Proximal Axon Research Articles

Extraglomerular Excitation of Rat Olfactory Bulb Mitral Cells by Depolarizing GABAergic Synaptic Input.

Principal cells in the olfactory bulb (OB), mitral and tufted cells, receive direct sensory input and generate output signals that are transmitted to downstream cortical targets. Excitatory input from glutamatergic receptor neurons are the primary known sources of rapid excitation to OB principal cells. Principal cells also receive inhibitory input from local GABAergic interneurons in both the glomerular and plexiform layers. Previous work suggests that the functional effect of these inhibitory inputs, including numerous dendrodendritic synapses with GABAergic granule cells, is to reduce firing probability. In this study, we use in vitro patch-clamp recordings to demonstrate that rat (of both sexes) OB mitral cells also can be excited by GABAergic synapses formed outside the glomerular layer. Depolarizing GABAergic responses to focal extracellular stimulation were revealed when fast ionotropic glutamate receptors were blocked, and occurred with short, monosynaptic latencies. These novel synaptic responses were abolished by gabazine, bicuculline, and picrotoxin, three structurally dissimilar GABAA receptor antagonists. The likely location of depolarizing GABAergic input to mitral cells was the proximal axon based on the actions of focally applied gabazine and GABA near this region. Excitatory GABAergic synaptic responses, commonly studied in cortical brain regions, have not been reported previously in OB principal cells. Excitatory GABAergic responses promote action potential firing and provide a mechanism for mitral cells to be excited independently of olfactory sensory input.SIGNIFICANCE STATEMENT Odor stimuli generate distinctive activity patterns in olfactory bulb neurons through a combination of excitatory and inhibitory synaptic interactions. Most of the excitatory drive to each principal cell is assumed to arise from a highly restricted subset of sensory neurons. This study describes a novel second source of synaptic excitation to principal cells to arise from GABAergic inputs to the proximal axon, a common site of action potential initiation. This new pathway provides a synaptic mechanism to excite OB principal cells that is independent of the canonical excitatory sensory input contained in the glomerular layer.

Sensory neurons have an axon initial segment that initiates spontaneous activity in neuropathic pain.

The axon initial segment is a specialized compartment of the proximal axon of CNS neurons where action potentials are initiated. However, it remains unknown whether this domain is assembled in sensory dorsal root ganglion neurons, in which spikes are initiated in the peripheral terminals. Here we investigate whether sensory neurons have an axon initial segment and if it contributes to spontaneous activity in neuropathic pain. Our results demonstrate that myelinated dorsal root ganglion neurons assemble an axon initial segment in the proximal region of their stem axon, enriched in the voltage-gated sodium channels Nav1.1 and Nav1.7. Using correlative immunofluorescence and calcium imaging, we demonstrate that the Nav1.7 channels at the axon initial segment are associated with spontaneous activity. Computer simulations further indicate that the axon initial segment plays a key role in the initiation of spontaneous discharges by lowering their voltage threshold. Finally, using a Cre-based mouse model for time-controlled axon initial segment disassembly, we demonstrate that this compartment is a major source of spontaneous discharges causing mechanical allodynia in neuropathic pain. Thus, an axon initial segment domain is present in sensory neurons and facilitates their spontaneous activity. This study provides a new insight in the cellular mechanisms that cause pathological pain and identifies a new potential target for chronic pain management.

Serotonin transporter binding in major depressive disorder: impact of serotonin system anatomy.

Serotonin transporter (5-HTT) binding deficits are reported in major depressive disorder (MDD). However, most studies have not considered serotonin system anatomy when parcellating brain regions of interest (ROIs). We now investigate 5-HTT binding in MDD in two novel ways: (1) use of a 5-HTT tract-based analysis examining binding along serotonergic axons; and (2) using the Copenhagen University Hospital Neurobiology Research Unit (NRU) 5-HT Atlas, based on brain-wide binding patterns of multiple serotonin receptor types. [11C]DASB 5-HTT PET scans were obtained in 60 unmedicated participants with MDD in a current depressive episode and 31 healthy volunteers (HVs). Binding potential (BPP) was quantified with empirical Bayesian estimation in graphical analysis (EBEGA). Within the [11C]DASB tract, the MDD group showed significantly lower BPP compared with HVs (p = 0.02). This BPP diagnosis difference also significantly varied by tract location (p = 0.02), with the strongest MDD binding deficit most proximal to brainstem raphe nuclei. NRU 5-HT Atlas ROIs showed a BPP diagnosis difference that varied by region (p < 0.001). BPP was lower in MDD in 3/10 regions (p-values < 0.05). Neither [11C]DASB tract or NRU 5-HT Atlas BPP correlated with depression severity, suicidal ideation, suicide attempt history, or antidepressant medication exposure. Future studies are needed to determine the causes of this deficit in 5-HTT binding being more pronounced in proximal axon segments and in only a subset of ROIs for the pathogenesis of MDD. Such regional specificity may have implications for targeting antidepressant treatment, and may extend to other serotonin-related disorders.

Bilateral wallerian degeneration of middle cerebellar peduncles following unilateral pontine infarction

Bilateral lesions of medial cerebellar peduncles are rare and are most frequently observed in acute cerebral infarction due to large-artery atherosclerosis, wallerian degeneration, neurodegenerative diseases (i.e. multiple system atrophy), inflammatory diseases (i.e. neuromyelitis optica, acute demyelinating encephalomyelitis), toxic encephalopathies (i.e. heroin abuse) and primary central nervous system lymphomas [3]. Wallerian degeneration regards progressive anterograde demyelination of the distal axons following injury to the proximal axon. As a result, fibrosis and atrophy of the affected fiber tracts occurs [2]. We present a case report of a patient with the rare finding of bilateral middle cerebral peduncle lesions, due to wallerian degeneration following pontine infarction. We further discuss differential diagnosis upon imaging findings and their clinical utility.

Intraneural Topography of Rat Sciatic Axons: Implications for Polyethylene Glycol Fusion Peripheral Nerve Repair.

Peripheral nerve injuries are the most common type of nerve trauma. We have been working with a novel repair technique using a plasmalemmal fusogen, polyethylene glycol (PEG), to re-fuse the membranes of severed axons. PEG-fusion repair allows for immediate re-innervation of distal targets, prevents axonal degeneration, and improves behavioral recovery. PEG-fusion of severed axons is non-specific, and we have previously reported that following injury and PEG-fusion misconnections between spinal motoneurons and their distal targets were present. Surprisingly, appropriately paired proximal and distal motor axons were observed in all PEG-fused animals. We hypothesized that a topographic organization of axons contributing to the sciatic nerve could explain the incidence of appropriate connections. We traced the course of specific axon populations contributing to the sciatic nerve in young adult male and female rats. Following intraneural injection of Fast Blue into the tibial branch, labeled axons were confined to a discrete location throughout the course of the nerve. Following intramuscular injection of cholera toxin-conjugated horseradish peroxidase into the anterior tibialis, labeled axons were confined to a smaller but still discrete location throughout the nerve. In both cases, the relative locations of labeled axons were consistent bilaterally within animals, as well as across animals and sexes. Thus, the relatively consistent location of specific axon populations could allow for realignment of appropriate populations of axons, and enhanced behavioral recovery seen in PEG-fused animals. Knowing the organization of axons within the sciatic nerve promotes accurate territory realignment during repair, therefore aiding in recovery outcomes.

Engineered neuronal microtissue provides exogenous axons for delayed nerve fusion and rapid neuromuscular recovery in rats.

Nerve injury requiring surgical repair often results in poor functional recovery due to the inability of host axons to re-grow long distances and reform meaningful connections with the target muscle. While surgeons can re-route local axon fascicles to the target muscle, there are no technologies to provide an exogenous source of axons without sacrificing healthy nerves. Accordingly, we have developed tissue engineered neuromuscular interfaces (TE-NMIs) as the first injectable microtissue containing motor and sensory neurons in an anatomically-inspired architecture. TE-NMIs provide axon tracts that are intended to integrate with denervated distal structures and preserve regenerative capacity during prolonged periods without host innervation. Following implant, we found that TE-NMI axons promoted Schwann cell maintenance, integrated with distal muscle, and preserved an evoked muscle response out to 20-weeks post nerve transection in absence of innervation from host axons. By repopulating the distal sheath with exogenous axons, TE-NMIs also enabled putative delayed fusion with proximal host axons, a phenomenon previously not achievable in delayed repair scenarios due to distal axon degeneration. Here, we found immediate electrophysiological recovery after fusion with proximal host axons and improved axon maturation and muscle reinnervation at 24-weeks post-transection (4-weeks following delayed nerve fusion). These findings show that TE-NMIs provide the potential to improve functional recovery following delayed nerve repair.

A mitochondria cluster at the proximal axon initial segment controls axodendritic TAU trafficking in rodent primary and human iPSC-derived neurons

Loss of neuronal polarity and missorting of the axonal microtubule-associated-protein TAU are hallmarks of Alzheimer’s disease (AD) and related tauopathies. Impairment of mitochondrial function is causative for various mitochondriopathies, but the role of mitochondria in tauopathies and in axonal TAU-sorting is unclear. The axon-initial-segment (AIS) is vital for maintaining neuronal polarity, action potential generation, and—here important—TAU-sorting. Here, we investigate the role of mitochondria in the AIS for maintenance of TAU cellular polarity. Using not only global and local mitochondria impairment via inhibitors of the respiratory chain and a locally activatable protonophore/uncoupler, but also live-cell-imaging and photoconversion methods, we specifically tracked and selectively impaired mitochondria in the AIS in primary mouse and human iPSC-derived forebrain/cortical neurons, and assessed somatic presence of TAU. Global application of mitochondrial toxins efficiently induced tauopathy-like TAU-missorting, indicating involvement of mitochondria in TAU-polarity. Mitochondria show a biased distribution within the AIS, with a proximal cluster and relative absence in the central AIS. The mitochondria of this cluster are largely immobile and only sparsely participate in axonal mitochondria-trafficking. Locally constricted impairment of the AIS-mitochondria-cluster leads to detectable increases of somatic TAU, reminiscent of AD-like TAU-missorting. Mechanistically, mitochondrial impairment sufficient to induce TAU-missorting results in decreases of calcium oscillation but increases in baseline calcium, yet chelating intracellular calcium did not prevent mitochondrial impairment-induced TAU-missorting. Stabilizing microtubules via taxol prevented TAU-missorting, hinting towards a role for impaired microtubule dynamics in mitochondrial-dysfunction-induced TAU-missorting. We provide evidence that the mitochondrial distribution within the proximal axon is biased towards the proximal AIS and that proper function of this newly described mitochondrial cluster may be essential for the maintenance of TAU polarity. Mitochondrial impairment may be an upstream event in and therapeutic target for AD/tauopathy.

Muscarinic Acetylcholine Receptor Localization on Distinct Excitatory and Inhibitory Neurons Within the ACC and LPFC of the Rhesus Monkey.

Acetylcholine (ACh) can act on pre- and post-synaptic muscarinic receptors (mAChR) in the cortex to influence a myriad of cognitive processes. Two functionally-distinct regions of the prefrontal cortex—the lateral prefrontal cortex (LPFC) and the anterior cingulate cortex (ACC)—are differentially innervated by ascending cholinergic pathways yet, the nature and organization of prefrontal-cholinergic circuitry in primates are not well understood. Using multi-channel immunohistochemical labeling and high-resolution microscopy, we found regional and laminar differences in the subcellular localization and the densities of excitatory and inhibitory subpopulations expressing m1 and m2 muscarinic receptors, the two predominant cortical mAChR subtypes, in the supragranular layers of LPFC and ACC in rhesus monkeys (Macaca mulatta). The subset of m1+/m2+ expressing SMI-32+ pyramidal neurons labeled in layer 3 (L3) was denser in LPFC than in ACC, while m1+/m2+ SMI-32+ neurons co-expressing the calcium-binding protein, calbindin (CB) was greater in ACC. Further, we found between-area differences in laminar m1+ dendritic expression, and m2+ presynaptic localization on cortico-cortical (VGLUT1+) and sub-cortical inputs (VGLUT2+), suggesting differential cholinergic modulation of top-down vs. bottom-up inputs in the two areas. While almost all inhibitory interneurons—identified by their expression of parvalbumin (PV+), CB+, and calretinin (CR+)—expressed m1+, the localization of m2+ differed by subtype and area. The ACC exhibited a greater proportion of m2+ inhibitory neurons compared to the LPFC and had a greater density of presynaptic m2+ localized on inhibitory (VGAT+) inputs targeting proximal somatodendritic compartments and axon initial segments of L3 pyramidal neurons. These data suggest a greater capacity for m2+-mediated cholinergic suppression of inhibition in the ACC compared to the LPFC. The anatomical localization of muscarinic receptors on ACC and LPFC micro-circuits shown here contributes to our understanding of diverse cholinergic neuromodulation of functionally-distinct prefrontal areas involved in goal-directed behavior, and how these interactions maybe disrupted in neuropsychiatric and neurological conditions.

Length impairments of the axon initial segment in rodent models of attention-deficit hyperactivity disorder and autism spectrum disorder

The axon initial segment (AIS) is a structural neuronal compartment of the proximal axon that plays key roles in sodium channel clustering, action potential initiation, and signal propagation of neuronal outputs. Mutations in constitutive genes of the AIS, such as ANK3, have been identified in patients with neurodevelopmental disorders. Nevertheless, morphological changes in the AIS in neurodevelopmental disorders have not been characterized. In this study, we investigated the length of the AIS in rodent models of attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). We observed abnormalities in AIS length in both animal models. In ADHD model rodents, we observed shorter AIS length in layer 2/3 (L2/3) neurons of the medial prefrontal cortex (mPFC) and primary somatosensory barrel field (S1BF). Further, we observed shorter AIS length in S1BF L5 neurons. In ASD model mice, we observed shorter AIS length in L2/3 and L5 neurons of the S1BF. These results suggest that impairments in AIS length are common phenomena in neurodevelopmental disorders such as ADHD and ASD and may be conserved across species. Our findings provide novel insight into the potential contribution of the AIS to the pathophysiology and pathogenesis of neurodevelopmental disorders.

Extensive Structural Remodeling of the Axonal Arbors of Parvalbumin Basket Cells during Development in Mouse Neocortex.

Parvalbumin-containing (PV+) basket cells are specialized cortical interneurons that regulate the activity of local neuronal circuits with high temporal precision and reliability. To understand how the PV+ interneuron connectivity underlying these functional properties is established during development, we used array tomography to map pairs of synaptically connected PV+ interneurons and postsynaptic neurons from the neocortex of mice of both sexes. We focused on the axon-myelin unit of the PV+ interneuron and quantified the number of synapses onto the postsynaptic neuron, length of connecting axonal paths, and their myelination at different time points between 2 weeks and 7 months of age. We find that myelination of the proximal axon occurs very rapidly during the third and, to a lesser extent, fourth postnatal weeks. The number of synaptic contacts made by the PV+ interneuron on its postsynaptic partner meanwhile is significantly reduced to about one-third by the end of the first postnatal month. The number of autapses, the synapses that PV+ interneurons form on themselves, however, remains constant throughout the examined period. Axon reorganizations continue beyond postnatal month 2, with the postsynaptic targets of PV+ interneurons gradually shifting to more proximal locations, and the length of axonal paths and their myelin becoming conspicuously uniform per connection. These continued microcircuit refinements likely provide the structural substrate for the robust inhibitory effects and fine temporal precision of adult PV+ basket cells.SIGNIFICANCE STATEMENT The axon of adult parvalbumin-containing (PV+) interneurons is highly specialized for fast and reliable neurotransmission. It is myelinated and forms synapses mostly onto the cell bodies and proximal dendrites of postsynaptic neurons for maximal impact. In this study, we follow the development of the PV+ interneuron axon, its myelination and synapse formation, revealing a rapid sequence of axonal reorganization, myelination of the PV+ interneuron proximal axon, and pruning of almost two-thirds of the synapses in an individual connection. This is followed by a prolonged period of axon refinement and additional myelination leading to a remarkable precision of connections in the adult mouse cortex, consistent with the temporal precision and fidelity of PV+ interneuron action.

Establishing the ground squirrel as a superb model for retinal ganglion cell disorders and optic neuropathies

Retinal ganglion cell (RGC) death occurs after optic nerve injury due to acute trauma or chronic degenerative conditions such as optic neuropathies (e.g., glaucoma). Currently, there are no effective therapies to prevent permanent vision loss resulting from RGC death, underlining the need for research on the pathogenesis of RGC disorders. Modeling human RGC/optic nerve diseases in non-human primates is ideal because of their similarity to humans, but has practical limitations including high cost and ethical considerations. In addition, many retinal degenerative disorders are age-related making the study in primate models prohibitively slow. For these reasons, mice and rats are commonly used to model RGC injuries. However, as nocturnal mammals, these rodents have retinal structures that differ from primates - possessing less than one-tenth of the RGCs found in the primate retina. Here we report the diurnal thirteen-lined ground squirrel (TLGS) as an alternative model. Compared to other rodent models, the number and distribution of RGCs in the TLGS retina are closer to primates. The TLGS retina possesses ~600,000 RGCs with the highest density along the equatorial retina matching the location of the highest cone density (visual streak). TLGS and primate retinas also share a similar interlocking pattern between RGC axons and astrocyte processes in the retina nerve fiber layer (RNFL). In addition, using TLGS we establish a new partial optic nerve injury model that precisely controls the extent of injury while sparing a portion of the retina as an ideal internal control for investigating the pathophysiology of axon degeneration and RGC death. Moreover, in vivo optical coherence tomography (OCT) imaging and ex vivo microscopic examinations of the retina in optic nerve injured TLGS confirm RGC loss precedes proximal axon degeneration, recapitulating human pathology. Thus, the TLGS retina is an excellent model, for translational research in neurodegeneration and therapeutic neuroprotection.This study characterizes the composition of the retinal ganglion cell (RGC) layer and the distribution of RGCs in the cone-dominant ground squirrel retina. The RGC density and the anatomical features of axon-astrocyte interaction in the ground squirrel resemble primates, rendering it an excellent alternative model for RGC neurodegeneration and neuroprotection.

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries.

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large (≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Although surgical procedures exist, patients often do not fully recover, and quality of life deteriorates. Peripheral nerves have limited regeneration, and it is usually mediated by Schwann cells and neurotrophic factors, like glial cell line-derived neurotrophic factor, as seen in Wallerian degeneration. Glial cell line-derived neurotrophic factor is a neurotrophic factor known to promote motor neuron survival and neurite outgrowth. Glial cell line-derived neurotrophic factor is upregulated in different forms of nerve injuries like axotomy, sciatic nerve crush, and compression, thus creating great interest to explore this protein as a potential treatment for peripheral nerve injuries. Exogenous glial cell line-derived neurotrophic factor has shown positive effects in regeneration and functional recovery when applied in experimental models of peripheral nerve injuries. In this review, we discuss the mechanism of repair provided by Schwann cells and upregulation of glial cell line-derived neurotrophic factor, the latest findings on the effects of glial cell line-derived neurotrophic factor in different types of peripheral nerve injuries, delivery systems, and complementary treatments (electrical muscle stimulation and exercise). Understanding and overcoming the challenges of proper timing and glial cell line-derived neurotrophic factor delivery is paramount to creating novel treatments to tend to peripheral nerve injuries to improve patients’ quality of life.

Stressed axons craving for glial sugar: links to regeneration?

Stressed axons craving for glial sugar: links to regeneration?

Voltage-Gated Potassium Channels Ensure Action Potential Shape Fidelity in Distal Axons.

The initiation and propagation of the action potential (AP) along an axon allows neurons to convey information rapidly and across distant sites. Although AP properties have typically been characterized at the soma and proximal axon, knowledge of the propagation of APs toward distal axonal domains of mammalian CNS neurons remains limited. We used genetically encoded voltage indicators (GEVIs) to image APs with submillisecond temporal resolution simultaneously at different locations along the long axons of dissociated hippocampal neurons from rat embryos of either sex. We found that APs became sharper and showed remarkable fidelity as they traveled toward distal axons, even during a high-frequency train. Blocking voltage-gated potassium channels (Kv) with 4-AP resulted in an increase in AP width in all compartments, which was stronger at distal locations and exacerbated during AP trains. We conclude that the higher levels of Kv channel activity in distal axons serve to sustain AP fidelity, conveying a reliable digital signal to presynaptic boutons.SIGNIFICANCE STATEMENT The AP represents the electrical signal carried along axons toward distant presynaptic boutons where it culminates in the release of neurotransmitters. The nonlinearities involved in this process are such that small changes in AP shape can result in large changes in neurotransmitter release. Since axons are remarkably long structures, any distortions that APs suffer along the way have the potential to translate into a significant modulation of synaptic transmission, particularly in distal domains. To avoid these issues, distal axons have ensured that signals are kept remarkably constant and insensitive to modulation during a train, despite the long distances traveled. Here, we uncover the mechanisms that allow distal axonal domains to provide a reliable and faithful digital signal to presynaptic terminals.

Ultrastructural characterization of peripheral denervation in a mouse model of Type III spinal muscular atrophy

Spinal muscular atrophy (SMA) is a heritable, autosomal recessive neuromuscular disorder characterized by a loss of the survival of motor neurons (SMN) protein, which leads to degeneration of lower motor neurons, and muscle atrophy. Despite SMA being nosographically classified as a motor neuron disease, recent advances indicate that peripheral alterations at the level of the neuromuscular junction (NMJ), involving the muscle, and axons of the sensory-motor system, occur early, and may even precede motor neuron loss. In the present study, we used a mouse model of slow progressive (type III) SMA, whereby the absence of the mouse SMN protein is compensated by the expression of two human genes (heterozygous SMN1A2G, and SMN2). This leads to late disease onset and prolonged survival, which allows for dissecting slow degenerative steps operating early in SMA pathogenesis. In this purely morphological study carried out at transmission electron microscopy, we extend the examination of motor neurons and proximal axons towards peripheral components, including distal axons, muscle fibers, and also muscle spindles. We document remarkable ultrastructural alterations being consistent with early peripheral denervation in SMA, which may shift the ultimate anatomical target in neuromuscular disease from the spinal cord towards the muscle. This concerns mostly mitochondrial alterations within distal axons and muscle, which are quantified here through ultrastructural morphometry. The present study is expected to provide a deeper knowledge of early pathogenic mechanisms in SMA.

Acute intermittent hypoxia enhances regeneration of surgically repaired peripheral nerves in a manner akin to electrical stimulation

The intrinsic repair response of injured peripheral neurons is enhanced by brief electrical stimulation (ES) at time of surgical repair, resulting in improved regeneration in rodents and humans. However, ES is invasive. Acute intermittent hypoxia (AIH) - breathing alternate cycles of regular air and air with ~50% normal oxygen levels (11% O2), considered mild hypoxia, is an emerging, promising non-invasive therapy that promotes motor function in spinal cord injured rats and humans. AIH can increase neural activity and under moderately severe hypoxic conditions improves repair of peripherally crushed nerves in mice. Thus, we posited an AIH paradigm similar to that used clinically for spinal cord injury, will improve surgically repaired peripheral nerves akin to ES, including an impact on regeneration-associated gene (RAG) expression–a predictor of growth states. Alterations in early RAG expression were examined in adult male Lewis rats that underwent tibial nerve coaptation repair with either 2 days AIH or normoxia control treatment begun on day 2 post-repair, or 1 h ES treatment (20 Hz) at time of repair. Three days post-repair, AIH or ES treatments effected significant and parallel elevated RAG expression relative to normoxia control at the level of injured sensory and motor neuron cell bodies and proximal axon front. These parallel impacts on RAG expression were coupled with significant improvements in later indices of regeneration, namely enhanced myelination and increased numbers of newly myelinated fibers detected 20 mm distal to the tibial nerve repair site or sensory and motor neurons retrogradely labeled 28 mm distal to the repair site, both at 25 days post nerve repair; and improved return of toe spread function 5–10 weeks post-repair. Collectively, AIH mirrors many beneficial effects of ES on peripheral nerve repair outcomes. This highlights its potential for clinical translation as a non-invasive means to effect improved regeneration of injured peripheral nerves.

Axonal TAU Sorting Requires the C-terminus of TAU but is Independent of ANKG and TRIM46 Enrichment at the AIS

Somatodendritic missorting of the axonal protein TAU is a hallmark of Alzheimer’s disease and related tauopathies. Rodent primary neurons and iPSC-derived neurons are used for studying mechanisms of neuronal polarity, including TAU trafficking. However, these models are expensive, time-consuming, and/or require the killing of animals. In this study, we tested four differentiation procedures to generate mature neuron cultures from human SH-SY5Y neuroblastoma cells and assessed the TAU sorting capacity. We show that SH-SY5Y-derived neurons, differentiated with sequential RA/BDNF treatment, are suitable for investigating axonal TAU sorting. These human neurons show pronounced neuronal polarity, axodendritic outgrowth, expression of the neuronal maturation markers TAU and MAP2, and, importantly, efficient axonal sorting of endogenous and transfected human wild-type TAU, similar to mouse primary neurons. We demonstrate that the N-terminal half of TAU is not sufficient for axonal targeting, as a C-terminus-lacking construct (N-term-TAUHA) is not axonally enriched in both neuronal cell models. Importantly, SH-SY5Y-derived neurons do not show the formation of a classical axon initial segment (AIS), indicated by the lack of ankyrin G (ANKG) and tripartite motif-containing protein 46 (TRIM46) at the proximal axon, which suggests that successful axonal TAU sorting is independent of classical AIS formation. Taken together, our results provide evidence that (i) SH-SY5Y-derived neurons are a valuable human neuronal cell model for studying TAU sorting readily accessible at low cost and without animal need, and that (ii) efficient axonal TAU targeting is independent of ANKG or TRIM46 enrichment at the proximal axon in these neurons.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

SummaryNeurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

Methylene blue enhances polyethylene glycol-fusion repair of completely severed rat sciatic nerves.

Complete transection of peripheral mixed nerves immediately produces loss of sensory perception, muscle contractions and voluntary behavior mediated by the severed distal axons. In contrast to natural regeneration (~1 mm/d) of proximal axons that may eventually reinnervate denervated targets, re-innervation is restored within minutes by PEG-fusion that consists of neurorrhaphy and a sequence of well specified hypo- and isotonic calcium-free or calcium-containing solutions, the anti-oxidant methylene blue (MB) and the membrane fusogen polyethylene glycol (PEG). In this study, we examined the relative efficacy of PEG-fusion with no MB (0%), 0.5% MB, or 1% MB on the recovery of voluntary behaviors by female Sprague-Dawley rats with a complete mid-thigh severance of their sciatic nerve bathed in extracellular fluid or calcium-containing isotonic saline. The recovery of voluntary behaviors is the most relevant measure of success of any technique to repair peripheral nerve injuries. We assessed recovery by the sciatic functional index, a commonly used measure of voluntary hindlimb behaviors following complete sciatic transections. We reported that both 1% MB and 0.5% MB in sterile distilled water in our PEG-fusion protocol with neurorrhaphy significantly increased the rate and extent of behavioral recovery compared to PEG plus neurorrhaphy alone. Furthermore, 0.5% MB was as effective as 1% MB in voluntary behavioral recovery as assessed by the sciatic functional index. Since sterile 1% MB is no longer clinically available, we therefore recommend that 0.5% MB be included in upcoming human clinical trials to evaluate the safety and efficacy of PEG-fusion. All animal procedures were approved by the University of Texas Institutional Animal Care and Use Committee (AUP-2019-00225) on September 9, 2020.

Myelination of parvalbumin interneurons shapes the function of cortical sensory inhibitory circuits

Myelination of projection neurons by oligodendrocytes is key to optimize action potential conduction over long distances. However, a large fraction of myelin enwraps the axons of parvalbumin-positive fast-spiking interneurons (FSI), exclusively involved in local cortical circuits. Whether FSI myelination contributes to the fine‐tuning of intracortical networks is unknown. Here we demonstrate that FSI myelination is required for the establishment and maintenance of the powerful FSI-mediated feedforward inhibition of cortical sensory circuits. The disruption of GABAergic synaptic signaling of oligodendrocyte precursor cells prior to myelination onset resulted in severe FSI myelination defects characterized by longer internodes and nodes, aberrant myelination of branch points and proximal axon malformation. Consequently, high-frequency FSI discharges as well as FSI-dependent postsynaptic latencies and strengths of excitatory neurons were reduced. These dysfunctions generated a strong excitation-inhibition imbalance that correlated with whisker-dependent texture discrimination impairments. FSI myelination is therefore critical for the function of mature cortical inhibitory circuits.