Brain Networks, the Matrix and the Mind

Nature does not seem to waste ideas. From the macrocosmos of the universe to themicrocosmos of the atom, everything appears to be comprised of matter and void as if this wasnature’s binary system. But the void is far from being empty or useless, in fact it is the theaterwhere atomic, electric or gravity forces interact, cementing the matter together.

The same principle is at work in biology: tissues are made of cells and the extracellular space.Again, the extracellular space is far from being empty or useless; it holds the tissue together,supports cellular communication and enables the function of organs. Likewise, the brain iscomprised of cells and the extracellular matrix (ECM). The ECM cements the organ together,supports signaling among cells and participates in engendering the mind.

The brain is made of cells and the extracellular matrix (ECM)

As we are getting well into the 21st century, it has become clearer that the mind is the productof the brain, just as the body movement is the product of the musculoskeletal system. With thesame token, it is clearer and clearer that psychiatric disorders are disruptions of cellular ormolecular communication in brain networks. In this context, studying the cellular cross-talkand connectivity in these networks offers the best modality of a brain-based understanding ofpsychiatric disorders.

Like any other organ, the brain can be currently studied at two levels of organization: cellularand molecular. These two realms follow different sets of rules, but complement each other ingenerating the mind.

Cellular Networks and the Neurovascular Unit (NVU)

In order to illustrate brain cellular networks, let’s take a stroll in a fascinating tropical forest. Aswe walk, we note the long, delicate and entangled branches stretching in every direction as faras we can see. The tree trunks are buzzing with activity as juices travel from the fertile groundto crowns far away. There is life and exuberance everywhere, the canopy is majestic, thick,knitted with intertwined branches that seem to be whispering to one another. The ground iswet because few sunbeams penetrate the narrow spaces between the entangled crowns. Thisforest is comprised of more than 100 billion neurons in addition to about as many glial cells,and you’d be surprised to learn that it fits in about 1200 cm3 of gelatinous matter, the brain(1).

The cellular level of tissue organization, is characterized by the “sovereignty” of the cellmembranes which establish cellular boundaries, connect cells into networks and preventspilling of intracellular content into the extracellular space.

In order to perform their job of producing the mind, the brain cells are organized in networks.Hebb named this architecture cell assemblies, and argued that repeated behavioral patternsstrengthen connections among cells in their corresponding assemblies, just like a frequentlyused hiking trail would eventually broaden. Hebb presumed that repetitive presynapticstimulation strengthens synapses (i.e. neurons that fire together wire together) (2).

At this point the analogy with the tropical forest needs to be broadened because the pictureneeds to accommodate about 600 km of brain microvessels composed of arterial and venouscapillaries accompanying each neuron at an average distance of 20 μm (3 ). Also large stellarcells, the astrocytes, need to be pictured with extensions that wrap the synapse and thecapillaries (4).

NVU, the building block of a complex cellular network comprised of neurons, glia and brain microvessels

Brain networks may be didactically divided into neuronal, glial or neuronal-glial networks,however practically such networks cannot exist without microvessels. Indeed, each brain cell isin immediate vicinity of an arterial and a venous capillary without which the networks could notbe functional. Therefore, all brain networks have three compartments: neuronal, glial andcapillary which render them complex cellular networks (CCN). In addition to their proximity toeach other, neurons, glia, endothelial cells of capillaries and pericytes engage in extensivecross-talk and together comprise the basic structure of information processing, theneurovascular unit (NVU).

Endothelial cells’ and pericytes’ cross-talk

The NVU is the basic building block of CCNs as well as the basic cellular assembly ofcomputation akin to a transistor. To illustrate the relationship of the NVU with CCNs let’spicture the CCN as a population of brain cells in which the NVU is a family. Likewise, to illustratethe same relationship in regards to computation, if the CCN is depicted as a microchip, the NVUwould represent a component transistor.

Hypothesis: the NVU, not the neuron, is the minimal cell assembly for information processingin the brain. It is hypothesized further that, within the NVU, all cells are involved ininformation processing.

Anatomically, the NVU can be described in terms of its component cells, howeverphysiologically, the NVU can be better comprehended as a whole. Likewise, a nephron, forexample can be anatomically discerned through its parts (i.e. glomerulus, Bowman’s capsuleand ducts), but its physiological function can be better grasped as a whole.

It is currently assumed that neuroimaging such as fMRI and BOLD reveal activation of neuronalnetworks. However, it is known that functional hyperemia (and oxygenated hemoglobin) do notcorrelate well with activation of neuronal networks (5)(6) (7) (8) (9) (10). Thus consideringneuronal networks activation in isolation from ECM, glial and vascular compartments should beavoided.

NVU- family as part of CCN population

On the other hand, if brain activation is fathomed as activation of CCNs comprised of numerousNVUs, this correlation can be positively established. The holistic understanding of the NVU as acompact assembly representing more than the sum of its cells can be discerned with moreprecision if examined from the molecular perspective.

Molecular Networks and the NVU

So far we have been strolling in the tropical forest by carefully stepping on the jungle floor,observing the trees, branches and crowns. It is time now to take an imaginary elevator onefloor down into the molecular realm and examine the nuts and bolts of life, the molecules. Ourdescent into the soil is even more fascinating, lo and behold the soil is alive, it is comprised ofintertwining roots (molecular networks) and ground water bathing them (ISF).

If the properties of matter could be summarized in one word, it would probably be “motion.”Indeed, matter and motion are always in tandem like the two faces of Janus. In biology, themolecules of life, the proteins, are endowed with motion of their subunits and conformationalchanges. One of the sine qua non aspects of life seems to be the indivisible marriage between

proteins conformational dynamics and their biological functions (11 ). Dynamic subunits ofmacromolecules can build on each other in “lego-like” fashion, self-assemble and disassemblein “Transformers’-like” manner, or fold and unfold like paper in the ancient Japanese art oforigami. In addition to their mechanical properties, or possibly because of them, proteins areendowed with electrical conductance (12)(13) and access to logicgates(14)(15)(16)(17)(18)(19)(20).

At the molecular level of brain organization we encounter a different world order in whichmolecular networks do not respect the boundaries of cell membranes, which themselves arecomprised of horizontal molecular networks (22). The proteins comprising the cellularcytoskeleton are known to assemble with membrane adhesion molecules such as integrins(23)(24)(25) which in turn bind ECM proteins generating global molecular networks (GMN)which crisscross the cells as well as the ECM, enmeshing the entire CNS (26).

The molecular networks should not be conceptualized as being static, since the ever-changingenvironment induces continuous fluctuations in the states of these molecules (i.e. adhesion vs.non-adhesion, assembly vs. disassembly, folding vs. unfolding). In the NVU those states arereflected in molecular switches that can turn “on” or “off” information processing in GMNs. Forexample when the integrin switch is “ON” adhesion is established between intra andextracellular molecular networks and the GMN is brought on-line. Subsequently, when thisswitch is “OFF”, there is loss of adhesion between intra and extracellular networks and theGMN is off-line.

Integrins link the intracellular and extracellular molecular networks into global molecular networks

Integrins are trans-membrane receptors composed of three domains: an intracellular domainwhich interacts with the cytoskeleton, a trans-membrane domain, and an extracellular domainthat interacts with the ECM macromolecules (27)(28). When a ligand binds to the cytoplasmicdomain, it causes elongation of the extracellular domain of the integrin molecule withsubsequent adhesion to ECM macromolecules (the switch is “ON”). Conversely, when a ligandbinds to the extracellular portion, the integrin shortens thus turning “OFF” the cytoskeleton-ECM adhesion (28)(29).

The molecular switching mechanisms endow the NVU with transistor-like access to Booleanlogic gates which are the building blocks of computation. Highly dynamic, shape-changingproteins like integrins or G-proteins are utilized as molecular switches throughout themolecular networks (30).

The switch aspect of proteins is not a new concept, indeed the epigenom consists of myriads ofswitches changing transcription status from activation to repression and vice versa in differentsets of genes without inducing changes of the underlying DNA sequence (31) .

A growing number of biophysical studies demonstrate how cytoskeletal macromolecules suchas actin filaments are able to act as genuine “electric cables” (32)(33). Both microtubules andactin filaments have highly charged surfaces that enable them to process both electric currentsand information (27) (28). In addition to conducting electronic signals, cytoskeletalmacromolecules respond to electromagnetic fields which may be able to induce structuralorganization of both actin filaments and microtubules (34)(35).

Information processing and decision making have been well documented in transcription-linkedmolecular networks, but recently it was demonstrated that individual proteins can performlogic operations as well (35). For example, performance of the logic gate AND by the actinregulatory protein N-WASP was described (36). Moreover, synthetic proteins based uponnaturally existing proteins have been constructed and shown to perform a number of differentlogic operations (37). Dendritic spines proteins were hypothesized to endow neuronal networkswith Boolean logic (38).

Like the skin, the brain derives from the ectoderm, and represents the interface between thebody and the unpredictable, ever-changing environment. Decreasing risk of injury and deathwas probably the driving force that led to the development of the mind. Making adequateplans, contingency plans and decisions in unpredictable situations was essential for survival.This is probably how the brain evolved the ability of “virtual reality”, that is creating a replica ofthe environment in its inner mental space where it could be studied, analyzed and a multitudeof risks, or reactions simultaneously assessed. Creating or disposing of the external worldmental image renders the mind is more a verb than a noun. The “virtual reality” aspect of themind allows enactment and evaluation of possible real-life situations, while eliminating theneed to live through each one of them. The mind enables other human attributes such asplanning, goal setting, assigning value to objects, individuals or ideas, and also findingfulfillment and meaning.

Some patterns of information processing may be transpersonal or species specific. It has beenknown that instead of being born “tabula rasa”, infants come prepared with built-in patterns ofinformation processing or archetypes, specific to human race (C.G. Jung ). Protein properties ofallostery, folding and conformational dynamics may offer a plausible explanation for thesepatterns of information processing. In the world of proteins, folding, for example, could occuralong innumerable lines, but like in origami, only one axis is chosen because it represent thelowest energy level (LEL) for that particular molecular network. Out of a multitude of possibleconformations that a receptor could take in the presence of ligands, it chooses the LEL, which isalso the biologically adaptive one. In order to illustrate this important aspect of proteins, let’simagine a blindfolded golfer, on a flat field; his chances to score are minimal, however if the

field is curved or funnel-shaped, his chances to score may approach 100%, regardless in whichdirection he aims.

Hypothesis: transpersonal patterns of information processing (i.e. archetypes) may representLEL of a particular molecular network.

Extracellular Matrix (ECM) within the NVU

In the NVU the ECM surrounds the cells, comprising the fourth brain compartment in which theother three: neuronal, glial and microvessels are embedded. This positions the ECM at thecenter of integration and synchronization of both cellular and molecular networks. In additionthe ECM also couples intra and extracellular molecular networks into GMNs, contributing to the“binding phenomenon”(22).

The ECM is comprised of a solid and a fluid phase (4). The solid phase contains hyaluronic acid,lecticans, hyaluronan, link proteins, and tenascins (39). The fluid phase of ECM is comprised ofinterstitial fluid (ISF) which represents the internal sea that bathes the cellular networksenabling both the glymphatic system clearance and volume transmission.

The volume of the ECM fluctuates during a 24 hours interval, being about 60% higher duringsleep (40). Volume fluctuations during sleep are believed to occur because of the glymphaticsystem exchange between CSF and ISF (41). However, this exchange may be enabled by the“OFF” position of integrin switches, characterized by shortening of integrines’ molecules (i.e.“loose” matrix)(42).

Hypothesis: the dynamic switching of integrins to non-adhesive state (“OFF”) occurs duringsleep and adhesive states (“ON”) during wakefulness. This may explain the increase in ECMvolume during sleep which empowers the glymphatic system to thoroughly removemolecular waste.

The perimeter of the NVU is demarcated by the arterial and venous capillary (a distance ofabout 40 μm. This space is filled with ECM in which the neuron, glia and both capillaries areembedded. This is the arena where the glymphatic system operates during sleep as the ECM is“loose”. This is also where the molecular switches operate, bringing on-line and off-line GMNs.For example, during sleep intra and extracellular molecular networks are “off line”, temporarilydisabling the GMN. It can be further hypothesized that the primary mental processesexperienced during dreaming reflect the “off line” status of intra and extracellular molecularnetworks. Conversely, rational, secondary mental processes require intracellular andextracellular molecular networks to be on-line (i.e synchronized). Interestingly, psychotic states,also characterized by primary mental processes are frequently triggered and/or accompaniedby sleep disturbances (43).

Other important molecular switches in the ECM of some NVUs are perineuronal nets (PNNs).They are well-organized, lattice-like structures that surround cell bodies, dendrites, and axons(44). It is believed that PNNs contribute to synaptic plasticity during the development, butswitch off plasticity during adulthood (45). This renders PNNs extremely interesting for bothphysical and memory rehabilitation, rendering ECM macromolecules possiblepsychopharmacological targets. For example It was reported that chronic treatment withfluoxetine causes restoration of synaptic plasticity especially in the dentate gyrus ofhippocampus (46)(47). In addition, it was demonstrated that enzymatic degradation of PNNs ortheir genetic deletion in mice leads to prolongation or restoration of synaptic plasticity (45).

PNNs seem to contribute to synaptic stabilization of parvalbumine inhibitory interneurons inthe hippocampus and cortex (49). Interestingly, these same neurons were involved inschizophrenia (50). A loss of PNNs throughout the medial temporal lobe has been reported inschizophrenic patients (51)(52). In addition, there is a growing body of evidence pointing to theinvolvement of ECM components such as reelin and chondroitin sulfate proteoglycans inschizophrenia (51)(52).

The ECM metalloproteinase (MMPs) have been heavily implicated in both cortical developmentand its psychopathology, for example a newly identified ADAM-10 metalloproteinase isinvolved in autism(53)(54), MMP-9 were involved in delirium ( 55), dementia (56 ) and PTSD(57) .

Looking at the Future

We started this road trip with a stroll in the tropical forest at the cellular level of brainorganization, than we took an imaginary elevator and descended one floor down to themolecular level. On January 6, 2014 the United States' Brookhaven National Laboratoryannounced the unprecedented ability to visualize chemical reactions at atomic level in real-time.

This sets the stage for studying the brain at yet another level, the nano level. A nanometer isone billionth of a meter; for visual perspective, a human hair has 100,000 times the diameter ofa carbon nanotube, which approximates the size of many biomolecules at work throughout theCNS. To be defined as "nano," the technology must have one dimension (length, width orheight) that is between 0.1 and 100 nanometers.

Nanoneuroscience is a new discipline which bridges neuroscience and nanotechnology, it usespotential nanomaterials (such as nanodiamonds or nanoparticles made from semiconductingmaterials) to diagnose neuropsychiatric disorders, to measure neurotransmitter levels orelectrical activity, or to stimulate in individual cells, and finally to build nanoscale molecularprosthetic devices that may restore activity patterns and cognitive function to brain cellular and

molecular networks. Assessment methods such as labeling macromolecules with luminescentnanorods have already been used to study self-assembly of microtubules (58), but in the future,they will be used as interventions, such as replacing protein subunits in enzymes, ECMmacromolecules or cellular cytoskeleton. Cytoskeletal orthopedics and prostetics will correctinformation processing in various networks, contributing to future treatments ofneurosychiatric disorders.

Conclusion

The intracellular and extracellular brain compartments are unified at the molecular level, wherethe main function of the brain, generation of the mind originates. By virtue of uniting the fourCNS compartments, the NVU is the smallest metabolic and computation unit of brain, thus thebuilding block of mind.

The brain could not function without the amazing properties of the building blocks of life, theproteins. They intimately connect the organic and inorganic realms of nature by coupling themechanical forces of motion with the biological actions in tissues. But proteins do more thancreate bridges between inorganic and organic chemistry, they are endowed with computationpower by virtue of their abilities: storage, transmission and processing of information. It canthus be emphatically stated that proteins represent the brain within the brain.

The CNS is situated at the interface between the environment and the body. The mind is thebrain’s adaptive response to nature’s unpredictability. Since the future events, such as life,injury or death cannot be predicted, the mind evolved to accomplish the next best: increasingthe odds of survival by an actuarial strive for lowering risk. The mind accomplishes this byplanning, analyzing possible scenarios, and managing the odds. This process is possible only bybringing the outside reality into the, inner, virtual space where it could be dissected, disposedof and recreated at will.

Nanoneuroscience is opening new technological possibilities not only for evaluation, but alsofor intervention in cellular and molecular networks with the purpose of correcting informationprocessing via novel proteomic methods such as replacing or activating enzymes, cytoskeletalproteins, or designing and applying individualized molecular prosthetic devices that may correctcellular signaling or recruit alternate networks to compensate for the dysfunctional ones.

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