Axonal pathology is a key contributor to long-term disability in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), but the mechanisms that underlie axonal pathology in MS remain elusive. Evidence suggests that axonal pathology is a direct consequence of demyelination, as we and others have shown that the node of Ranvier disassembles following loss of myelin. In contrast to the node of Ranvier, we now show that the axon initial segment (AIS), the axonal domain responsible for action potential initiation, remains intact following cuprizone-induced cortical demyelination. Instead, we find that the AIS is disrupted in the neocortex of mice that develop experimental autoimmune encephalomyelitis (EAE) independent of local demyelination. EAE-induced mice demonstrate profound compromise of AIS integrity with a progressive disruption that corresponds to EAE clinical disease severity and duration, in addition to cortical microglial reactivity. Furthermore, treatment with the drug didox results in attenuation of AIS pathology concomitantly with microglial reversion to a less reactive state. Together, our findings suggest that inflammation, but not demyelination, disrupts AIS integrity and that therapeutic intervention may protect and reverse this pathology. GLIA 2016;64:1190-1209.

The ''master clock'' in the suprachiasmatic nucleus (SCN) of the hypothalamus controls most behavioral, physiological, and molecular circadian rhythms in mammals. However, there are other, still unidentified, circadian oscillators that are able to carry out some SCN functions. Here we show that one of these, the methamphetaminesensitive circadian oscillator (MASCO), which generates behavioral rhythms in the absence of the SCN, is based on an entirely different molecular mechanism. We tested mice lacking, or with mutations of, genes that form the canonical circadian machinery. In all cases, animals that were arrhythmic as a consequence of genetic defect expressed circadian locomotor rhythms when treated with methamphetamine. These results strongly support the hypothesis that the mechanism generating MASCO does not involve the molecular feedback loops that underlie canonical circadian rhythmicity. The properties of MASCO may provide insight into the evolution of circadian mechanisms. Importantly, MASCO may play a role in addiction to psychostimulants.mouse ͉ suprachiasmatic nucleus ͉ psychostimulant T he dominant circadian pacemaker in the mammalian brain is contained within the suprachiasmatic nucleus (SCN) of the hypothalamus (1, 2). However, if the SCN is ablated by lesion or suppressed by constant bright light, resulting in arrhythmic locomotor behavior, circadian rhythmicity can be reconstituted with methamphetamine (3, 4). Arrhythmic, SCN-lesioned rats (SCNX) (3) and mice (4) given ad libitum access to drinking water containing a low dose of methamphetamine produce clear, robust locomotor rhythms with circadian periods that are considerably longer than those of intact animals drinking pure water. Such methamphetamine-induced rhythms continue under constant conditions as long as the drug is present in the drinking water and often for several cycles after its removal (4). These experiments, together with the appropriate controls (3, 4), demonstrate that these rhythms are not the result of a non-oscillating ''hourglass'' but rather constitute clear evidence of the existence of an effective circadian pacemaker outside the SCN. We have called this pacemaker the methamphetamine-sensitive circadian oscillator (MASCO) and are studying its properties.As yet very little is known about MASCO despite overwhelming evidence of its existence. Its location is unknown, although it would be surprising if it were not somewhere in the brain, perhaps associated with dopaminergic and/or serotonergic pathways. Parsimony would suggest that it might be generated by the same molecular feedback loops that produce circadian rhythmicity in the SCN itself and in many peripheral cells, tissues, and organs. However there is reason to believe that this may not be the case. Mice carrying the Clock⌬19 gene mutation and others in which the 2 cryptochromes have been knocked out have arrhythmic phenotypes in constant conditions but become rhythmic when given methamphetamine (5, 6). These data suggest that the molecular events that underlie MASCO...

The axon initial segment (AIS) is the site of action potential (AP) initiation, thus a crucial regulator of neuronal activity. In excitatory pyramidal neurons, the high density of voltage-gated sodium channels (NaV1.6) at the distal AIS regulates AP initiation. A surrogate AIS marker, ankyrin-G (ankG) is a structural protein regulating neuronal functional via clustering voltage-gated ion channels. In neuronal circuits, changes in presynaptic input can alter postsynaptic output via AIS structural-functional plasticity. Recently, we showed experimental mild traumatic brain injury (mTBI) evokes neocortical circuit disruption via diffuse axonal injury (DAI) of excitatory and inhibitory neuronal systems. A key finding was that mTBI-induced neocortical electrophysiological changes involved non-DAI/ intact excitatory pyramidal neurons consistent with AIS-specific alterations. In the current study we employed Thy1-yellow fluorescent protein (YFP)-H mice to test if mTBI induces AIS structural and/or functional plasticity within intact pyramidal neurons 2 days after mTBI. We used confocal microscopy to assess intact YFP+ pyramidal neurons in layer 5 of primary somatosensory barrel field (S1BF), whose axons were continuous from the soma of origin to the subcortical white matter (SCWM). YFP+ axonal traces were superimposed on ankG and NaV1.6 immunofluorescent profiles to determine AIS position and length. We found that while mTBI had no effect on ankG start position, the length significantly decreased from the distal end, consistent with the site of AP initiation at the AIS. However, NaV1.6 structure did not change after mTBI, suggesting uncoupling from ankG. Parallel quantitative analysis of presynaptic inhibitory terminals along the postsynaptic perisomatic domain of these same intact YFP+ excitatory pyramidal neurons revealed a significant decrease in GABAergic bouton density. Also within this non-DAI population, patch-clamp recordings of intact YFP+ pyramidal neurons showed AP acceleration decreased 2 days post-mTBI, consistent with AIS functional plasticity. Simulations of realistic pyramidal neuron computational models using experimentally determined AIS lengths showed a subtle decrease is NaV1.6 density is sufficient to attenuate AP acceleration. Collectively, these findings highlight the complexity of mTBI-induced neocortical circuit disruption, involving changes in extrinsic/presynaptic inhibitory perisomatic input interfaced with intrinsic/postsynaptic intact excitatory neuron AIS output.

Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50–100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair.

Studies on a variety of highly regenerative tissues, including the central nervous system (CNS) in non-mammalian vertebrates, have consistently demonstrated that tissue damage induces the formation of an ionic current at the site of injury. These injury currents generate electric fields (EF) that are 100-fold increased in intensity over that measured for uninjured tissue. In vitro and in vivo experiments have convincingly demonstrated that these electric fields (by their orientation, intensity and duration) can drive the migration, proliferation and differentiation of a host of cell types. These cellular behaviors are all necessary to facilitate regeneration as blocking these EFs at the site of injury inhibits tissue repair while enhancing their intensity promotes repair. Consequently, injury-induced currents, and the EFs they produce, represent a potent and crucial signal to drive tissue regeneration and repair. In this review, we will discuss how injury currents are generated, how cells detect these currents and what cellular responses they can induce. Additionally, we will describe the growing evidence suggesting that EFs play a key role in regulating the cellular response to injury and may be a therapeutic target for inducing regeneration in the mammalian CNS.