NON-SYNAPTIC TRANSMISSION AND THE FOUNDATIONS OF AFFECT

Neuroscience has overwhelmingly and understandably focused on the synaptic modality of signal transmission. There is evidence, however, that from an evolutionary perspective, non-synaptic transmission (NST) preceded synaptic signaling. Moreover, in modern nervous systems, NST coexists and extensively interacts with synaptic transmission modifying neuronal dynamics. In fact, NST remains widespread in complex animals, especially within the interoceptive system where the dearth of insulating barriers such as myelin sheaths and the blood-brain barrier enhances the communication between neural and non-neural tissues mediated by NST. We suggest that this physiological arrangement makes a fundamental contribution to interoception⎯the process of sensing visceral states⎯which is an essential underpinning of the capacity to feel and the foundation of affective processing. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 24 January 2019 © 2019 by the author(s). Distributed under a Creative Commons CC BY license. doi:10.20944/preprints201901.0252.v1

Signal transmission can be found in rudimentary and ancient organisms.In fact, most modern neurotransmitters arose and functioned as signaling agents long before the appearance of neurons in evolution.For example, in the sponges that make up the phylum Porifera, GABA, glutamate, acetylcholine and serotonin operate in paracrine * mode, in the absence of synapses and neurons 2 .
Genetic, anatomical and biochemical analyses indicate that the paracrine pathways of Porifera are evolutionarily related to certain synaptic elements present in metazoans, although it remains unclear whether sponges preceded the evolutionary appearance of neurons or underwent a loss of neural cell types 2 .The origin of the molecules commonly referred to as "neurotransmitters" dates even farther back than Porifera or even the animal kingdom.In fact, most of the main neurotransmitters found in the mammalian nervous system play a central role in organisms *The term Paracrine designates signaling involving a molecule secreted by a cell and interacting with receptors on neighboring cells; the term Intracrine refers to intracellular signaling by a molecule acting in the cell where it is produced; Autocrine signaling involves a molecule secreted by a cell and interacting with receptors on that same cell; Endocrine signaling involves a molecule secreted by a cell and interacting with receptors on distant cells.
devoid of nervous systems, such as unicellular eukaryotes and even bacteria 3,4 .Acetylcholine, for example, may date as far back as 3 billion years 5 while animals are only thought to have arisen ~600 million years ago and nervous systems even later 6 .Neurotransmitters were not "neuro" for the vast majority of their existence.
One theory that has been advanced to account for the evolution of signal transmission proposes that metabolites initially functioned in an autocrine or intracrine manner, in bacteria.
Later, in unicellular eukaryotes, they operated as messengers within and between unicellular organisms 2,[7][8][9] .Even later, in multicellular organisms, these messengers continued to fulfill their inter-cellular function, but now within the perimeter of a single organism 10,11 .Thus in early

II. NST IN MODERN NERVOUS SYSTEMS
In spite of the advantages conferred by the synapse, the rudimentary NST precursor was not replaced and has remained prevalent.NST is prominent in numerous invertebrate phyla, including species where it coexists with synaptic signaling, and remains widespread in the vertebrate nervous system 11,[12][13][14] , where nonsynaptic receptors may actually outnumber synaptic ones 15,16 .In fact, most neurotransmitters can act nonsynaptically, and in many cases do so predominantly 15,17,18 .For example, a large fraction of acetylcholinergic, serotonergic and noradrenergic varicosities are nonsynaptic; in some areas of the nervous system, as many as 80-90% do not show the structural specializations that characterize synapses and are therefore thought to release the neurotransmitters for diffusion in the extracellular compartment, thereby activating more or less remote receptors 19,[20][21][22][23] .This scenario is supported by fact that the monoaminergic (dopamine, noradrenaline and serotonin) fiber terminals from which the transmitters are released are often not adjacent to the respective receptors [24][25][26] , indicating that following their release the molecules must diffuse in the extracellular space.The numerous examples of monoamine involvement in NST include the vertebrate retina (where dopamine may play a role in light-dark adaptation 28 ) and the amygdala 27 .Dopamine is also believed to operate non-synaptically in the spinal cord and in the cortical microvasculature where it helps regulate blood flow 26,29,30 .Even neurotransmitters typically associated with synaptic transmission, such as glutamate and GABA, have been shown to operate less conventionally and signal via extrasynaptic pathways, e.g. in the cerebellum and hippocampus (34, 35).
The scope of NST extends far beyond canonical neurotransmitters.ATP and neuropeptides also have a well-established non-synaptic role (31, 32, 33).Examples of peptide signaling include cytokines such as interleukin-1b(IL-1b) (42), and ß-endorphin, which can be detected in cortical areas distant from the cell bodies where it originates and is thought to migrate long distances via diffusion in the extracellular fluid and cerebrospinal fluid (36).In fact, all hormones, peptide and otherwise, can be said to function via nonsynaptic signaling.Endocrine signaling, the diffusion of signaling molecules via the bloodstream, can be seen as a modality of NST.
ATP and neuropeptides also have a well-established role in NST 31,32,33 .Even neurotransmitters typically associated with canonical synaptic transmission, such as glutamate and GABA, have been shown to play less conventional roles and signal via extrasynaptic pathways, e.g. in the cerebellum and hippocampus 34,35 .Yet another example of a diffusing substance in the CNS is β-endorphin, which can be detected in cortical areas distant from the cell bodies where it originates; it is thought to migrate long distances via diffusion in the extracellular fluid and cerebrospinal fluid 36 .
Gases and ions can also mediate NST.For example, nitric oxide (NO) is a widespread paracrine transmitter (neuro-and otherwise) with well-established roles in a plethora of physiological and pathological phenomena such as vasodilation, gastro-intestinal smooth muscle relaxation, learning and memory, and the immune response 37 .Carbon monoxide (CO) is another example of a gas involved in non-synaptic signaling.CO is produced in vascular smooth muscle cells and plays a role in the regulation of blood vessel tone.It may also function as a neurotransmitter 38 .NO and CO can cross cell membranes and diffuse away from their production sites, ultimately acting on cell surface and intracellular targets alike 35 .As for ions, extracellular potassium (K + ) produced as a result of impulse transmission and synaptic currents can also modulate neuron signal transmission and glial function 39 .Additionally, protons (H + ) modulate membrane excitability, synaptic function and gap-junction conductancefor example, H + can bind NMDA receptors 40 .Finally, receptors for Ca 2+ are present in the cell membranes of nerve terminals in several brain regions 41 .
NST is prevalent across most areas of the nervous system.In the CNS, instances of NST can be found at virtually every level of the neuraxis, especially in subcortical regions, namely the spinal cord, brain stem, basal ganglia and some hypothalamic nuclei.NST may play a particularly prominent role in limbic structures 11 .In the peripheral nervous system, chemosensitivity has been widely documented along the axonal trunks of peripheral sensory fibers and of the vagus nerve [43][44][45][46][47] , strongly pointing to a role for NST in interoception/visceroception.NST also seems widespread in peripheral ganglia.It can be observed in roughly 90% of spinal ganglion neurons 48 and has also been reported in the nodose ganglion of the vagus 49 .Yet its physiological roles and mechanisms have remained unclear.
NST in peripheral ganglia is thought to be neurotransmitter-mediated, although the specific transmitter in question has not been identified [49][50][51] .The extensive crosstalk observed among neighboring cell bodies elicits changes in membrane potential and firing threshold, which in turn control the likelihood of action potential propagation along sensory axons 52 .Thus DRG somas have the potential to act as sensory pathway gatekeepers and may operate as a synchronization mechanism.NST is also prevalent in the autonomic nervous system.Examples include 1) autonomic neuroeffector junctions 53 and 2) the enteric nervous system, where noradrenaline released from sympathetic nerves acts on neighboring cholinergic terminals to prevent acetylcholine release, in spite of the fact that no synaptic contacts exist between the two types of fibers 35 .
Given its ubiquity in modern nervous systems, NST is increasingly recognized as a major modality of neurotransmission on a par with the canonic synaptic variety 15 .
Of note, the physiological role of NST extends far beyond the nervous system.Unlike synapses, which connect neurons to each other or to cells such as myocytes or glia, NST has virtually no cell type restriction.Examples of NST between non-neuronal cells abound 26 .
Certain endocrine hormones, such as insulin or cortisol, are obvious examples.A paracrine role in the pancreas has also been described for glutamate, acetylcholine and GABA.The latter is secreted by pancreatic -cells and binds membrane receptors on the surface of neighboring islet cells, possibly regulating insulin and glucagon secretion 54,55 .Acetylcholine is thought to be a nearly universal signaling molecule, playing a paracrine, non-neuronal role in the lung bronchial epithelium, in erythrocytes, in ovarian endocrine cells and the immune system, among others 5,56,57 .These observations demonstrate that NST establishes communication pathways involving neuronal and non-neuronal cells alike.

III. THE DISTINCTIVE PHYSIOLOGY OF NST
By comparison to synaptic transmission, NST presents a number of striking differences.NST entails an agent (neurotransmitter, neuropeptide, ion or gas) being released by a cell, diffusing across the extracellular space and exerting its effects by reaching one or more target cells where it can act by e.g.binding to extra-synaptic membrane receptors.However, unlike the case of synapses, where the point of communication is physically well defined, NST does not require terminal varicosities or boutons and the two or more cells involved in the communication need not be adjacent.The signaling agent diffuses three-dimensionally in the extracellular space, predominantly in the intercellular interstitium but in some instances also in the cerebrospinal fluid and even in the blood stream.Signals travel over variable distances as a consequence of energy gradients 58,59 .For example, the monoamines dopamine, noradrenaline and serotonin are prominent NST messengers involved mostly in short-range transmission, on the order of micrometers 60 .Dopamine gets released into the extracellular fluid, where its receptors are often located in the non-synaptic regions of dendrites, axons and cell somas 61,62 .These receptors have an effective radius of 2-8μm 63 , i.e. 2-3 orders of magnitude larger than the typical synaptic cleft width of ~12-20nm 64 .In sharp contrast, neuropeptides (beta-endorphin, oxytocin, etc) are typical long-distance signalers, covering millimeters or even meters in the extreme cases of ghrelin, leptin and insulin, which are produced in the gastro-intestinal system and can act as far away as the CNS 65 .
NST is affected by the myriad physicochemical factors that have an impact on extracellular diffusion.These include electrical potentials and concentration, pressure, and temperature  66,67 .Extasynaptic neurotransmitters are also more vulnerable to inactivating mechanisms such as degrading enzymes present in the extracellular fluid and plasma membrane transporters.The latter play an especially intriguing role, since in addition to their uptake of extracellular transmitters -which reduces the concentration of diffusing molecules -they can operate in "reverse mode" by releasing transmitters from the neuronal cytoplasm into the surrounding extracellular space and thereby enabling NST 35 .Together these elements determine the clearance and half-life of non-synaptic agents and therefore effectively and dynamically regulate NST.

IV. THE FUNCTIONAL ADVANTAGES OF NST
The widespread distribution of NST in the nervous system and its role in myriad biological processes, from synaptic plasticity to behavior, strongly indicate that NST is more than a mere evolutionary relic.Another powerful argument in favor of the physiological relevance of NST is its remarkable structural conservation.For example, the architecture of central monoaminergic neurons (with its neurotransmitter-releasing terminals typically distant from the receptor sites) is identical across several mammalian species, indicating functional relevance 67 .
The persistence of NST suggests a substantial evolutionary benefit.In fact, although at first glance it may appear metabolically wasteful compared to the targeted and geographically confined synapse, NST may provide advantages in specific circumstances.While synapses clearly are a more direct and faster way to transmit a signal between two specific cells i.e. "oneto-one" transmission, NST becomes energy-efficient in instances of "one-to-many" communication, in which one cell can potentially influence hundreds or thousands of others without the need for the elaborate wiring network of axons that synaptic signaling would require to achieve the same effect 68,69 .NST may also allow faster regulation 70 .For example, in a local context where one cell secretes a messenger, e.g. a neuropeptide, hundreds of neighbors are potentially exposed to it, but their receptivity to the signal is determined individually by the expression of the respective membrane receptor.An unresponsive subpopulation of cells can be rapidly turned "on" merely by upregulating the expression of one protein, the membrane receptor in question (Fig. 1).Conversely, switching the cell "off" requires only that the receptor be internalized or downregulated.In contrast, synaptic transmission requires entire axons/synapses to be built anew, a far more energy-and time-consuming process 34 .Thus NST may allow a population of cells exposed to one or many signals to regulate signal transmission quickly and with energetic efficiency 71 .Synapses and NST are thus likely to be complementary, contextdependent methods of signal transmission 68 .Synaptic signaling provided an evolutionary advantage in cases in which specific information is relayed between two well-defined points in a time-sensitive manner (e.g.movement, reflexes, perception), while its predecessor, NST, remained the approach of choice for instances of one-to-many transmission, with fewer time constraints.diffuse back into the synaptic compartment of neighboring synapses, a phenomenon known as intersynaptic "spillover" (Fig. 2).All different permutations of this scenario have been reported 34 .Whether of synaptic or nonsynaptic origin, neurotransmitters can affect membrane potential, thereby modulating the likelihood of action potential firing.In the context of the synapse these ionic changes are known as post-synaptic potentials (PSPs) and can be excitatory or inhibitory, depending on whether the post-synaptic membrane undergoes depolarization or hyperpolarization, respectively increasing or decreasing the likelihood of the neuron reaching threshold 74 .Nonsynaptic transmitters can have similar effects 48 .In fact, nonsynaptically generated PSPs can have exceptionally long durations 35 , probably due to the longer-lasting and additive, this represents a potential mechanism for synaptic/non-synaptic signal integration.Thus a given neuron's likelihood of firing is determined by the integration of the mechanisms acting upon it and those mechanisms are both synaptic and non-synaptic, both excitatory and inhibitory.
In addition to serving parallel and complementary functions, there is some evidence that ST and NST can, on occasion, fulfill overlapping roles.For instance, in Parkinson's disease, which is due to loss of dopaminergic neurons in the substantia nigra, one physiological compensatory strategy involves a potentiation of NST 26 .Moreover, the therapeutic effect of L-Dopa, the key treatment of Parkinsonism, is attributable to NST since L-Dopa is converted to dopamine by non-dopaminergic neurons and released non-synaptically, allowing it to reach its target cells by diffusion 26,75 .

VI. THE BRAIN-BODY INTERFACE, NST AND THE PHYSIOLOGY OF AFFECT
There is a striking affinity between NST and unmyelinated neuronal fibers.Unmyelinated axons were followed by an evolutionary specialization⎯the myelin sheath⎯that enhanced the speed of signal transmission, but as is the case with NST, unmyelinated fibers were not simply replaced by the more modern, faster variety.They remain widely represented in complex nervous systems, including those of humans.Myelination is a metabolically costly process but that fact alone is unlikely to explain the less than universal adoption of myelin 76 .The topographical overlap of NST and unmyelinated axons is another sign of their affinity.For example, unmyelinated, tightly packed fibers are more prone to ephaptic interactions, which depend on NST 77 .Moreover, unmyelinated axons of the peripheral nervous system are sensitive to extrasynaptic neurotransmitters such as acetylcholine, serotonin, norepinephrine and ATP, even if these molecules only reach the axonal trunk, away from the nerve terminals [44][45][46]78 . In he CNS, the subcortical nervous system is home to a substantial proportion of unmyelinated structures, and is also the origin of many nonsynaptic transmitters 22 .Specifically, medial structures of the neuraxis such as those in the limbic system and brain stem, which are prominently involved in the regulation of homeostasis and affect (drives, emotions, feelings), are especially rich in unmyelinated fibers and monoaminergic/peptidergic neurons and are thought to signal prominently via NST 79,80 .In contrast, the more lateral domains of the mammalian brain (including the main sensorymotor systems, the association cortices and the cerebellum) are especially rich in well myelinated axons 11,81 .Finally, the interoceptive nervous system-a key physiological contributor to the phenomena of feeling-exhibits a high concentration of unmyelinated fibers, as well as numerous instances of NST 45,49,50,78 .The structures where NST is prevalent tend to have abundant denuded axons, whereas structures relying mostly on synaptic communication-those involved in movement, cognition, sensory perception-appear to be predominantly served by myelinated fibers.This bimodal distribution matches the contrasting temporal scales of the related neural process.Myelination is an improvement that mainly serves time-sensitive synaptic transmission, whereas unmyelinated fibers and NST pertain largely to processes that can afford slower and less precise transmission, which is the case with certain aspects of interoceptive information and feelings 76,82 .
The conspicuous overlap between NST and unmyelinated fibers is likely to have functional relevance.In effect, myelin is a physical obstacle to NST, the myelin sheath acting as a potential block to both secretion and reception of the messenger [83][84][85] .Unmyelinated fibers are comparatively exposed and thus able to sense and emit circulating molecules, including neurotransmitters.
Another striking feature of the interoceptive nervous system are the localized gaps in the blood-brain barrier (BBB)-or blood-nerve barrier, as it is commonly designated in the peripheral nervous system.The BBB exhibits particularly conspicuous gaps in spinal ganglia.
Each ganglion is divided into two regions, one housing most cell bodies and another containing mainly nerve fibers.The former is greatly enriched for vascularization, with a dense network of capillaries intimately wrapped around the sensory neuron somas.In contrast, the nerve fiber-rich area of the ganglion, as well as the adjacent nerves, are almost entirely devoid of blood vessels 86 .Moreover, the endothelium of the capillaries supplying the ganglia shows large fenestrations and is permeable to blood-borne agents of high molecular weight, properties reminiscent of areas of the CNS lacking a BBB, such as the area postrema, and also observable in autonomic sympathetic ganglia and the enteric nervous system 87 .In sharp contrast, the capillaries present within nerve trunks tend to be impermeable.Presumably as a result of this selective vulnerability, blood-borne toxins preferentially affect the sensory pathways, and indeed can accumulate in spinal ganglia, whereas motor pathways are comparatively spared 86 .This selectivity is revealed by intra-venous administration of labeled albumin, subsequently found to accumulate in the extravascular, peri-neuronal space of the cell body-rich, but not the nerve fiber-rich area of spinal ganglia.This strongly indicates that the dense network of capillaries enveloping the sensory cell bodies bear a specific and topographically conscribed gap in the BBB, presumably a result of the epithelial fenestrations and molecular differences in tight junction composition 88 .The trigeminal ganglion is also devoid of BBB 89,90 , suggesting that this feature is a general characteristic of peripheral sensory ganglia.
Stripped of a BBB and thus constitutively exposed to blood-borne factors, spinal ganglia may operate as chemosensors, responding to blood pH or concentration of metabolites or neuroactive substances 91  The three characteristic features of the interoceptive nervous system highlighted abovepredominant use of NST, lack of myelin sheath and presence of gaps in the BBB-are structurally and functionally connected.The dearth of physical barriers such as BBB and myelin effectively exposes the denuded neuronal structures to chemical factors present in the blood stream (in the case of spinal ganglion neuronal cell bodies) and the internal milieu (in the case of unmyelinated axonal trunks of PNS), providing them with an unparalleled ability to sense circulating molecules and detecting metabolic changes.These denuded, highly sensitized regions of the nervous system constitute an interface between neural and non-neural tissue, akin to the circumventricular organs but peripheral and dispersed throughout the organism rather than central.In all likelihood these regions are more capable of detecting subtler, incipient and localized chemical changes, potentially placing these structures at the very root of interoceptive sensing and, ultimately, of the feelings that allow us to become cognizant of varied states of homeostasis.
The contrasting anatomical and physiological features of synapses and NST (Table 1) suggest a theoretical framework.NST appears ideal to operate the molecular and visceral phenomena expressed in affective experiences 15,92,93 , in full agreement with the previously proposed role for unmyelinated axons in the processing of affect 76 .Whereas movement and perception require precision and gain from speed, the world of affect-moods, and plenty of feelings-can tolerate some vagueness and slowness, perhaps even benefit from them.Affective experiences originate in the graded, modulated transitions from one visceral configuration to another.This would be in keeping with the fact that NST occurs over relatively long timescales-seconds, minutes or even longer-, as opposed to the milli-or even microsecond range involved in synaptic transmission 94,26 .Also in support of this notion, the neurotransmitters involved in volume transmission originate mainly in subcortical structures 11,22 , which have been implicated as primary central neural substrates of feeling and subjectivity 76 .Thus synapses may mediate the "what" of neural function (consisting of processes such as sensory perception, skeletal muscle contraction and higher cognition) and NST leads in "how" functions such as affect and arousal 15,70,81,95 .This view has notable implications for the elucidation of the neurophysiology of feelings and, ultimately, for a comprehensive understanding of human mental processes.
SIGNAL TRANSMISSIONSynapses mediate and finely regulate nerve impulse conduction throughout the nervous system, playing a central role in physiological responses, movement and thought.Not surprisingly, synapses are commonly regarded as biological marvels and fundamental pillars of neural function.Nonetheless, several lines of evidence suggest that the intricately sophisticated synapse stems from comparatively humble beginnings, and that, no less importantly, exemplars of these earlier forms continue to play crucial roles in modern nervous systems.1 animals inter-cellular communication was largely mediated by signals released into the extracellular matrix (i.e.paracrine signaling).During the development of Metazoa, evolutionary forces may have favored more direct and faster communication modalities, leading to changes in cellular morphologythe development of axons and dendritesand to the compartmentalization of the neurotransmitter release channels.These changes would have facilitated a gradual transition from dispersed release to an open synaptic architecture, and, finally, to closed synapses, thus effecting a transition from paracrine to electrochemical signal transduction 2 .This conceivable evolutionary path would have substantially increased channel selectivity and speed in point-to-point transmission, an obvious advantage in the case of movement and, eventually, of perception and cognitive abilities such as memory and the manipulation of percepts, actual or recalled.In this perspective, synapses are an evolutionary specialization of nonsynaptic transmission (NST) offering refinement and efficiency.
coexist in an intricate interactive balance of extensive crosstalk and mutual regulation.How are these two signal transmission modes integrated and translated into neural function?What are the molecular mechanisms of their crosstalk?Transmembrane receptor heteromers appear to play a central role.Ion channels and G-protein coupled receptors (GPCRs) act as sensors of synaptic and non-synaptic signals, and exhibit direct, allosteric interactions which integrate distinct extracellular signals into common downstream molecular pathways 60,72,73 .Another integration mechanism involves the diffusion of neurotransmitters from the synaptic compartment to the surrounding extracellular space.Leaked synaptic transmitters can (a) activate nonsynaptic membrane receptors, effectively merging synaptic transmission and NST, or (b)

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, thereby serving as major sites of convergence of neural and humoral signals originating in the body, a role that has typically been associated with more rostral structures in the CNS.Blood-borne chemical signals crossing blood vessel walls and gaining direct access to neuronal somas may bind surface receptors and trigger changes in membrane potential, thereby impacting cellular excitability and action potential propagation, effectively governing the function of spinal ganglia and, consequently, interoceptive processing. Preprints(

Table 1 .
General characteristics of synaptic and nonsynaptic transmission modes.