Tripartite Mechanism of Neural Memory: Proof-of-Concept with Neuromimetic Impedence Electrodes

The idea that neural signaling is the basis of mental processes has a long history. We graphically summarize salient developments in the neurobiology of signaling, as a Timeline. In particular, we review the “tripartite mechanism” of neural memory, which centers on the interactions between a neuron with its surrounding extracellular matrix (nECM) doped with metals and neurotransmitters (NTs). Essentially, the neuron employs the nECM as its “memory material”, wherein it uses dopants to encode cognitive units of information (termed “cuinfo”). The NTs, which elicit bodily reactions (feelings), also encode past feelings as emotions, which “color” mental states in real-time and in memory. In the interest of developing experimental tests of the tripartite mechanism, impedance glass electrodes were covalently coated with an exemplar NT (oxytocin) or a sulfated tetra-saccharide analog of the nECM, were constructed and tested. The two types of coated, neuro-mimetic electrodes, termed “neulectrodes”, were capable of detecting metals, such as Hg, Pb, Cd, Cu, and Zn with very high selectivity and sensitivity. The “neulectrodes” demonstrated that the chemodynamic interactions of metal cations with NTs or nECM-saccharide analogues can translate into electrodynamic signals. They experimentally validate the concept of the tripartite mechanism that underlies the chemo-electric encoding of neural memory.


Background
Neurons are chemo-and electro-dynamically linked cells that express a talent of mentation and somehow encode cognitive information as the basis for neural memory. But details are lacking regarding the physicality of the neural code and where the memory trace (engram) is located. Many presume that memory is somehow stored in the synaptic gaps between neurons (Cajal, 1911;Hebbs, 1949  Some researchers developed nano-or microdevices to enable simultaneous, long-term, multi-site, intracellular electrical recordings from single or many neurons (Spira & Hai, 2013). While they explored the electrophysiologic aspects of synaptic signaling using sensing electrodes, they did not address the issue of the mental states achieved by neurons. Other workers suggested that memory is stored in the nucleus (Kandel, 2001;Josselyn & Frankland, 2015), though the kinetics of nuclear processes appear to be too slow and require high energy.
The computer metaphor for neural memory exerts a strong influence on the field of cognitive neurobiology. Many see a direct parallel between a computer and the brain (Turing, 1943;McCulloch & Pitts, 1943;von Neumann, 1958;Arbib, 1987Arbib, , 2000Piccinini G. 2006;Giudolin et al, 2011).
"Computational systems are useful to describe brain processes mathematically".
-Giudolin et al, 2011 But the metaphor is incomplete. Aside from the quite different energy expenditures by a brain compared to a supercomputer (i.e. 20 Watts vs 250 megaWatts), it does not account for emotive states achieved by neural systems, for which there are no digital or electrodynamic equivalents.
The idea of neural signaling as the basis of mental processes has a long history beginning with Galvani through Golgi, Cajal, Hebb and Kandel, as we graphically summarize in the Timeline presented below ( Figure  1). How is an emotive mental state encoded, stored and recalled from memory?

Tripartite mechanism of neural memory
We have proposed a tripartite mechanism for neural memory based on the formation of metal-centered complexes that attract neurotransmitters (NTs) (Marx & Gilon, 2012. Neurotransmitters (NTs) elicit psychic states parallel to physiologic reactions. Chemically, NTs bind to the metals anchored in the nECM. Effectively, they combine to establish the neuron's code for emotive states ( Figure 2).

Figure 2.
Icons of an electron-rich nECM metal-confinement "address", capable of complexing with metals and neurotransmitters (NTs), thereby encoding a cognitive unit of information (cuinfo). Each NT endows the cuinfo (singular & plural) with an emotive quality not available in binary code.
To encapsulate: The "neuron" marshals the components available to it, notably the surrounding extracellular matrix (nECM) and the dopants (metals and NTs) which the neuron accumulates within vesicles. The neuron employs these to encode cognitive units of information, called "cuinfo", metal-centered complexes within the nECM, described by a chemographic notation ( Figure 2). This mechanism is universal, in that it applies to the recall function of all neural creatures, from C. elegans (302 neurons) to homo sapien (10 15 neurons).
The term "chemotronic" refers to an intersection disciplines, such as those of chemistry with electronics or optics (see Khrustalev & Rozhitskii, 2001). Here, we propose that memory links biochemical processes to psychological states (i.e. emotions). For example, hydrogels have been studied as structural entities, in terms of shear thinning, stretching, selfhealing and breaking strength. (Zhang & Khadehosseini, 2017), but have not been studied for "chemotronic" signaling potential for encoding "psycho-chemical" states.
In this regard, consider the nECM as a 3-D hydrogel comprising a lattice of sulfated glycosamino-glycans (GAGs) (such as hyaluronate, chondroitins and heparans) which bind metal cations. The degree of sulfation of the GAG's is a major factor impinging on its metal binding affinity. Though a number of sulfation-enzymes have been identified

Results and Discussion
Following the idea of McCulloch & Pitts (Figure 1) that described a single neutron in mathematical terms, we propose that the impedence electrode is a model for a chemo-electric neural receptor. Inspired by these ideas, our colleagues embarked on a program to fabricate various neuromimetic impedence electrodes ("neulectrodes") coated with materials available to the neuron, namely a neurotransmitter (oxytocin) or a sulfated tetrasaccharide analogue of the nECM. These electrodes could be tested for their binding affinity and selectivity to different metal cations.
We review two published examples that use Electrochemical Impedance Spectroscopy (EIS) as a method to detect changes in the electrochemical properties of coatings of "tripartite" components. We call such neuromimetic electrodes, "neulectrodes". EIS is highly sensitive to interactions of the electrode sensing layer with analytes, especially to conformational changes that result from metal binding to the surface. It does not depend on fluorescence, luminescence or light absorbance techniques frequently used to study metal binding by biopolymers.

"Neulectrode" type 1: NT-coated impedance sensor
An impedence electrode was modified in step-wise manner to covalently coat the sensing surface with oxytocin (OT), as schematically illustrated in Figure 3. Each neural sensor has a unique specificity for a given NT ligand (Neumann, 2007). Thus, the OT-coated electrode, could be described as "neuro-mimetic" and might become diagnostically useful in a clinical setting to test for trace Cu +2 and Zn +2 in the circulating blood or lymph fluid of patients with multiple sclerosis, Alzheimer disorder, Parkinson disorder and autism.

"Neulectrode" type 2: Sulfated oligo-Saccharide coated impedance sensor
Uniquely sulfated tetra-saccharides were prepared (Table 1)  . While hyaluron is not generally sulfated, its tetra-saccharides serves as a good analogue of the generally sulfated chondroitins (glucosamine N-Ac-Glucuronic acid; GluNAc-GlcA)n (n≥ 2). The process for covalently coating the electrode with the tetra-hyluron azides (Table 1) was similar to that developed for oxytocin (Figure 2), using tetra-hyluron azides instead of oxytocin azide (Alshanski et al, 2019).  The high sensitivity of the electrode coated with sulfated HA4-GCE to metals such as Hg +2 , Pb +2 , and Cd +2 depended on the degree of sulfation, indicating that the interactions of sulfated oligo-saccharides with metal ions, especially heavy metal ones, are dictated by the sulfation patterns rather than by the core saccharide. Moreover, it corroborates our hypothesis that the sulfation pattern of the GAGs in the nECM determines the pattern of the metal distribution that is used for coding the neural memory. We hypothesized that NT (like oxytocin) could form ternary complexes with the metals embedded in the sulfated GAGs in the nECM, to be used for coding emotive memory. We also hypothesized that many thousands of sensors (i.e., GPCR, K channels, receptors, integrins), presented by the neural surface serve to "read" the cognitive information encoded by the metal -NT complexes in the nECM around the neurons.

Conclusions
The operational details of neural memory remain the focus of intense theoretical, modeling, basic and applied research. We have proposed that cognitive information received by the neuron is encoded as metal-centered complexes in the nECM surrounding the neurons. This implies that the neuron can form defined patterns of dopant complexes (metals and NTs) in the nECM. Moreover, it is capable of chemodynamic sensing such a pattern of metalcentered complexes and translating this information into electrodynamic signaling to the neural circuit ( Figure 5).

Figure 5.
Conceptual model of a synthetic neuron with a variety of "neulectrodes" embedded in a lipid membrane. The sensory output of all would be directed to an electronic central processing circuit (CPU), permitting a brain-computer interface.
The "neulectrodes" are interesting from both fundamental and technological perspectives. They are truly neuro-mimetic in that they employ components available to the neuron. They may be useful as diagnostic tools or as implants for monitoring or controlling neural activity.
The two types of "neulectrodes" reviewed here demonstrate chemoelectric effects. Namely, that the chemo-dynamic interactions of metal cations with NTs or sulfated analogs of the nECM GAG can translate into electrodynamic signals. They experimentally validate the concept of the tripartite mechanism of neural memory. We are pleased that we inspired and instigated this work by the research groups headed by Professor Shlomo Yitzchaik and Dr. Mattan Hurevich (Hebrew University) that fabricated and studied the coated electrodes, cited and discussed here.

Conflict of Interest
GM is a founder of MX Biotech Ltd., with the commercial goal to develop new classes of "memory materials" and devices.
CG is emeritus professor at the Institute of Chemistry, The Hebrew University of Jerusalem. He is active in developing technologies for the conversion of peptides and active regions of proteins into orally available drug leads.
This work did not receive any external funding support. Notwithstanding, the ideas forwarded here are scientifically genuine and presented in good faith, without commercial clouding of the concepts expressed herein.