would not contribute, but they occur on a time scale far too slow to yield useful indications of membrane potential. The authors' observation that PSR correlates negatively with T2 in their experiments is also consistent with this explanation, given the inverse relationship usually observed (and mechanistically expected) between these two parameters. If the authors could show a tight correspondence between millisecond-scale membrane potential changes and MRI contrast, their argument for a causal connection or a useful correlational relationship between membrane potential and image contrast would be much stronger. As it is, however, the article does not succeed in demonstrating that membrane potential changes can be detected by MRI.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This paper examines changes in relaxation time (T1 and T2) and magnetization transfer parameters that occur in a model system and in vivo when cells or tissue are depolarized using an equimolar extracellular solution with different concentrations of the depolarizing ion K+. The motivation is to explain T2 changes that have previously been observed by the authors in an in vivo model with neural stimulation (DIANA) and to try provide a mechanism to explain those changes.

Strengths:

The authors argue that the use of various concentrations of KCL in the extracellular fluid depolarize or hyperpolarize the cell pellets used and that this change in membrane potential is the driving force for the T2 (and T1-supplementary material) changes observed. In particular, they report an increase in T2 with increasing KCL concentration in the extracellular fluid (ECF) of pellets of SH-SY5Y cells. To offset the increasing osmolarity of the ECF due to the increase in KCL, the NaCL molarity of the ECF is proportionally reduced. The authors measure the intracellular voltage using patch clamp recordings, which is a gold standard. With 80 mM of KCL in the ECF, a change in T2 of the cell pellets of ~10 ms is observed with the intracellular potential recorded as about -6 mv. A very large T1 increase of ~90 ms is reported under the same conditions. The PSR (ratio of hydrogen protons on macromolecules to free water) decreases by about 10% at this 80 mM KCL concentration. Similar results are seen in a Jurkat cell line and similar, but far smaller changes are observed in vivo, for a variety of reasons discussed. As a final control, T1 and T2 values are measured in the various equimolar KCL solutions. As expected, no significant changes in T1 and T2 of the ECF were observed for these concentrations.

Weaknesses:

While the concepts presented are interesting, and the actual experimental methods seem to be nicely executed, the conclusions are not supported by the data for a number of reasons. This is not to say that the data isn't consistent with the conclusions, but there are other controls not included that would be necessary to draw the conclusion that it is membrane potential that is driving these T1 and T2 changes. Unfortunately for these authors, similar experiments conducted in 2008 (Stroman et al. Magn. Reson. in Med. 59:700-706) found similar results (increased T2 with KCL) but with a different mechanism, that they provide definite proof for. This study was not referenced in the current work.

It is well established that cells swell/shrink upon depolarization/hyperpolarization. Cell swelling is accompanied by increased light transmittance in vivo, and this should be true in the pellet system as well. In a beautiful series of experiments, Stroman et al. (2008) showed in perfused brain slices that the cells swell upon equimolar KCL depolarization and the light transmittance increases. The time course of these changes is quite slow, of the order of many minutes, both for the T2-weighted MRI signal and for the light transmittance. Stroman et al. also show that hypoosmotic changes produce the exact same timecourse as the KCL depolarization changes (and vice versa for the hyperosmotic changes - which cause cell shrinkage). Their conclusion, therefore, was that cell swelling (not membrane potential) was the cause of the T2-weighted changes observed, and that these were relatively slow (on the scale of many minutes).

What are the implications for the current study? Well, for one, the authors cannot exclude cell swelling as the mechanism for T2 changes, as they have not measured that. It is however well established that cell swelling occurs during depolarization, so this is not in question. Water in the pelletized cells is in slow/intermediate exchange with the ECF, and the solutions for the two compartment relaxation model for this are well established (see Menon and Allen, Magn. Reson. in Med. 20:214-227 (1991). The T2 relaxation times should be multiexponential (see point (3) further below). The current work cannot exclude cell swelling as the mechanism for T2 changes (it is mentioned in the paper, but not dealt with). Water entering cells dilutes the protein structures, changes rotational correlation times of the proteins in the cell and is known to increase T2. The PSR confirms that this is indeed happening, so the data in this work is completely consistent with the Stroman work and completely consistent with cell swelling associated with depolarization. The authors should have performed light scattering studies to demonstrate the presence or absence of cell swelling. Measuring intracellular potential is not enough to clarify the mechanism.

We appreciate the reviewer’s comments. We agree that changes in cell volume due to depolarization and hyperpolarization significantly contribute to the observed changes in T2, PSR, and T1, especially in pelletized cells. For this reason, we already noted in the Discussion section of the original manuscript that cell volume changes influence the observed MR parameter changes, though this study did not present the magnitude of the cell volume changes. In this regard, we thank the reviewer for introducing the work by Stroman et al. (Magn Reson Med 59:700-706, 2008). When discussing the contribution of the cell volume changes to the observed MR parameter changes, we will additionally discuss the work of Stroman et al. in the revised manuscript.

In addition, we acknowledge that the title and main conclusion of the original manuscript may be misleading, as we did not separately consider the effect of cell volume changes on MR parameters. To more accurately reflect the scope and results of this study and to consider the reviewer 2’s suggestion, we will adjust the title to “Responses to membrane potential-modulating ionic solutions measured by magnetic resonance imaging of cultured cells and in vivo rat cortex” and will also revise the relevant phrases in the main text.

Finally, when [K+]-induced membrane potential changes are involved, there seems to be factors other than cell volume changes also appear to influence T2 changes. Our ongoing study shows that there are differences in T2 changes (for the same volume changes) between two different situations: pure osmotic volume changes vs. [K+]-induced volume changes (e.g., hypoosmotic vs. depolarization). Furthermore, this study suggests that mechanisms such as changes in free (primarily intracellular) and bound water within a voxel play an important role in generating this T2 difference. Our group is preparing a manuscript for this follow-up study and will report on it shortly.

So why does it matter whether the mechanism is cell swelling or membrane potential? The reason is response time. Cell swelling due to depolarization is a slow process, slower than hemodynamic responses that characterize BOLD. In fact, cell swelling under normal homeostatic conditions in vivo is virtually non-existent. Only sustained depolarization events typically associated with non-naturalistic stimuli or brain dysfunction produce cell swelling. Membrane potential changes associated with neural activity, on the other hand, are very fast. In this manuscript, the authors have convincingly shown a signal change that is virtually the same as what was seen in the Stroman publication, but they have not shown that there is a response that can be detected with anything approaching the timescale of an action potential. So one cannot definitely say that the changes observed are due to membrane potential. One can only say they are consistent with cell swelling, regardless of what causes the cell swelling.

For this mechanism to be relevant to explaining DIANA, one needs to show that the cell swelling changes occur within a millisecond, which has never been reported. If one knows the populations of ECF and pellet, the T2s of the ECF and pellet and the volume change of the cells in the pellet, one can model any expected T2 changes due to neuronal activity. I think one would find that these are minuscule within the context of an action potential, or even bulk action potential.

In the context of cell swelling occurring at rapid response times, if we define cell swelling simply as an “increase in cell volume,” there are several studies reporting transient structural (or volumetric) changes (e.g., ~nm diameter change over ~ms duration) in neuron cells during action potential propagation (Akkin et al., Biophys J 93:1347-1353, 2007; Kim et al., Biophys J 92:3122-3129, 2007; Lee et al., IEEE Trans Biomed Eng 58:3000-3003, 2011; Wnek et al., J Polym Sci Part B: Polym Phys 54:7-14, 2015; Yang et al., ACS Nano 12:4186-4193, 2018). These studies show a good correlation between membrane potential changes and cell volume changes (even if very small) at the cellular level within milliseconds.

As mentioned in the Response 1 above, this study does not address rapid dynamic membrane potential changes on the millisecond scale, which we explicitly discussed as one of the limitations in the Discussion section of the original manuscript. For this reason, we do not claim in this study that we provide the reader with definitive answers about the mechanisms involved in DIANA. Rather, as a first step toward addressing the mechanism of DIANA, this study confirms that there is a good correlation between changes in membrane potential and measurable MR parameters (e.g., T2 and PSR) when using ionic solutions that modulate membrane potential. Identifying T2 changes that occur during millisecond-scale membrane potential changes due to rapid neural activation will be further addressed in future studies.

There are a few smaller issues that should be addressed.

(1) Why were complicated imaging sequences used to measure T1 and T2? On a Bruker system it should be possible to do very simple acquisitions with hard pulses (which will not need dictionaries and such to get quantitative numbers). Of course, this can only be done sample by sample and would take longer, but it avoids a lot of complication to correct the RF pulses used for imaging, which leads me to the 2nd point.

We appreciate the reviewer’s suggestion regarding imaging sequences. We would like to clarify that dictionaries were used for fitting in vivo T2 decay data, not in vitro data. Sample-by-sample nonlocalized acquisition with hard pulses may be applicable for in vitro measurements. However, for in vivo measurements, a slice-selective multi-echo spin-echo sequence was necessary to acquire T2 maps within a reasonable scan time. Our choice of imaging sequence was guided by the need to spatially resolve MR signals from specific regions of interests while balancing scan time constraints.

(2) Figure S1 (H) is unlike any exponential T2 decay I have seen in almost 40 years of making T2 measurements. The strange plateau at the beginning and the bump around TE = 25 ms are odd. These could just be noise, but the fitted curve exactly reproduces these features. A monoexponential T2 decay cannot, by definition, produce a fit shaped like this.

The T2 decay curves in Figure S1(H) indeed display features that deviate from a simple monoexponential decay. In our in vivo experiments, we used a multi-echo spin-echo sequence with slice-selective excitation and refocusing pulses. In such sequences, the echo train is influenced by stimulated echoes and imperfect slice profiles. This phenomenon is inherent to the pulse sequence rather than being artifacts or fitting errors (Hennig, Concepts Magn Reson 3:125-143, 1991; Lebel and Wilman, Magn Reson Med 64:1005-1014, 2010; McPhee and Wilman, Magn Reson Med 77:2057-2065, 2017). Therefore, we fitted the T2 decay curve using the technique developed by McPhee and Wilman (2017).

(3) As noted earlier, layered samples produce biexponential T2 decays and monoexponential T1 decays. I don't quite see how this was accounted for in the fitting of the data from the pellet preparations. I realize that these are spatially resolved measurements, but the imaging slice shown seems to be at the boundary of the pellet and the extracellular media and there definitely should be a biexponential water proton decay curve. Only 5 echo times were used, so this is part of the problem, but it does mean that the T2 reported is a population fraction weighted average of the T2 in the two compartments.

We understand the reviewer’s concern regarding potential biexponential decay due to the presence of different compartments. In our experiments, we carefully positioned the imaging slice sufficiently remote from the pellet-media interface. This approach ensures that the signal predominantly arises from the cells (and interstitial fluid), excluding the influence of extracellular media above the cell pellet. We will clearly describe the imaging slice in the revised manuscript. As mentioned in our Methods section, for in vitro experiments, we repeated a single-echo spin-echo sequence with 50 difference echo times. While Figure 1C illustrates data from five echo times for visual clarity, the full dataset with all 50 echo times was used for fitting. We will clarify this point in the revised manuscript to avoid any misunderstanding.

(4) Delta T1 and T2 values are presented for the pellets in wells, but no absolute values are presented for either the pellets or the KCL solutions that I could find.

As requested by the reviewer, we will include the absolute values in the revised manuscript.

Reviewer #2 (Public review):

Summary:

Min et al. attempt to demonstrate that magnetic resonance imaging (MRI) can detect changes in neuronal membrane potentials. They approach this goal by studying how MRI contrast and cellular potentials together respond to treatment of cultured cells with ionic solutions. The authors specifically study two MRI-based measurements: (A) the transverse (T2) relaxation rate, which reflects microscopic magnetic fields caused by solutes and biological structures; and (B) the fraction or "pool size ratio" (PSR) of water molecules estimated to be bound to macromolecules, using an MRI technique called magnetization transfer (MT) imaging. They see that depolarizing K+ and Ba2+ concentrations lead to T2 increases and PSR decreases that vary approximately linearly with voltage in a neuroblastoma cell line and that change similarly in a second cell type. They also show that depolarizing potassium concentrations evoke reversible T2 increases in rat brains and that these changes are reversed when potassium is renormalized. Min et al. argue that this implies that membrane potential changes cause the MRI effects, providing a potential basis for detecting cellular voltages by noninvasive imaging. If this were true, it would help validate a recent paper published by some of the authors (Toi et al., Science 378:160-8, 2022), in which they claimed to be able to detect millisecond-scale neuronal responses by MRI.

Strengths:

The discovery of a mechanism for relating cellular membrane potential to MRI contrast could yield an important means for studying functions of the nervous system. Achieving this has been a longstanding goal in the MRI community, but previous strategies have proven too weak or insufficiently reproducible for neuroscientific or clinical applications. The current paper suggests remarkably that one of the simplest and most widely used MRI contrast mechanisms-T2 weighted imaging-may indicate membrane potentials if measured in the absence of the hemodynamic signals that most functional MRI (fMRI) experiments rely on. The authors make their case using a diverse set of quantitative tests that include controls for ion and cell type-specificity of their in vitro results and reversibility of MRI changes observed in vivo.

Weaknesses:

The major weakness of the paper is that it uses correlational data to conclude that there is a causational relationship between membrane potential and MRI contrast. Alternative explanations that could explain the authors' findings are not adequately considered. Most notably, depolarizing ionic solutions can also induce changes in cellular volume and tissue structure that in turn alter MRI contrast properties similarly to the results shown here. For example, a study by Stroman et al. (Magn Reson Med 59:700-6, 2008) reported reversible potassium-dependent T2 increases in neural tissue that correlate closely with light scattering-based indications of cell swelling. Phi Van et al. (Sci Adv 10:eadl2034, 2024) showed that potassium addition to one of the cell lines used here likewise leads to cell size increases and T2 increases. Such effects could in principle account for Min et al.'s results, and indeed it is difficult to see how they would not contribute, but they occur on a time scale far too slow to yield useful indications of membrane potential. The authors' observation that PSR correlates negatively with T2 in their experiments is also consistent with this explanation, given the inverse relationship usually observed (and mechanistically expected) between these two parameters. If the authors could show a tight correspondence between millisecond-scale membrane potential changes and MRI contrast, their argument for a causal connection or a useful correlational relationship between membrane potential and image contrast would be much stronger. As it is, however, the article does not succeed in demonstrating that membrane potential changes can be detected by MRI.

We appreciate the reviewer’s comments. We agree that changes in cell volume due to depolarization and hyperpolarization significantly contribute to the observed MR parameter changes. For this reason, we have already noted in the Discussion section of the original manuscript that cell volume changes influence the observed MR parameter changes. In this regard, we thank the reviewer for introducing the work by Stroman et al. (Magn Reson Med 59:700-706, 2008) and Phi Van et al. (Sci Adv 10:eadl2034, 2024). When discussing the contribution of the cell volume changes to the observed MR parameter changes, we will additionally discuss both work of Stroman et al. and Phi Van et al. in the revised manuscript.

In addition, this study does not address rapid dynamic membrane potential changes on the millisecond scale, which we explicitly discussed as one of the limitations of this study in the Discussion section of the original manuscript. For this reason, we do not claim in this study that we provide the reader with definitive answers about the mechanisms involved in DIANA. Rather, as a first step toward addressing the mechanism of DIANA, this study confirms that there is a good correlation between changes in membrane potential and measurable MR parameters (although on a slow time scale) when using ionic solutions that modulate membrane potential. Identifying T2 changes that occur during millisecond-scale membrane potential changes due to rapid neural activation will be further addressed in future studies.

Together, we acknowledge that the title and main conclusion of the original manuscript may be misleading. To more accurately reflect the scope and results of this study and to consider the reviewer’s suggestion, we will adjust the title to “Responses to membrane potential-modulating ionic solutions measured by magnetic resonance imaging of cultured cells and in vivo rat cortex” and will also revise the relevant phrases in the main text.