New signaling mechanism in catecholaminergic neurons

Ferritin has electrical properties that make it both useful in engineered electronic components and intriguing with its presence in the mammalian brain. A novel function of biological ferritin has been proposed for neural processing in catecholaminergic neurons. The presence of ferritin and neuromelanin in catecholaminergic neurons may allow electron tunneling or similar functions to be used in neural processing. Electron tunneling behavior can be observed over distances as great as 12 nm through individual ferritin particles (1) and even long-distance tunneling (> 80 microns) has been observed in ferritin structures similar to those found in these tissues (2). Evidence of tunneling associated with cellular ferritin is found in the retina, the cochlea, macrophages, mitochondria and other tissues (3). Electron tunneling can also be induced in fixed human SNc tissue (4), suggesting that ferritin mediated electron tunneling may be widely-occurring phenomenon in cellular tissues. Together these findings are consistent with the hypothesis that neuronal ferritin may modulate or mediate novel forms of neural processing in vivo. If so, there is a potential to better understand this novel function in neuronal circuits and explore the potential for developing ferritin-optimized therapies and diagnostics. For example, it would not be difficult to test live neural tissue to determine whether electron tunneling in ferritin is present using quantum dot or nanodiamond fluorophores. The proposed test would involve using fluorophores that are conjugated with anti-ferritin on live rodent neural tissue slices to look for emitted photons caused by tunneling electrons. A negative result would indicate that the hypothesized intercellular communications mechanism is not present. A positive test would provide justification for further testing, such as with polymer-fiber-coupled field-effect sensors (5) or other suitable methods. While ferritin-mediated neural processing may seem a bold hypothesis, the opportunities to test for its presence are relatively simple… And the opportunities to optimize therapies and brain sensing based upon ferritin tunneling are immense.

  1. Rourk, Christopher J. Ferritin and neuromelanin “quantum dot” array structures in dopamine neurons of the substantia nigra pars compacta and norepinephrine neurons of the locus coeruleus." Biosystems 171 (2018): 48-58
  2. Rourk, Christopher, et al. Indication of Strongly Correlated Electron Transport and Mott Insulator in Disordered Multilayer Ferritin Structures (DMFS). Materials 14.16 (2021): 4527.
  3. Perez, Ismael Diez, et al. Electron tunneling in ferritin and associated biosystems. IEEE Transactions on Molecular, Biological and Multi-Scale Communications (2023).
  4. Rourk,Christopher J. Indication of quantum mechanical electron transport in human substantia nigra tissue from conductive atomic force microscopy analysis. Biosystems 179 (2019): 30-38
  5. Guo, Yuanyuan, et al. “Polymer-fiber-coupled field-effect sensors for label-free deep brain recordings.” Plos one 15.1 (2020): e0228076)

As I understand, your hypothesis is that there are neuronal pathways between neurons that involve Ferritin. So Iron ions inside those complex would act as charges pathways?
To test we would need fluorophores that are specific to those Ferritin proteins and would change fluorescence upon voltage changes around those proteins. Is that a correct summary?

The pathway involves electron tunneling as opposed to the movement of ions. Figs. 6 (a,b) of reference 3) shows examples of the ferritin structures in macrophages and SNc glial cells (which are both phagoctyes) that have been shown to be capable of sequential tunneling over distances as great as 80 microns in reference (2). The electrons have been shown to cause release of Fe2+, which influences iron homeostasis in a way that has been shown to result in calcium release (see e.g. Hidalgo, Cecilia, and Marco T. Núñez. “Calcium, iron and neuronal function.” IUBMB life 59.4‐5 (2007): 280-285), which contributes to action potential generation in certain large SNc neurons. The fluorophores should be able to detect electron tunneling if they are close enough to the ferritin to be involved in the tunneling process. This brief summary leaves out a lot of details, of course.

In case you are unfamiliar with electron tunneling through proteins, I ran a search in Google scholar and it returned ~12,900 papers. Some are rather old. I think there is a general bias against anything dealing with quantum mechanics in neuroscience, at least that has been my experience.

First, no bias here.
All questions are good. Good science can come from all places.

Are these fluorophores developed? Developing new sensors can be quite a complex task. OpenScope is well positioned to test things but I don’t know if we can develop new sensors. Can you just give a little more background on what those fluorophores are? Has there been in vivo use of these previously?

QD fluorophores tagged to ferritin can detect electron tunneling, based on some preliminary proof of concept work that was done for me by Dr. Suman Singh using the amine-functionalized graphene quantum dots reported in her paper - Garg, Mayank, et al. “Amine-functionalized graphene quantum dots for fluorescence-based immunosensing of ferritin.” ACS Applied Nano Materials 4.7 (2021): 7416-7425. QD fluorophores conjugated to anti-ferritin can be purchased from various suppliers, such as Thermo-Fisher. Nanodiamond fluorophores conjugated to anti-ferritin should also work and are also commercially available.

I just checked your 2018 review. I wonder if testing that component first would be easier in brain slices as a first step. In vivo experiments (as those we do for OpenScope currently) might not be entirely conclusive on their own and you will need to provide ample controls in slice to support the effect. Even calcium reporters are still heavily debated, despite decades of efforts in validation. Have you consider that option?

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Yes - I looked into OpenScope at the suggestion of a neuroscience professor who I met at a conference, but brain slices would be a good first step. I discussed a brain slice project with an electrophysiologist who does research on SNc neurons at UTSW, and he said he would need $3 million to do that, which is well beyond my ability to fund! So, I am looking for research that will not cost me that much. The UTSW researcher spent some time with me looking for a professor at a university who would agree to be a principal investigator on an NSF/NIH grant application, but while we found a lot of people who were interested in working on the project, nobody felt comfortable or had the time to be a PI. I have pretty much given up on that route until I find someone who is doing similar research. I am instead pursuing a deep brain stimulation project to see if the unusual electrical properties of the SNc tissue that I observed in my CAFM tests can be detected with a standard DBS probe, and if so, used to improve treatment outcomes. That has a much greater commercial potential than tissue slice tests and could potentially allow me to fund tissue slice testing on my own. However, even if that DBS project is successful, it will not shed much light on how the neurons are using that mechanism. If you have any recommendations on someone who might be interested in tissue slice testing, I would appreciate that information!

So it is hard for me to speak for them, but I think you are trying to develop a new sensor here. So you could get more interest among labs that are developing new biosensors. Many of these already have pipelines in place.

Andre Berndt comes to mind Andre Berndt | UW Bioengineering
If you are willing to provide support to test components on their system, they could be interested.

Thanks, I reached out to Prof. Berndt last Friday but he has not responded. The odds are about 20:1 against a response to an unsolicited email, based on my experience.

I would be interested in your thoughts on the 2018 review - I have been thinking about updating it to include my subsequent research that provides evidence of numerous predictions that were made in it, such as 1) the prediction that macroscopic electron tunneling would be detected in SNc tissue, 2) evidence that long-distance electron tunneling occurs through ferritin structures, 3) evidence of a switching mechanism that is provided by ferritin structures, and 4) evidence that the SNc mediates action selection, as shown from research by Prof. Pascal Kaeser at Harvard Medical School (Liu, Changliang, Pragya Goel, and Pascal S. Kaeser. “Spatial and temporal scales of dopamine transmission.” Nature Reviews Neuroscience 22.6 (2021): 345-358). I suspect that most neuroscientists just dismiss the 2018 paper as being Orchestrated Objective Reduction or one of the other “quantum consciousness” ideas, based on feedback I have received from such reviewers during peer review (after I explained why it is unrelated to those ideas, they changed their recommendation from “do not publish” to “publish.”) It seems like a hypothesis that makes accurate predictions should be given some consideration, but my experience is that many people just dismiss it out of hand. I can’t say I blame them - there are a lot of whacky “quantum consciousness” ideas, but this is not one of them.

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So I am very interested in consciousness questions and this debate is very strong in our community. I am not an expert by any mean on it. Perhaps the core of the debate is a lack of agreed upon strong set of definitions of what a neuronal correlate of consciousness is. There are ideas and hypothesis but those have not gained complete community agreement as far as I can tell. I have seen hypothesis that starts from higher network levels, to mitochondria down to very fundamental levels of physics, like how matter is organized.
By contrast, neuronal pathways are very clearly defined and are usually testable at the lowest level, down to the chemistry and signaling of single molecules.
Making a jump between these two levels is very hard and I understand why this is being pushed back. Whatever is proposed should be testable with clearly defined experiments as you said. I would focus on establishing the lower levels pathways first if I were approaching this and let the consciousness debate mature independently of how it could be supported.

It’s not clear why electron tunneling through intracellular ferritin nanoparticles should be significant from a physiological point of view. Electrons flowing across membranes change the membrane potential, which then affects voltage sensitive channels, but electrons moving within the cell do not have a direct physiological significance. So a mechanism that allow electrons to move between ferritin structures is not obviously important for neuron function, other than potentially altering the intracellular resistance. It was not clear from the review how large an effect one expects that to be from a physical modeling perspective. Nonetheless, to show that tunneling was important, you need a way to quickly and reversibly interfere with tunneling. I think this basic form of necessity experiment is easier and a better first step before trying to measure ferritin tunneling in vivo.

Knockouts of ferritin will take a long time to translate into reduced protein levels, and we know iron is important for neuron metabolism so any effects on physiology could be attributed to that, so that’s not a great option. You would like a small molecule that you could show first in in vitro experiments blocks electron tunneling between ferritin particles by altering the electronic structure of the macromolecule, then show that it is tolerated by neurons that don’t have ferritin, then show that it changes the physiology of neurons that due have ferritin. This is akin to the first experiments showing that certain ion channels are necessary for the observations of certain physiological currents by developing and applying selective blockers of those channels. These are all basic cellular physiology questions that should addressed before trying to show its importance in higher level phenomena and manipulations that haven’t been verified to work via the hypothesized mechanisms.

Thanks - there are some researchers who are investigating action selection and its relationship to consciousness. Cognitive processing is certainly an important part of the function of the brain and action selection, but there is a lot of evidence that this occurs in numerous parallel processes that do not result in phenomenal consciousness of action selection until after action has been selected, see, e.g. Humphries, Mark D., and Kevin Gurney. “Making decisions in the dark basement of the brain: A look back at the GPR model of action selection and the basal ganglia.” Biological Cybernetics 115.4 (2021): 323-329. The phenomenon of readiness potential is well known, but there are many consciousness ideas that ignore the fact that we are not consciously aware of cognitive processing associated with an action selection until after that processing has generated signals that cause an action selection to be made. The action selection mechanism that results in a specific action has not been identified - the GPR model has identified ways that competing channels are blocked after an action is selected, see, e.g. Codol, Olivier, Paul L. Gribble, and Kevin N. Gurney. “Differential Dopamine Receptor-Dependent Sensitivity Improves the Switch Between Hard and Soft Selection in a Model of the Basal Ganglia.” Neural Computation 34.7 (2022): 1588-1615, but the mechanism is incapable of preventing seizures from multiple simultaneous selections. The neural communications mechanism that is hypothesized to be associated with the unusual concentrations of ferritin and neuromelanin in the SNc is a physical structure that could provide an action selection mechanism, as well as a binding mechanism for the different channels of cognitive signals and sensory inputs that are delivered to that structure and that relate to the phenomenal experience of consciousness. More work would be needed to determine whether it does and to explain how, but I have been unable to find a compelling explanation that anything else can provide those functions.

It is difficult to test the function of large SNc neurons, but Kaeser has done so and has found that they exhibit unusual action potential behavior that is also consistent with the 2018 hypothesis, see Liu, Changliang, et al. “An action potential initiation mechanism in distal axons for the control of dopamine release.” Science 375.6587 (2022): 1378-1385. The videos in the supplemental materials are particularly interesting. The ectopic action potentials observed by Kaeser are created by afferent cortical signals at striatal dendrites - this was predicted in my 2018 paper. Again, what prevents that mechanism from causing multiple simultaneous activations and seizures? The hypothesized signaling mechanism could do that.

As mentioned, electron tunneling through intracellular ferritin could result in iron release in large dopamine neuron soma, which could contribute to the generation of calcium release and calcium-induced calcium release, which is known to be involved with action potential generation in large SNc dopamine neurons. See e.g. Hidalgo, Cecilia, and Marco T. Núñez. “Calcium, iron and neuronal function.” IUBMB life 59.4‐5 (2007): 280-285 (cited above) and Riegel, Arthur C., and John T. Williams. “CRF facilitates calcium release from intracellular stores in midbrain dopamine neurons.” Neuron 57.4 (2008): 559-570. It would not be a rapid mechanism, which would help to prevent high frequency oscillations in the action selection mechanism and seizures.

The source of the electrons for the mechanism is hypothesized to be chemiexcitation resulting from dopamine metabolism and the interaction of the associated metabolites with ferritin and neuromelanin in the SNc neurons, see Brash, Douglas E., Leticia CP Goncalves, and Etelvino JH Bechara. “Chemiexcitation and its implications for disease.” Trends in molecular medicine 24.6 (2018): 527-541. Anesthetics like propofol are dopamine antagonists and interfere with dopamine metabolism, and it is also known that stimulation of the VTA (but not the SNc) can help to induce reanimation from propofol Solt, Ken, et al. “Electrical stimulation of the ventral tegmental area induces reanimation from general anesthesia.” Anesthesiology 121.2 (2014): 311-319.

Based on the structure of ferritin, electron tunneling appears to result from chiral induced spin selectivity and the interaction of the chiral ferritin proteins and the iron oxide core, see Koplovitz, Guy, et al. “Single domain 10 nm ferromagnetism imprinted on superparamagnetic nanoparticles using chiral molecules.” Small 15.1 (2019): 1804557. If so, then a small molecule that could block electron tunneling without destroying the physical structure of ferritin might not exist. In that case, it would be easier to interfere with the source of the electrons as opposed to the ferritin (and that has already been done with propofol, but there may be other molecules that interfere with dopamine metabolism that are not anesthetics). It is also possible that QD fluorophores would divert electron tunneling from transiting between soma, providing both evidence of tunneling and evidence that tunneling is associated with action selection.

In case it is of interest, I found a good example of how quantum tunneling is different from ionic interactions. In Fig. 1 of Broome, M. A., et al. “Mapping the chemical potential landscape of a triple quantum dot.” Physical Review B 94.5 (2016): 054314, the conductance of an arrangement of quantum dots as a function of voltage is shown. The quantum dots are “gates” of a single electron transistor (SET), which is like a switch that the gates turn on or off, and the colored graph shows how different voltages on the gates influence the change in current through the SET as a function of voltage across the SET. The dark blue lines at -45 degrees are where no current flows as a function of changes in the applied voltage across the SET.

If the gate voltages were chemical redox potentials, then there would be states where electrons are permitted to tunnel and states where they are not permitted. In a chemical reaction involving electron tunneling from ferritin, the situation is more complex in part because the distances between the ferritin electron donor and the chemical electron recipient are constantly changing, but the concept is the same – there will be potential states where electrons can tunnel and other states where tunneling is prohibited. There is also a probability component that is not shown in Fig. 1 of the paper, and that will effect reaction rates. The net effect is the same, though – when the electron moves the chemical reaction takes place (e.g. neutralization of an ROS). It looks like it could be a chemical reaction, but it makes no sense, like how ferritin is overexpressed in response to ROS that is not generated by Fe2+. By itself, ferritin should have no effect on such ROS reduction, but in conjunction with an antioxidant electron donor, it can receive the electron from the donor, store it for some period of time, and donate the electron to the ROS by tunneling when it is in the correct spatial position. There are more spatial positions for that reaction than for direct interaction between ROS and the antioxidant, so ferritin acts as a catalyst and improves the rate at which the antioxidants neutralize the ROS (that has been demonstrated, see Alkhateeb, Ahmed A., Bing Han, and James R. Connor. “Ferritin stimulates breast cancer cells through an iron-independent mechanism and is localized within tumor-associated macrophages.” Breast cancer research and treatment 137 (2013): 733-744. (“Moreover, this proliferative effect was independent of the iron content of ferritin and did not increase intracellular iron levels in cancer cells indicating a novel iron-independent function for this protein. Together, these findings suggest that the release of ferritin by infiltrating macrophages in breast tumors may represent an inflammatory effector mechanism by which ferritin directly stimulates tumorigenesis.”)). That is why macrophages evolved to provide ferritin to cells to help protect them from ROS. When the ferritin particles are close enough to each other, then electrons can tunnel between them, too.