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The axo-myelinic synapse
Opinion
The axo-myelinic synapse Peter K. Stys Department of Clinical Neurosciences, Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada
Axons have evolved to acquire myelination, enabling denser packing and speedier transmission. Although myelin is considered a passive insulator, recent reports suggest a more dynamic role. Axons, in turn, are endowed with neurotransmitter release and uptake systems along their trunks. Based on these observations, I argue that there may exist a new type of chemical synapse between axon and myelin, one that supports activity-dependent communication between the two. This raises intriguing possibilities of dynamic fine-tuning of the myelin sheath even in adulthood, efficient recruitment of resources for myelin maintenance and bi-directional signaling, whereby the axon informs its myelinating cell of its metabolic needs proportionally to the electrical traffic it is transmitting. This would also have implications for de- and dysmyelinating diseases should this axo-myelinic synapse become dysfunctional. Introduction Axons play the key role of transmitting electrical impulses to, from and within the nervous system. To remain responsive and to preserve well-coordinated afferent and efferent signaling by minimizing conduction latency, transmission velocities along fibers had to keep pace: the simplest solution for unmyelinated axons was to increase their diameter, resulting in a conduction velocity that varies directly with the square root of fiber diameter [1–3]. As organisms and their nervous systems became more complex, the bandwidth of afferent, efferent and internal [i.e. between different regions of the central nervous system (CNS)] information flow increased, which imposed two fundamental limitations: first, in an attempt to reduce latency, axonal diameter could not increase indefinitely as there was insufficient space to house bundles containing millions of fibers, if each was half a millimeter in diameter or more, as in the case of the squid giant axon. Second, continuous (non-saltatory) action potential propagation is relatively energy inefficient; therefore, packing a high density of such axons would require an inordinate continuous supply of energy that would quickly exceed the capacity of a nervous system to deliver such energy reliably. A different solution was thus needed. Myelination: an elegant solution for high-speed energyefficient information flow Enlarging an unmyelinated axon increases its conduction velocity. Another way of improving conduction velocity without excessively increasing fiber diameter, is to limit resistive and capacitive leak currents across the Corresponding author: Stys, P.K. ([email protected])
axolemma. Wrapping an axon in multiple layers of lipidrich low-conductance membrane (i.e. myelination) solves two problems: first, the high resistivity of myelin membranes helps reduce transmembrane ohmic current leakage (although more detailed experiments suggest that myelin is not as resistive as once assumed [4,5]). Second, and perhaps more importantly, the reduction in capacitance of the axonal membrane achieved by increasing the thickness of the equivalent dielectric with each myelin layer, reduces capacitive current leaks across the axolemma. The result is a 10–100-fold improvement in conduction velocity of myelinated axons over unmyelinated fibers of the same diameter [3,6]. Additionally, conduction velocity now rises linearly with diameter, rather than as the square root [1,7], so more rapidly conducting fibers can be packed into a smaller volume. Moreover, not only does myelination greatly increase conduction velocity and save space at the same time, but action potential propagation in myelinated fibers is also more energy efficient [6], thereby reducing the metabolic demands in high-density central nervous systems. The myelinating event is so important in more advanced nervous systems, that it is under complex and tightly orchestrated control by numerous signaling pathways (reviewed in [6,8–16]). Moreover, it is now well established that functional properties of axons can be modulated even in the mature nervous system, which might underlie essential plasticity in the CNS through deliberate tuning of action potential timing [17]. Taken together, myelination during development, and possibly even modulation of structural and functional properties of both axon and myelin throughout adult life, might be cardinal events whose importance for the normal and pathological functioning of the CNS might have been underestimated. As such, in this Opinion, I speculate on how and why the axon might modulate its myelin sheath in an activity-dependent manner via the proposed axo-myelinic synapse, whose arrangement exhibits striking parallels to traditional interneuronal synapses (Figure 1). Neurotransmitter receptors in myelin Cells have evolved a complex array of mechanisms for communicating among themselves over short and long distances. Generally, these modes include electrical coupling via gap junctions between adjacent cells for instance [18], and chemical transmission, whereby a diffusible substance is released and acts on another cell. Probably the most spatiotemporally precise and specific cell–cell communication occurs between neurons via the chemical synapse (Figure 1a). Thus, when receptors are found on a membrane, one might suspect that chemical transmission
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Figure 1. Comparison between conventional interneuronal chemical synapses and the proposed axo-myelinic synapse. (a) In the former, the presynaptic terminal is invaded by an action potential, leading to Ca2+-dependent fusion of transmittercontaining vesicles with the presynaptic terminal membrane and release of transmitter into the synaptic cleft (indicated by *). Receptors located on both the pre- and postsynaptic membranes respond to the released transmitter, which is then rapidly taken up by Na+-coupled transporters on perisynaptic astrocytic processes (green cell), as well as on the pre- and postsynaptic neuron. (b) The proposed axo-myelinic synapse exhibits striking parallels. An action potential transits the internodal axon (AX). Fusion and release of transmitter-containing vesicles has been shown to occur in unmyelinated axons [53,54], and might also occur in myelinated fibers. Transmitter is released into the confined periaxonal space (indicated by *), which is analogous to the synaptic cleft. Receptors exist on the presynaptic (the internodal axolemma) and postsynaptic [the inner loops of myelin (MY)] membranes, together with transmitter uptake systems, paralleling the conventional synapse.
is operational; a search for the other components of a synapse would further strengthen this suspicion. Glial cells of all types express a variety of ligand-gated ionotropic and metabotropic receptors [19–24]. By contrast, myelin was, until very recently, considered to be a relatively inert passive structure that, once laid down, remained invariant throughout the lifespan of an organism, serving to insulate against leakage currents and to support saltatory conduction. Studies over the past decade have demonstrated that myelin sheaths express various ionotropic glutamate receptor (GluR) subunits. In 2000, it was reported that mature CNS myelin expresses GluR4 AMPA receptor subunits [25], an observation that was subsequently confirmed and extended to include the KA2 kainate receptor subunit [26,27]. These observations 394
raised the possibility that mature myelin per se is the target of released neurotransmitter. Subsequently, three complementary papers reported the functional expression of NMDA receptors in mature oligodendrocytes, their processes and the myelin sheath itself [28–30]. Interestingly, the soma exhibits currents carried by both AMPA and NMDA receptors, whereas the processes and myelin appear to be subject mainly to NMDA receptor-mediated ion fluxes. These findings underscore important differences at not only the structural level, but also functional protein expression in oligodendroglia and their unique subdomains. In the myelin sheath per se, most of the specific immunoreactivity for NMDA receptor subunits is found along the inner and outer myelin loops [29] (Figure 2a), although surprisingly, some signal is also detected in the middle of the compact sheath itself [28,29]. More recently, using transgenic mice and a technique specifically optimized for myelin Ca2+ imaging using 2-photon laser scanning microscopy [31], it was reported [32] that a large fraction of NMDA receptor-mediated myelin Ca2+ fluctuations are mediated by an unusual ‘glycine-only NMDA receptor’ comprised of NR1 and NR3 subunits [33,34]. Traditional NMDA receptors composed of NR1 and NR2 subunits are somewhat unique in that they require dual coagonists (NMDA or glutamate at the binding site within the NR2 subunit, together with glycine or D-serine binding at the NR1 subunit) for activation. Interestingly, ‘NMDA’ receptors composed of NR1 and the more recently discovered NR3 subunits, but devoid of NR2, form functional excitatory receptors requiring only glycine or D-serine binding at the NR1 and NR3 subunits for activation. These ‘glycine-only’ receptors exhibit unique properties compared with traditional NMDA receptors, most notably reduced Ca2+ permeability and diminished sensitivity to Mg2+ block (reviewed in [35,36]). Taken together, these observations strongly suggest that mature myelin expresses functional receptors of the NMDA class, and possibly also of the AMPA class. Glial cells also express a broad repertoire of purinergic receptors, both ionotropic (P2X) and metabotropic (P2Y), including oligodendrocytes at various stages of maturity [22,24,37]. The ionotropic and highly Ca2+-permeable P2X7 receptor is particularly well represented in the oligodendrocyte soma and, interestingly, also in the mature myelin sheath [38–40]. Close inspection of published immunoelectron micrographs [39,40] indicates that, similar to myelinic NMDA receptors [29], the P2X7 receptors are also mainly segregated to the inner and outer myelin loops, with little if any signal in the compact myelin per se (Figure 2b). In support of potential signaling mechanisms contained within myelin, biochemical analysis reveals distinct microdomains in both central and peripheral myelin (e.g. lipid rafts), domains typically associated with signaling molecules such as transmitter receptors (reviewed in [41]). The presence of receptors with relatively low affinities for their ligands (e.g. AMPA receptors, which require hundreds of micromolar of glutamate, and P2X7 receptors requiring millimolar ATP) raises an interesting question of whether such concentrations would ever be reached physiologically in the extracellular space. However, the periaxonal space is very small, similar in width to the synaptic
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Figure 2. Example of neurotransmitter receptor clusters in central nervous system (CNS) myelin (My) of the adult rat optic nerve. (a) Upper panels: transverse sections immunostained for neurofilament (NF160, red) denoting axon (Ax) cylinders. Although the myelin is not labeled, all axons from mature rat optic nerve are myelinated; therefore, regions immediately outside the axon cylinders represent myelin. All three main NMDA receptor subunits (NR1, NR2 and NR3A) are expressed in clusters in myelin (green). Scale bars = 2 mm. Lower panels: immunogold labeling reveals that most of the NMDA receptor subunits are clustered at the inner or outer myelin loops. It is conceivable that the protein at the outer loops is in transit to its final place of insertion at the inner mesaxon. Scale bars = 50 nm. Adapted, with permission, from [29]. (b) P2X7 purinergic receptors have a similar myelinic distribution, being concentrated at the outer and inner mesaxon. Scale bars = 300 nm. Adapted, with permission, from [39].
cleft; if chemical axo-myelinic transmission occurs across this space as hypothesized here, concentrations of ligand could transiently reach high levels, just as they do in the traditional synaptic cleft. These recent data are beginning to redefine the view of the myelin sheath as a much more active structure capable of dynamic signaling, which repurposes many of the same receptors that are used by neurons for synaptic communication. In addition, recent reports indicate internodal glutamate receptors of the AMPA and kainate (but probably not NMDA [29,42]) classes expressed as clustered macromolecular nanocomplexes on the internodal axolemma under the myelin sheath [42–45]. Because I focus here on unidirectional signaling from axon to myelin, axonal receptors are not considered further; the interested reader is referred to a recent review [46]. Notably, these axonal receptors parallel presynaptic receptors found at many interneuronal synapses (Figure 1). Receptors are not enough These emerging data suggest, but in no way prove, that myelin is the target for ‘neurotransmission’ across a putative axo-myelinic synapse. In such a scenario, the internodal axon cylinder forms the presynaptic cell, the periaxonal space the ‘‘synaptic cleft’’, and the inner myelin loop expressing adaxonal myelinic receptors, the post-synaptic equivalent (Figure 1b). Chemical interneuronal synapses require more components before they can be assumed to be functional, not the least of which are: (i) transmitter release machinery on the presynaptic side (in this case, the axon, far removed from the terminal); and (ii)
transmitter uptake systems to terminate the signal. Axons typically serve the role of transmitting information that ends by invading a terminal, then communicating to the next cell via a traditional synapse. However, recent reports indicate that chemical transmission between axons and a non-neuronal target cell also occurs along the course of the fiber, well away from the terminal specialization. Activitydependent receptor-mediated signaling from axon to glial cell was shown decades ago in squid giant axons and later in vertebrate fibers [47–49]. Recently, more in-depth investigations were carried out in the mammalian CNS in an attempt to identify the precise sources and targets of this axo-glial communication. Akin to neuron-glia synapses [50] (reviewed in [51,52]), two groups described in 2007 how unmyelinated callosal axons form synaptic specializations with oligodendrocyte precursor cells (OPCs) and exhibit functional vesicular glutamatergic axo-glial transmission [53,54]. Notably, these axo-glial synapses form along the trunks of unmyelinated fibers en passant, far away from the presynaptic terminals. So far, these observations establish that unmyelinated fibers are able to release transmitter in a vesicular manner to signal adjacent glia. Can myelinated axons do the same? A recent report indicates that myelinated axons of the rodent optic nerve can also signal OPCs via AMPA and purinergic receptors by releasing glutamate and ATP in an activity-dependent manner [55]. The mechanism or locus of transmitter release is not clear, although this probably occurs at or near nodes of Ranvier, given the intimate association between OPCs (NG2 cells) and these nodes. Moreover, it has also been shown that premyelinated 395
Opinion central axons contain glutamate-laden vesicles, voltagegated Ca2+ channels and machinery traditionally found at synapses necessary for vesicular release [56]; whether these components persist in mature axons after full myelination is unknown. Taken together, these recent observations indicate that unmyelinated axons have discrete specializations along their course, far removed from their terminals, designed to release transmitter. In addition, these data also provide tantalizing hints that myelinated axons might also be able to release transmitter in a vesicular manner. Whether this occurs internodally, where axomyelinic transmission is expected to occur, awaits further investigation. Transmitter uptake systems, typically coupled to the transmembrane Na+ gradient, play the essential roles of terminating neurotransmission and confining the potent effects of released messengers spatially and temporally. Mammalian CNS white matter tracts express a variety of Na+-dependent uptake transporters for glutamate, glycine and noradrenaline, variously distributed on astrocytes, oligodendrocyte cell bodies, the axon membrane (particularly on the nodal axolemma) and even myelin itself [31,57–59]. These white matter-resident transporters can release toxic amounts of transmitter under pathological conditions [31,57–60]. This is analogous to reverse uptake in gray matter under ischemic conditions [61]. Undoubtedly, these transporters serve an important physiological role, in particular, in transmitter uptake. Myelin Ca2+ imaging studies have indicated that pharmacological inhibition of Na+-dependent glutamate or glycine transport in resting uninjured rodent white matter promotes a small but significant increase in myelin Ca2+ levels [31]. This was found to occur only in myelin, and not adjacent oligodendrocytes [31]. These results suggest that these transporters normally operate in the transmitter uptake mode, limiting the exposure of myelinic glutamate receptors to ambient transmitter. This also implies that there is a continuous basal release of transmitter for the transporters to be taking up. Based on the observations from previous studies discussed above, one can speculate that physiological (as opposed to pathophysiological) transmitter release occurs by vesicular release, probably into the confined periaxonal space under the myelin. Moreover, this release might be expected to be activity-dependent and accelerated in the face of increased action potential traffic conducted by the parent axon. Definitive proof of this prediction must await detailed, and technically very difficult, experiments, probably using myelin Ca2+ fluctuations as the readout for internodally released transmitter. Is myelin electrically polarized? A key requirement for synaptic signaling is for both the pre- and postsynaptic membranes to be electrically polarized. This permits gating of channels, flow of ions down electrochemical gradients and, thus, transduction of signals from electrical to chemical, then back to either electrical (in the case of ionotropic receptors) or chemical (for metabotropic receptors) at the target cell. In the proposed axo-myelinic synapse, the presynaptic cell (the internodal axon) is polarized, with an estimated resting potential of approximately 80 mV [62,63]. What about the postsyn396
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aptic element, the myelin sheath? Admittedly, myelin is a very unusual structure composed of numerous wraps of closely apposed lipid-rich membrane, with minuscule cytoplasmic and extracellular spaces, each approximately 4–5 nm wide [64,65]. There are two key requirements for a cell to become electrically polarized: (i) it must express ion-motive ATPases so that cellular energy can be used to transport ions against their electrochemical gradients; and (ii) it must insert relatively selective ion channels into its membrane, that possess some permeability at rest, so that a Nernstian potential can be created in the face of transmembrane concentration gradients established by the iontransporting ATPases. Analysis of the myelin proteome reveals the presence of all four of the known Na+-K+-ATPase a subunits (1–4) as well as two b subunits [66,67]. However, the presence of these proteins does not necessarily prove functional ATPdependent ion translocation in myelin. Moreover, given the tight adherence of the inner myelin loops with the axolemma, the question remains whether this is an axonal contaminant. Earlier biochemical and immunohistochemical studies strongly suggested that Na+-K+-ATPase detected from isolated myelin fractions is a myelin-resident enzyme rather than a contaminant [68]. Moreover, ultracytochemical experiments looking for ouabain- and K+-sensitive ATPase activity (probably Na+-K+-ATPase) revealed reaction product in myelin (including in the compact myelin), which was particularly abundant in the paranodal loops [69–71]. Taken together, it seems highly probable, therefore, that the mature compact myelin sheath supports ATP-dependent Na+ and K+ transporting activity. If, in addition, ionophoric proteins are inserted into myelin membranes, this Na+ and K+ pumping activity would also lead to electrical polarization of this structure, satisfying the second requirement needed to make myelin a functional postsynaptic element. Interestingly, there are two potential candidates. Proteolipid protein (PLP) is the most abundant protein of CNS myelin and is thought to play an important structural role in the formation and maintenance of compact myelin [72,73]. Could this major myelin protein play a role other than a structural one? Extraction of myelin proteolipid and incorporation into artificial lipid bilayers induces a K+ conductance in these membranes with discrete single-channel fluctuations [74] and patch-clamp recording from PLP reconstituted in proteoliposomes also revealed single channel-like activity [75– 77]. Another myelin protein that is a potential candidate for forming ionic conductance is plasmolipin. Similar to PLP, this 18-kDa proteolipid protein also forms relatively K+-selective ion channels in lipid bilayers [78,79]. However, unlike PLP, which is restricted to compact myelin, plasmolipin is found both in compact and peri-axolemmal myelin and, curiously, also in the axolemma [76,80]. The peri-axolemmal myelin localization of plasmolipin is particularly interesting as it might indicate that the inner myelin loop is electrically polarized, precisely where transmitter-gated channels appear to be preferentially expressed [29,39] (Figure 2). An important caveat surrounding such studies involves the constant danger of protein contaminants (such as ion
Opinion channels unrelated to PLP or plasmolipin) from other cell constituents, which would lead to misinterpretation of any putative electrical function of these two proteolipid proteins. Nonetheless, it is interesting that both PLP and plasmolipin are part of the tetraspan protein family, a collection of proteins characterized by four transmembrane segments. Many ligand-gated ion channels and the group of two-pore domain background leak K+ channels (those thought to be responsible for establishing resting membrane potential in neurons and glia) are also tetraspan proteins [81], adding further credence to the argument that PLP and/or plasmolipin might function as membrane potential-setting ionophores. Finally, if one posits that Ca2+-permeable (e.g. NMDA or P2X7) receptors are functionally active in myelin, as they are in neurons, mechanisms to buffer the Ca2+ transients to terminate the signal and prevent toxic Ca2+ overload are essential. Proteomic and immunochemical studies have revealed that myelin expresses two isoforms of the Na+– Ca2+ exchanger (NCX1 and 2) [66,82], a Ca2+ transporter widely expressed in excitable tissues [83]. Notably, Na+– Ca2+ exchange requires an established transmembrane Na+ gradient, again arguing in favor of functional Na+K+-ATPase activity. In addition, two isoforms of plasma membrane Ca2+-ATPase are also found in myelin [67], with cytochemical evidence of activity mainly on the outer and paranodal loops [84–86]. These Ca2+-buffering systems are typical of excitable cells where Ca2+ transients and intracellular levels need to be tightly controlled. To date, there is no conclusive evidence that myelin is electrically polarized. Definitive proof will probably require detailed optical methods using potentiometric dyes, because this structure is not amenable to conventional electrophysiological approaches. Nevertheless, the experimental findings summarized above suggest that myelin is electrically polarized, which would position the sheath as a potentially excitable postsynaptic element. Speculations on the role of the axo-myelinic synapse The intriguing question is, what role might such an axomyelinic communication serve? The arrangement would suggest an elegant system to allow activity-dependent signaling from the axon to its sheath. One possibility involves an optimization of myelin sheath thickness or composition. Although g-ratios (i.e. ratio of inner to total diameter of a myelinated axon) are maintained on average at approximately 0.6–0.7, there is a fair amount of spread around this value in the CNS [87]. Perhaps this is random variation or, alternatively, by design, where space is at a premium. It is conceivable that a historically active axon has signaled its myelin sheath (and, by extension, the process of the parent oligodendrocyte, which itself might be responsible for ensheathing dozens of fibers) in a dynamic fashion to divert more resources to its branch, to build up a thicker sheath. This would help reduce energy consumption of that fiber as its electrical characteristics could be optimized. By contrast, an axon that historically has supported lower frequency traffic might be allowed to remain suboptimally myelinated, so that net space and energy utilization is optimized in the white matter tract as a whole. Recent evidence is emerging in support of such
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activity-dependent plasticity in the white matter of the CNS. Diffusion tensor imaging studies have shown that training in specialized tasks induces changes in white matter in specific regions of the brain [88–90], indicating changes in axon caliber and/or myelination. An axo-myelinic synapse would be ideally positioned to transduce such plasticity. An intriguing bidirectional signaling might also be supported by activity-dependent axo-myelinic communication. Experiments indicate that lactate derived from glycogen, and released by white matter astrocytes, can sustain function of myelinated axons during periods of limited energy availability or high-frequency impulse transmission [91– 93]. It was proposed that this transfer takes place from an astrocyte to an axon at the node of Ranvier, but definitive evidence for such direct astrocytic–axonal transfer is lacking. Oligodendrocytes and their processes express glucose transporters [94], as well as monocarboxylate (e.g. lactate) transporters, with the latter also expressed in myelin [95]. In addition, astrocytes are coupled to oligodendrocytes via gap junctions [96–98], setting the stage for possible transfer of metabolites, such as lactate, from astrocyte to oligodendrocyte, then finally to the internodal axon, where metabolites can be more widely presented rather than just at the very restricted nodal gap. Such a two-stage transfer of metabolites has been suggested in a recent review [99]; I propose here that activity-dependent communication via an axo-myelinic synapse might signal the oligodendrocyte (and indirectly possibly even the supplying astrocyte) to upregulate the transfer of energy metabolites to fuel a more electrically active fiber. It has been known for some time that axons contain ribosomes that support local protein synthesis far removed from the parent neuronal soma (reviewed in [100,101]). This makes sense as it would be far more efficient for long fibers to renew proteins locally rather than relying on synthesis in the distant soma; indeed, the time it would take to transport somatically synthesized proteins to the end of a long spinal axon might result in arrival of ‘expired’ material, given the limited functional half-lives of many essential proteins [99]. Recently, it was reported than myelinating Schwann cells in the peripheral nervous system (PNS) locally supply their axons with mRNA-loaded polyribosomes by intercellular transfer [102]. This raises intriguing questions of whether and how the axon might signal its myelinating glial cells to import ribosomes. Given the potentially rich variety of glial-derived mRNAs transferred by this mechanism [103], even more intriguing is the possibility that the axon might somehow signal its glial cells, via myelinic receptors, to supply certain transcripts over others, in response to physiological stimuli or pathological perturbations. Indeed, it is well known that myelin is essential to support the health of axons and that demyelination eventually leads to axonal degeneration, although the mechanisms are not clear. It is conceivable that, through loss of its postsynaptic partner by demyelination, the internodal axon finds itself deprived of an essential source of ribosomes that are required for local replenishment of important structural and functional proteins, eventually leading to degeneration, even in the absence of a direct insult aimed at the denuded fiber. 397
Opinion Box 1. The axo-myelinic synapse and neuropsychiatric diseases It is conceivable that dysfunction of the axo-myelinic synapse might even underlie some neuropsychiatric disorders. The concept of transmitter-dependent axo-myelinic signaling could extend thinking about diseases traditionally thought to be related to defects in certain neurotransmitter systems and, therefore by default, considered to affect only gray matter and synaptic elements. In schizophrenia for instance, observations using diffusion tensor imaging point to subtle abnormalities of white matter in certain brain regions in these patients [104,105], as do ultrastructural data suggesting myelin defects [106,107]. It has been speculated that, as a result, the characteristic symptoms might be due to subtle aberrations of impulse timing, potentially caused by myelination defects, in key pathways linking certain brain regions [108]. Interestingly, changes in resting membrane potential of oligodendrocytes (and, by extension, also possibly of the myelin sheath) have been shown to modulate conduction velocity in the axons that they myelinate by up to 10% [109,110], possibly enough to either correctly synchronize impulse propagation in healthy states, or to desynchronize electrical traffic along key white matter tracts in disease states. Moreover, schizophrenia is also hypothesized to involve an aberration in NMDA receptor signaling [111]. One can speculate whether aberrations in NMDA receptor-dependent communication at the level of the axo-myelinic synapse might be responsible for these subtle alterations, either by modulating the electrical polarization of myelin (and thus in turn modulating the conduction velocity of the ensheathed axon) and/or by altering the structure (e.g. chemical composition or number of wraps; for a recent in-depth review, see [17]) of the sheath, thus imparting longterm influences on conduction velocity and by extension, on timing relationships between key brain regions. Other mental health disorders, such as major depression and autism, have also been found to harbor subtle white matter abnormalities [112,113]. This further raises questions about the sites of action of many currently used psychoactive drugs, most of which are aimed at modulating neurotransmitter systems. Perhaps some of the actions occur at the axo-myelinic unit in the white matter, and this might be one reason why the beneficial effects are often delayed by weeks or more, as the axo-myelinic relationship is slowly remodeled under the influence of these agents.
Concluding remarks Although at this time, the axo-myelinic synapse is a concept, it is one borne of rapidly accumulating evidence in support of a mechanism that would allow an axon to communicate with its overlying myelin sheath in a manner proportional to the amount of electrical traffic being conducted along each individual fiber. One can only speculate about the purpose of such a putative synapse. The proposal of an axo-myelinic synapse dependent on conventional neurotransmitters, receptors and transporters might explain how defects in transmitter systems could also lead to potentially important aberrations in CNS white matter that might contribute to clinical disease (Box 1). Moreover, such a concept underscores the dynamic nature of axons and their ensheathing myelin, which might continue to undergo significant plasticity not only during early development, but also throughout adult life. Acknowledgments Work in the author’s laboratory has been supported by grants from the Alberta Heritage Foundation for Medical Research, Canadian Institutes of Health Research, Canada Foundation for Innovation, National Institutes of Health, Heart and Stroke Foundation and the Multiple Sclerosis Society. The author thanks S. Baltan, A. Brown, B. Ransom and R. Turner for helpful discussions.
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Opinion 86 Maxwell, W.L. et al. (1995) Cytochemical evidence for redistribution of membrane pump calcium-ATPase and ecto-Ca-ATPase activity, and calcium influx in myelinated nerve fibres of the optic nerve after stretch injury. J. Neurocytol. 24, 925–942 87 Hildebrand, C. and Mohseni, S. (2005) The structure of myelinated axons in the CNS. In Multiple Sclerosis as a Neuronal Disease (Waxman, S.G., ed.), pp. 1–28, Elsevier Academic Press 88 Bengtsson, S.L. et al. (2005) Extensive piano practicing has regionally specific effects on white matter development. Nat. Neurosci. 8, 1148–1150 89 Paus, T. et al. (1999) Structural maturation of neural pathways in children and adolescents: in vivo study. Science 283, 1908–1911 90 Scholz, J. et al. (2009) Training induces changes in white-matter architecture. Nat. Neurosci. 12, 1370–1371 91 Wender, R. et al. (2000) Astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter. J. Neurosci. 20, 6804–7610 92 Brown, A.M. et al. (2005) Astrocyte glycogen metabolism is required for neural activity during aglycemia or intense stimulation in mouse white matter. J. Neurosci. Res. 79, 74–80 93 Brown, A.M. et al. (2004) Energy transfer from astrocytes to axons: the role of CNS glycogen. Neurochem. Int. 45, 529–536 94 Yu, S. and Ding, W.G. (1998) The 45 kDa form of glucose transporter 1 (GLUT1) is localized in oligodendrocyte and astrocyte but not in microglia in the rat brain. Brain Res. 797, 65–72 95 Rinholm, J.E. et al. (2011) Regulation of oligodendrocyte development and myelination by glucose and lactate. J. Neurosci. 31, 538–548 96 Kamasawa, N. et al. (2005) Connexin-47 and connexin-32 in gap junctions of oligodendrocyte somata, myelin sheaths, paranodal loops and Schmidt-Lanterman incisures: implications for ionic homeostasis and potassium siphoning. Neuroscience 136, 65–86 97 Orthmann-Murphy, J.L. et al. (2007) Two distinct heterotypic channels mediate gap junction coupling between astrocyte and oligodendrocyte connexins. J. Neurosci. 27, 13949–13957 98 Kleopa, K.A. et al. (2010) Gap junction disorders of myelinating cells. Rev. Neurosci. 21, 397–419
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Trends in Neurosciences August 2011, Vol. 34, No. 8 99 Nave, K.A. (2010) Myelination and the trophic support of long axons. Nat. Rev. Neurosci. 11, 275–283 100 Giuditta, A. et al. (2008) Local gene expression in axons and nerve endings: the glia–neuron unit. Physiol. Rev. 88, 515–555 101 Twiss, J.L. and Fainzilber, M. (2009) Ribosomes in axons: scrounging from the neighbors? Trends Cell Biol. 19, 236–243 102 Court, F.A. et al. (2008) Schwann cell to axon transfer of ribosomes: toward a novel understanding of the role of glia in the nervous system. J. Neurosci. 28, 11024–11029 103 Willis, D.E. and Twiss, J.L. (2011) Profiling axonal mRNA transport. Methods Mol. Biol. 714, 335–352 104 McIntosh, A.M. et al. (2008) White matter tractography in bipolar disorder and schizophrenia. Biol. Psychiatry 64, 1088–1092 105 Peters, B.D. et al. (2010) Diffusion tensor imaging in the early phase of schizophrenia: what have we learned? J. Psychiatr. Res. 44, 993–1004 106 Chance, S.A. et al. (1999) Fiber content of the fornix in schizophrenia: lack of evidence for a primary limbic encephalopathy. Am. J. Psychiatry 156, 1720–1724 107 Uranova, N. et al. (2001) Electron microscopy of oligodendroglia in severe mental illness. Brain Res. Bull. 55, 597–610 108 Whitford, T.J. et al. (2010) Schizophrenia, myelination, and delayed corollary discharges: a hypothesis. Schizophr. Bull. DOI: 10.1093/ schbul/sbq105 109 Yamazaki, Y. et al. (2010) Oligodendrocytes: facilitating axonal conduction by more than myelination. Neuroscientist 16, 11–18 110 Yamazaki, Y. et al. (2007) Modulatory effects of oligodendrocytes on the conduction velocity of action potentials along axons in the alveus of the rat hippocampal CA1 region. Neuron Glia Biol. 3, 325–334 111 Banerjee, A. et al. (2010) Neuregulin 1-erbB4 pathway in schizophrenia: from genes to an interactome. Brain Res. Bull. 83, 132–139 112 Heng, S. et al. (2010) White matter abnormalities in bipolar disorder: insights from diffusion tensor imaging studies. J. Neural Transm. 117, 639–654 113 White, T. et al. (2008) Diffusion tensor imaging in psychiatric disorders. Top. Magn. Reson. Imaging 19, 97–109
The axo-myelinic synapse Peter K. Stys Department of Clinical Neurosciences, Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada
Axons have evolved to acquire myelination, enabling denser packing and speedier transmission. Although myelin is considered a passive insulator, recent reports suggest a more dynamic role. Axons, in turn, are endowed with neurotransmitter release and uptake systems along their trunks. Based on these observations, I argue that there may exist a new type of chemical synapse between axon and myelin, one that supports activity-dependent communication between the two. This raises intriguing possibilities of dynamic fine-tuning of the myelin sheath even in adulthood, efficient recruitment of resources for myelin maintenance and bi-directional signaling, whereby the axon informs its myelinating cell of its metabolic needs proportionally to the electrical traffic it is transmitting. This would also have implications for de- and dysmyelinating diseases should this axo-myelinic synapse become dysfunctional. Introduction Axons play the key role of transmitting electrical impulses to, from and within the nervous system. To remain responsive and to preserve well-coordinated afferent and efferent signaling by minimizing conduction latency, transmission velocities along fibers had to keep pace: the simplest solution for unmyelinated axons was to increase their diameter, resulting in a conduction velocity that varies directly with the square root of fiber diameter [1–3]. As organisms and their nervous systems became more complex, the bandwidth of afferent, efferent and internal [i.e. between different regions of the central nervous system (CNS)] information flow increased, which imposed two fundamental limitations: first, in an attempt to reduce latency, axonal diameter could not increase indefinitely as there was insufficient space to house bundles containing millions of fibers, if each was half a millimeter in diameter or more, as in the case of the squid giant axon. Second, continuous (non-saltatory) action potential propagation is relatively energy inefficient; therefore, packing a high density of such axons would require an inordinate continuous supply of energy that would quickly exceed the capacity of a nervous system to deliver such energy reliably. A different solution was thus needed. Myelination: an elegant solution for high-speed energyefficient information flow Enlarging an unmyelinated axon increases its conduction velocity. Another way of improving conduction velocity without excessively increasing fiber diameter, is to limit resistive and capacitive leak currents across the Corresponding author: Stys, P.K. ([email protected])
axolemma. Wrapping an axon in multiple layers of lipidrich low-conductance membrane (i.e. myelination) solves two problems: first, the high resistivity of myelin membranes helps reduce transmembrane ohmic current leakage (although more detailed experiments suggest that myelin is not as resistive as once assumed [4,5]). Second, and perhaps more importantly, the reduction in capacitance of the axonal membrane achieved by increasing the thickness of the equivalent dielectric with each myelin layer, reduces capacitive current leaks across the axolemma. The result is a 10–100-fold improvement in conduction velocity of myelinated axons over unmyelinated fibers of the same diameter [3,6]. Additionally, conduction velocity now rises linearly with diameter, rather than as the square root [1,7], so more rapidly conducting fibers can be packed into a smaller volume. Moreover, not only does myelination greatly increase conduction velocity and save space at the same time, but action potential propagation in myelinated fibers is also more energy efficient [6], thereby reducing the metabolic demands in high-density central nervous systems. The myelinating event is so important in more advanced nervous systems, that it is under complex and tightly orchestrated control by numerous signaling pathways (reviewed in [6,8–16]). Moreover, it is now well established that functional properties of axons can be modulated even in the mature nervous system, which might underlie essential plasticity in the CNS through deliberate tuning of action potential timing [17]. Taken together, myelination during development, and possibly even modulation of structural and functional properties of both axon and myelin throughout adult life, might be cardinal events whose importance for the normal and pathological functioning of the CNS might have been underestimated. As such, in this Opinion, I speculate on how and why the axon might modulate its myelin sheath in an activity-dependent manner via the proposed axo-myelinic synapse, whose arrangement exhibits striking parallels to traditional interneuronal synapses (Figure 1). Neurotransmitter receptors in myelin Cells have evolved a complex array of mechanisms for communicating among themselves over short and long distances. Generally, these modes include electrical coupling via gap junctions between adjacent cells for instance [18], and chemical transmission, whereby a diffusible substance is released and acts on another cell. Probably the most spatiotemporally precise and specific cell–cell communication occurs between neurons via the chemical synapse (Figure 1a). Thus, when receptors are found on a membrane, one might suspect that chemical transmission
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Figure 1. Comparison between conventional interneuronal chemical synapses and the proposed axo-myelinic synapse. (a) In the former, the presynaptic terminal is invaded by an action potential, leading to Ca2+-dependent fusion of transmittercontaining vesicles with the presynaptic terminal membrane and release of transmitter into the synaptic cleft (indicated by *). Receptors located on both the pre- and postsynaptic membranes respond to the released transmitter, which is then rapidly taken up by Na+-coupled transporters on perisynaptic astrocytic processes (green cell), as well as on the pre- and postsynaptic neuron. (b) The proposed axo-myelinic synapse exhibits striking parallels. An action potential transits the internodal axon (AX). Fusion and release of transmitter-containing vesicles has been shown to occur in unmyelinated axons [53,54], and might also occur in myelinated fibers. Transmitter is released into the confined periaxonal space (indicated by *), which is analogous to the synaptic cleft. Receptors exist on the presynaptic (the internodal axolemma) and postsynaptic [the inner loops of myelin (MY)] membranes, together with transmitter uptake systems, paralleling the conventional synapse.
is operational; a search for the other components of a synapse would further strengthen this suspicion. Glial cells of all types express a variety of ligand-gated ionotropic and metabotropic receptors [19–24]. By contrast, myelin was, until very recently, considered to be a relatively inert passive structure that, once laid down, remained invariant throughout the lifespan of an organism, serving to insulate against leakage currents and to support saltatory conduction. Studies over the past decade have demonstrated that myelin sheaths express various ionotropic glutamate receptor (GluR) subunits. In 2000, it was reported that mature CNS myelin expresses GluR4 AMPA receptor subunits [25], an observation that was subsequently confirmed and extended to include the KA2 kainate receptor subunit [26,27]. These observations 394
raised the possibility that mature myelin per se is the target of released neurotransmitter. Subsequently, three complementary papers reported the functional expression of NMDA receptors in mature oligodendrocytes, their processes and the myelin sheath itself [28–30]. Interestingly, the soma exhibits currents carried by both AMPA and NMDA receptors, whereas the processes and myelin appear to be subject mainly to NMDA receptor-mediated ion fluxes. These findings underscore important differences at not only the structural level, but also functional protein expression in oligodendroglia and their unique subdomains. In the myelin sheath per se, most of the specific immunoreactivity for NMDA receptor subunits is found along the inner and outer myelin loops [29] (Figure 2a), although surprisingly, some signal is also detected in the middle of the compact sheath itself [28,29]. More recently, using transgenic mice and a technique specifically optimized for myelin Ca2+ imaging using 2-photon laser scanning microscopy [31], it was reported [32] that a large fraction of NMDA receptor-mediated myelin Ca2+ fluctuations are mediated by an unusual ‘glycine-only NMDA receptor’ comprised of NR1 and NR3 subunits [33,34]. Traditional NMDA receptors composed of NR1 and NR2 subunits are somewhat unique in that they require dual coagonists (NMDA or glutamate at the binding site within the NR2 subunit, together with glycine or D-serine binding at the NR1 subunit) for activation. Interestingly, ‘NMDA’ receptors composed of NR1 and the more recently discovered NR3 subunits, but devoid of NR2, form functional excitatory receptors requiring only glycine or D-serine binding at the NR1 and NR3 subunits for activation. These ‘glycine-only’ receptors exhibit unique properties compared with traditional NMDA receptors, most notably reduced Ca2+ permeability and diminished sensitivity to Mg2+ block (reviewed in [35,36]). Taken together, these observations strongly suggest that mature myelin expresses functional receptors of the NMDA class, and possibly also of the AMPA class. Glial cells also express a broad repertoire of purinergic receptors, both ionotropic (P2X) and metabotropic (P2Y), including oligodendrocytes at various stages of maturity [22,24,37]. The ionotropic and highly Ca2+-permeable P2X7 receptor is particularly well represented in the oligodendrocyte soma and, interestingly, also in the mature myelin sheath [38–40]. Close inspection of published immunoelectron micrographs [39,40] indicates that, similar to myelinic NMDA receptors [29], the P2X7 receptors are also mainly segregated to the inner and outer myelin loops, with little if any signal in the compact myelin per se (Figure 2b). In support of potential signaling mechanisms contained within myelin, biochemical analysis reveals distinct microdomains in both central and peripheral myelin (e.g. lipid rafts), domains typically associated with signaling molecules such as transmitter receptors (reviewed in [41]). The presence of receptors with relatively low affinities for their ligands (e.g. AMPA receptors, which require hundreds of micromolar of glutamate, and P2X7 receptors requiring millimolar ATP) raises an interesting question of whether such concentrations would ever be reached physiologically in the extracellular space. However, the periaxonal space is very small, similar in width to the synaptic
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Figure 2. Example of neurotransmitter receptor clusters in central nervous system (CNS) myelin (My) of the adult rat optic nerve. (a) Upper panels: transverse sections immunostained for neurofilament (NF160, red) denoting axon (Ax) cylinders. Although the myelin is not labeled, all axons from mature rat optic nerve are myelinated; therefore, regions immediately outside the axon cylinders represent myelin. All three main NMDA receptor subunits (NR1, NR2 and NR3A) are expressed in clusters in myelin (green). Scale bars = 2 mm. Lower panels: immunogold labeling reveals that most of the NMDA receptor subunits are clustered at the inner or outer myelin loops. It is conceivable that the protein at the outer loops is in transit to its final place of insertion at the inner mesaxon. Scale bars = 50 nm. Adapted, with permission, from [29]. (b) P2X7 purinergic receptors have a similar myelinic distribution, being concentrated at the outer and inner mesaxon. Scale bars = 300 nm. Adapted, with permission, from [39].
cleft; if chemical axo-myelinic transmission occurs across this space as hypothesized here, concentrations of ligand could transiently reach high levels, just as they do in the traditional synaptic cleft. These recent data are beginning to redefine the view of the myelin sheath as a much more active structure capable of dynamic signaling, which repurposes many of the same receptors that are used by neurons for synaptic communication. In addition, recent reports indicate internodal glutamate receptors of the AMPA and kainate (but probably not NMDA [29,42]) classes expressed as clustered macromolecular nanocomplexes on the internodal axolemma under the myelin sheath [42–45]. Because I focus here on unidirectional signaling from axon to myelin, axonal receptors are not considered further; the interested reader is referred to a recent review [46]. Notably, these axonal receptors parallel presynaptic receptors found at many interneuronal synapses (Figure 1). Receptors are not enough These emerging data suggest, but in no way prove, that myelin is the target for ‘neurotransmission’ across a putative axo-myelinic synapse. In such a scenario, the internodal axon cylinder forms the presynaptic cell, the periaxonal space the ‘‘synaptic cleft’’, and the inner myelin loop expressing adaxonal myelinic receptors, the post-synaptic equivalent (Figure 1b). Chemical interneuronal synapses require more components before they can be assumed to be functional, not the least of which are: (i) transmitter release machinery on the presynaptic side (in this case, the axon, far removed from the terminal); and (ii)
transmitter uptake systems to terminate the signal. Axons typically serve the role of transmitting information that ends by invading a terminal, then communicating to the next cell via a traditional synapse. However, recent reports indicate that chemical transmission between axons and a non-neuronal target cell also occurs along the course of the fiber, well away from the terminal specialization. Activitydependent receptor-mediated signaling from axon to glial cell was shown decades ago in squid giant axons and later in vertebrate fibers [47–49]. Recently, more in-depth investigations were carried out in the mammalian CNS in an attempt to identify the precise sources and targets of this axo-glial communication. Akin to neuron-glia synapses [50] (reviewed in [51,52]), two groups described in 2007 how unmyelinated callosal axons form synaptic specializations with oligodendrocyte precursor cells (OPCs) and exhibit functional vesicular glutamatergic axo-glial transmission [53,54]. Notably, these axo-glial synapses form along the trunks of unmyelinated fibers en passant, far away from the presynaptic terminals. So far, these observations establish that unmyelinated fibers are able to release transmitter in a vesicular manner to signal adjacent glia. Can myelinated axons do the same? A recent report indicates that myelinated axons of the rodent optic nerve can also signal OPCs via AMPA and purinergic receptors by releasing glutamate and ATP in an activity-dependent manner [55]. The mechanism or locus of transmitter release is not clear, although this probably occurs at or near nodes of Ranvier, given the intimate association between OPCs (NG2 cells) and these nodes. Moreover, it has also been shown that premyelinated 395
Opinion central axons contain glutamate-laden vesicles, voltagegated Ca2+ channels and machinery traditionally found at synapses necessary for vesicular release [56]; whether these components persist in mature axons after full myelination is unknown. Taken together, these recent observations indicate that unmyelinated axons have discrete specializations along their course, far removed from their terminals, designed to release transmitter. In addition, these data also provide tantalizing hints that myelinated axons might also be able to release transmitter in a vesicular manner. Whether this occurs internodally, where axomyelinic transmission is expected to occur, awaits further investigation. Transmitter uptake systems, typically coupled to the transmembrane Na+ gradient, play the essential roles of terminating neurotransmission and confining the potent effects of released messengers spatially and temporally. Mammalian CNS white matter tracts express a variety of Na+-dependent uptake transporters for glutamate, glycine and noradrenaline, variously distributed on astrocytes, oligodendrocyte cell bodies, the axon membrane (particularly on the nodal axolemma) and even myelin itself [31,57–59]. These white matter-resident transporters can release toxic amounts of transmitter under pathological conditions [31,57–60]. This is analogous to reverse uptake in gray matter under ischemic conditions [61]. Undoubtedly, these transporters serve an important physiological role, in particular, in transmitter uptake. Myelin Ca2+ imaging studies have indicated that pharmacological inhibition of Na+-dependent glutamate or glycine transport in resting uninjured rodent white matter promotes a small but significant increase in myelin Ca2+ levels [31]. This was found to occur only in myelin, and not adjacent oligodendrocytes [31]. These results suggest that these transporters normally operate in the transmitter uptake mode, limiting the exposure of myelinic glutamate receptors to ambient transmitter. This also implies that there is a continuous basal release of transmitter for the transporters to be taking up. Based on the observations from previous studies discussed above, one can speculate that physiological (as opposed to pathophysiological) transmitter release occurs by vesicular release, probably into the confined periaxonal space under the myelin. Moreover, this release might be expected to be activity-dependent and accelerated in the face of increased action potential traffic conducted by the parent axon. Definitive proof of this prediction must await detailed, and technically very difficult, experiments, probably using myelin Ca2+ fluctuations as the readout for internodally released transmitter. Is myelin electrically polarized? A key requirement for synaptic signaling is for both the pre- and postsynaptic membranes to be electrically polarized. This permits gating of channels, flow of ions down electrochemical gradients and, thus, transduction of signals from electrical to chemical, then back to either electrical (in the case of ionotropic receptors) or chemical (for metabotropic receptors) at the target cell. In the proposed axo-myelinic synapse, the presynaptic cell (the internodal axon) is polarized, with an estimated resting potential of approximately 80 mV [62,63]. What about the postsyn396
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aptic element, the myelin sheath? Admittedly, myelin is a very unusual structure composed of numerous wraps of closely apposed lipid-rich membrane, with minuscule cytoplasmic and extracellular spaces, each approximately 4–5 nm wide [64,65]. There are two key requirements for a cell to become electrically polarized: (i) it must express ion-motive ATPases so that cellular energy can be used to transport ions against their electrochemical gradients; and (ii) it must insert relatively selective ion channels into its membrane, that possess some permeability at rest, so that a Nernstian potential can be created in the face of transmembrane concentration gradients established by the iontransporting ATPases. Analysis of the myelin proteome reveals the presence of all four of the known Na+-K+-ATPase a subunits (1–4) as well as two b subunits [66,67]. However, the presence of these proteins does not necessarily prove functional ATPdependent ion translocation in myelin. Moreover, given the tight adherence of the inner myelin loops with the axolemma, the question remains whether this is an axonal contaminant. Earlier biochemical and immunohistochemical studies strongly suggested that Na+-K+-ATPase detected from isolated myelin fractions is a myelin-resident enzyme rather than a contaminant [68]. Moreover, ultracytochemical experiments looking for ouabain- and K+-sensitive ATPase activity (probably Na+-K+-ATPase) revealed reaction product in myelin (including in the compact myelin), which was particularly abundant in the paranodal loops [69–71]. Taken together, it seems highly probable, therefore, that the mature compact myelin sheath supports ATP-dependent Na+ and K+ transporting activity. If, in addition, ionophoric proteins are inserted into myelin membranes, this Na+ and K+ pumping activity would also lead to electrical polarization of this structure, satisfying the second requirement needed to make myelin a functional postsynaptic element. Interestingly, there are two potential candidates. Proteolipid protein (PLP) is the most abundant protein of CNS myelin and is thought to play an important structural role in the formation and maintenance of compact myelin [72,73]. Could this major myelin protein play a role other than a structural one? Extraction of myelin proteolipid and incorporation into artificial lipid bilayers induces a K+ conductance in these membranes with discrete single-channel fluctuations [74] and patch-clamp recording from PLP reconstituted in proteoliposomes also revealed single channel-like activity [75– 77]. Another myelin protein that is a potential candidate for forming ionic conductance is plasmolipin. Similar to PLP, this 18-kDa proteolipid protein also forms relatively K+-selective ion channels in lipid bilayers [78,79]. However, unlike PLP, which is restricted to compact myelin, plasmolipin is found both in compact and peri-axolemmal myelin and, curiously, also in the axolemma [76,80]. The peri-axolemmal myelin localization of plasmolipin is particularly interesting as it might indicate that the inner myelin loop is electrically polarized, precisely where transmitter-gated channels appear to be preferentially expressed [29,39] (Figure 2). An important caveat surrounding such studies involves the constant danger of protein contaminants (such as ion
Opinion channels unrelated to PLP or plasmolipin) from other cell constituents, which would lead to misinterpretation of any putative electrical function of these two proteolipid proteins. Nonetheless, it is interesting that both PLP and plasmolipin are part of the tetraspan protein family, a collection of proteins characterized by four transmembrane segments. Many ligand-gated ion channels and the group of two-pore domain background leak K+ channels (those thought to be responsible for establishing resting membrane potential in neurons and glia) are also tetraspan proteins [81], adding further credence to the argument that PLP and/or plasmolipin might function as membrane potential-setting ionophores. Finally, if one posits that Ca2+-permeable (e.g. NMDA or P2X7) receptors are functionally active in myelin, as they are in neurons, mechanisms to buffer the Ca2+ transients to terminate the signal and prevent toxic Ca2+ overload are essential. Proteomic and immunochemical studies have revealed that myelin expresses two isoforms of the Na+– Ca2+ exchanger (NCX1 and 2) [66,82], a Ca2+ transporter widely expressed in excitable tissues [83]. Notably, Na+– Ca2+ exchange requires an established transmembrane Na+ gradient, again arguing in favor of functional Na+K+-ATPase activity. In addition, two isoforms of plasma membrane Ca2+-ATPase are also found in myelin [67], with cytochemical evidence of activity mainly on the outer and paranodal loops [84–86]. These Ca2+-buffering systems are typical of excitable cells where Ca2+ transients and intracellular levels need to be tightly controlled. To date, there is no conclusive evidence that myelin is electrically polarized. Definitive proof will probably require detailed optical methods using potentiometric dyes, because this structure is not amenable to conventional electrophysiological approaches. Nevertheless, the experimental findings summarized above suggest that myelin is electrically polarized, which would position the sheath as a potentially excitable postsynaptic element. Speculations on the role of the axo-myelinic synapse The intriguing question is, what role might such an axomyelinic communication serve? The arrangement would suggest an elegant system to allow activity-dependent signaling from the axon to its sheath. One possibility involves an optimization of myelin sheath thickness or composition. Although g-ratios (i.e. ratio of inner to total diameter of a myelinated axon) are maintained on average at approximately 0.6–0.7, there is a fair amount of spread around this value in the CNS [87]. Perhaps this is random variation or, alternatively, by design, where space is at a premium. It is conceivable that a historically active axon has signaled its myelin sheath (and, by extension, the process of the parent oligodendrocyte, which itself might be responsible for ensheathing dozens of fibers) in a dynamic fashion to divert more resources to its branch, to build up a thicker sheath. This would help reduce energy consumption of that fiber as its electrical characteristics could be optimized. By contrast, an axon that historically has supported lower frequency traffic might be allowed to remain suboptimally myelinated, so that net space and energy utilization is optimized in the white matter tract as a whole. Recent evidence is emerging in support of such
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activity-dependent plasticity in the white matter of the CNS. Diffusion tensor imaging studies have shown that training in specialized tasks induces changes in white matter in specific regions of the brain [88–90], indicating changes in axon caliber and/or myelination. An axo-myelinic synapse would be ideally positioned to transduce such plasticity. An intriguing bidirectional signaling might also be supported by activity-dependent axo-myelinic communication. Experiments indicate that lactate derived from glycogen, and released by white matter astrocytes, can sustain function of myelinated axons during periods of limited energy availability or high-frequency impulse transmission [91– 93]. It was proposed that this transfer takes place from an astrocyte to an axon at the node of Ranvier, but definitive evidence for such direct astrocytic–axonal transfer is lacking. Oligodendrocytes and their processes express glucose transporters [94], as well as monocarboxylate (e.g. lactate) transporters, with the latter also expressed in myelin [95]. In addition, astrocytes are coupled to oligodendrocytes via gap junctions [96–98], setting the stage for possible transfer of metabolites, such as lactate, from astrocyte to oligodendrocyte, then finally to the internodal axon, where metabolites can be more widely presented rather than just at the very restricted nodal gap. Such a two-stage transfer of metabolites has been suggested in a recent review [99]; I propose here that activity-dependent communication via an axo-myelinic synapse might signal the oligodendrocyte (and indirectly possibly even the supplying astrocyte) to upregulate the transfer of energy metabolites to fuel a more electrically active fiber. It has been known for some time that axons contain ribosomes that support local protein synthesis far removed from the parent neuronal soma (reviewed in [100,101]). This makes sense as it would be far more efficient for long fibers to renew proteins locally rather than relying on synthesis in the distant soma; indeed, the time it would take to transport somatically synthesized proteins to the end of a long spinal axon might result in arrival of ‘expired’ material, given the limited functional half-lives of many essential proteins [99]. Recently, it was reported than myelinating Schwann cells in the peripheral nervous system (PNS) locally supply their axons with mRNA-loaded polyribosomes by intercellular transfer [102]. This raises intriguing questions of whether and how the axon might signal its myelinating glial cells to import ribosomes. Given the potentially rich variety of glial-derived mRNAs transferred by this mechanism [103], even more intriguing is the possibility that the axon might somehow signal its glial cells, via myelinic receptors, to supply certain transcripts over others, in response to physiological stimuli or pathological perturbations. Indeed, it is well known that myelin is essential to support the health of axons and that demyelination eventually leads to axonal degeneration, although the mechanisms are not clear. It is conceivable that, through loss of its postsynaptic partner by demyelination, the internodal axon finds itself deprived of an essential source of ribosomes that are required for local replenishment of important structural and functional proteins, eventually leading to degeneration, even in the absence of a direct insult aimed at the denuded fiber. 397
Opinion Box 1. The axo-myelinic synapse and neuropsychiatric diseases It is conceivable that dysfunction of the axo-myelinic synapse might even underlie some neuropsychiatric disorders. The concept of transmitter-dependent axo-myelinic signaling could extend thinking about diseases traditionally thought to be related to defects in certain neurotransmitter systems and, therefore by default, considered to affect only gray matter and synaptic elements. In schizophrenia for instance, observations using diffusion tensor imaging point to subtle abnormalities of white matter in certain brain regions in these patients [104,105], as do ultrastructural data suggesting myelin defects [106,107]. It has been speculated that, as a result, the characteristic symptoms might be due to subtle aberrations of impulse timing, potentially caused by myelination defects, in key pathways linking certain brain regions [108]. Interestingly, changes in resting membrane potential of oligodendrocytes (and, by extension, also possibly of the myelin sheath) have been shown to modulate conduction velocity in the axons that they myelinate by up to 10% [109,110], possibly enough to either correctly synchronize impulse propagation in healthy states, or to desynchronize electrical traffic along key white matter tracts in disease states. Moreover, schizophrenia is also hypothesized to involve an aberration in NMDA receptor signaling [111]. One can speculate whether aberrations in NMDA receptor-dependent communication at the level of the axo-myelinic synapse might be responsible for these subtle alterations, either by modulating the electrical polarization of myelin (and thus in turn modulating the conduction velocity of the ensheathed axon) and/or by altering the structure (e.g. chemical composition or number of wraps; for a recent in-depth review, see [17]) of the sheath, thus imparting longterm influences on conduction velocity and by extension, on timing relationships between key brain regions. Other mental health disorders, such as major depression and autism, have also been found to harbor subtle white matter abnormalities [112,113]. This further raises questions about the sites of action of many currently used psychoactive drugs, most of which are aimed at modulating neurotransmitter systems. Perhaps some of the actions occur at the axo-myelinic unit in the white matter, and this might be one reason why the beneficial effects are often delayed by weeks or more, as the axo-myelinic relationship is slowly remodeled under the influence of these agents.
Concluding remarks Although at this time, the axo-myelinic synapse is a concept, it is one borne of rapidly accumulating evidence in support of a mechanism that would allow an axon to communicate with its overlying myelin sheath in a manner proportional to the amount of electrical traffic being conducted along each individual fiber. One can only speculate about the purpose of such a putative synapse. The proposal of an axo-myelinic synapse dependent on conventional neurotransmitters, receptors and transporters might explain how defects in transmitter systems could also lead to potentially important aberrations in CNS white matter that might contribute to clinical disease (Box 1). Moreover, such a concept underscores the dynamic nature of axons and their ensheathing myelin, which might continue to undergo significant plasticity not only during early development, but also throughout adult life. Acknowledgments Work in the author’s laboratory has been supported by grants from the Alberta Heritage Foundation for Medical Research, Canadian Institutes of Health Research, Canada Foundation for Innovation, National Institutes of Health, Heart and Stroke Foundation and the Multiple Sclerosis Society. The author thanks S. Baltan, A. Brown, B. Ransom and R. Turner for helpful discussions.
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