Frontiers in Cellular Neuroscience

Myelinating Schwann cell (SC)- dorsal root ganglion (DRG) neuron cocultures have been an important technique over the last four decades in understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. While methods using rat SCs and rat DRG neurons are commonplace, there are no established protocols in the field describing the use of mouse SCs with mouse DRG neurons in dissociated myelinating cocultures. There is a great need for such a protocol as this would allow the use of cells from many different transgenic mouse lines. Here we describe a protocol to coculture dissociated mouse SCs and DRG neurons and induce robust myelination. Use of microfluidic chambers permits fluidic isolation for drug treatments, allows cultures to be axotomised to study injury responses, and cells can readily be transfected with lentiviruses to permit live imaging. We used this model to quantify the rate of degeneration after traumatic axotomy in the presence and absence of myelinating SCs and axon aligned SCs that were not induced to myelinate. We find that SCs, irrespective of myelination status, are axo-protective and delay axon degeneration early on. At later time points after injury, we use live imaging of cocultures to show that once axonal degeneration has commenced SCs break up, ingest, and clear axonal debris. Summary statementA novel compartmentalised dissociated mouse myelinating SC-DRG coculture system reveals distinct axo-protective and axo-destructive phases of Schwann cells on axon integrity after trauma.

Social discrimination in rats relies on vasopressin cells (VPCs) intrinsic to the olfactory bulb (OB). We had observed that VPCs responded to electrical stimulation of the olfactory nerve in acute OB slices with inhibitory postsynaptic potentials, and that the neuromodulator acetylcholine could revert these responses to excitation, resulting in action potentials (AP). Moreover, in behaving rats that were exposed to conspecifics, more VPCs were immunopositive to the neural activity marker pERK (pERK+ VPC) than in control rats. However, it is unclear whether these two observations, the increased ERK activation in vivo, and the generation of APs in the presence of ACh in vitro, can be actually mapped onto each other. Here we investigated ERK activation in acute OB slices from transgenic VP-eGFP rats upon either chemical stimulation or tetanic olfactory nerve stimulation. Both KCl and NMDA stimulation resulted in substantial pERK induction across bulbar layers and caused VPC spiking in whole-cell recordings, but only NMDA slightly increased pERK+ VPC percentage. Tetanic olfactory nerve stimulation yielded localized, column-like ERK activation of neurons across bulbar layers. The presence of ACh during tetanic stimulation substantially and specifically increased percentages of pERK+ VPCs within columns, indicating that columnar pERK+ VPCs were preferentially activated by coincident cholinergic neuromodulation and synaptic input. Our results validate pERK induction as a tool to monitor synaptic VPC excitation in the OB, and imply that depolarization of VPCs alone is insufficient to activate ERK. We propose that synaptically evoked APs are a prerequisite for pERK induction in VPCs.

Perineuronal nets (PNNs) are extracellular matrix structures consisting of proteoglycans crosslinked to hyaluronan. They wrap around subgroups of individual neurons in the brain, primarily parvalbumin positive inhibitory neurons. The nets have been found to affect conductances and activation curves of certain ion channels, including Ca2+-activated K+ and high-voltage-activated Ca2+ channels. We studied how PNN related parameters affected the firing rate of one-compartment neuron models. We found that the direct effect of the CaHVA current on firing rate was small, while it had a much larger indirect effect on firing rate through initiation of the SK current. Upregulation of the SK conductance similarly had a pronounced effect on the firing rate. The SK currents therefore acted as the main determinant of firing rate out of these two mechanisms. We shifted the CaHVA channel activation by 14.5 mV and increased the SK conductance by a factor of 3.337, consistent with experimental findings on PNN breakdown in the literature. We studied this in nine different models and found a reduction in firing rate in some, but not all, of these models.

When baseline activity in a neuronal network is modified by external challenges, a set of mechanisms is prompted to homeostatically restore activity levels. These homeostatic mechanisms are thought to be profoundly important in the maturation of the network. We have previously shown that 2-day blockade of either excitatory GABAergic or glutamatergic transmission in the living embryo transiently blocks the movements generated by spontaneous network activity (SNA) in the spinal cord. However, by 2 hours of persistent receptor blockade embryonic movements begin to recover, and by 12 hours we observe a complete homeostatic recovery in vivo. Compensatory changes in voltage-gated conductances in motoneurons were observed by 12 hours of blockade, but not changes in synaptic strength. It was unclear whether changes in voltage-gated conductances were observed by 2 hours of blockade when the recovery actually begins. Further, compensatory changes in voltage-gated conductances were not observed following glutamatergic blockade where embryonic movements were blocked but then recovered in a similar manner to GABAergic blockade. In this study, we discover a mechanism for homeostatic recovery in these first hours of neurotransmitter receptor blockade. In the first 6 hours of GABAergic or glutamatergic blockade there was a clear depolarization of resting membrane potential in both motoneurons and interneurons. These changes reduced action potential threshold and were mainly observed in the continued presence of the antagonist. Therefore, it appears that fast changes in resting membrane potential represent a key fast homeostatic mechanism for the maintenance of network activity in the living embryonic nervous system.\n\nSignificanceHomeostatic plasticity represents a set of mechanisms that act to recover cellular or network activity following a challenge to that activity and is thought to be critical for the developmental construction of the nervous system. The chick embryo afforded us the opportunity to observe in a living developing system the timing of the homeostatic recovery of network activity following 2 distinct perturbations. Because of this advantage, we have identified a novel homeostatic mechanism that actually occurs as the network recovers and is therefore likely to contribute to nervous system homeostasis. We found that a depolarization of the resting membrane potential in the first hour of the perturbations enhances excitability and supports the recovery of embryonic spinal network activity.

Insects provide useful models for investigating evolutionarily conserved mechanisms underlying electrical events associated with brain injury and death. Spreading depolarizations (SD) are transient events that propagate through neuropil whereas the negative ultraslow potential (NUP) is sustained and reflects accumulating damage in the tissue. We used the locust, Locusta migratoria, to investigate ion homeostasis at the blood brain barrier (BBB) during SD and NUP induced by treatment with the Na+/K+-ATPase inhibitor, ouabain. We found that sustained SD caused by the metabolic inhibitor, sodium azide, was associated with a large reduction of K+ efflux through the BBB at ganglia (= grey matter) but not at connectives (= white matter). This was accompanied by a large increase in tissue resistivity but no conductance changes of identified motoneuron dendrites in the neuropil. Males recovered more slowly from ouabain-induced SD, as previously described for anoxic SD. Impairment of barrier functions of the BBB pharmacologically with cyclosporin A or DIDS, or by cutting nerve roots, accelerated the NUP, thus promoting earlier and more frequent SD, but had no effect on the temporal parameters of SD. We conclude that the mechanisms underlying onset and recovery of SD are minimally affected by the damage associated with the NUP. We suggest that future research using tissue-specific genetic approaches in Drosophila to target identified molecular structures of the BBB are likely to be fruitful. New and NoteworthyInhibition of the sodium pump in the locust CNS causes repetitive spreading depolarization (SD) and a negative ultraslow potential (NUP) providing a model for investigation of phenomena relevant to human health. We show that impairment of the blood brain barrier accelerates the NUP but has no impact on the trajectory of SD events. Hence, rapid mechanisms of onset and recovery of ion homeostasis occur against a background of slowly increasing neural damage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/648410v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1d8174corg.highwire.dtl.DTLVardef@1e1a57aorg.highwire.dtl.DTLVardef@13719a9org.highwire.dtl.DTLVardef@75b641_HPS_FORMAT_FIGEXP M_FIG C_FIG

NG2 chondroitin sulfate proteoglycan positive oligodendrocyte progenitor cells (OPCs) reside throughout the brain. They divide asymmetrically and differentiate into myelinating oligodendrocytes throughout adulthood. OPCs have been successfully isolated from rodents using several techniques including magnetic beads, immunopanning and exploiting differential centripetal adhesion. Whereas rat OPCs are relatively simple to propagate in vitro, it has been difficult to expand mouse OPCs. Therefore, we evaluated the effects of oxygen levels, growth factors and extracellular matrix components to produce a simple and reproducible method to prepare large numbers of nearly homogenous cultures of primary mouse OPCs from postnatal day 0-2 mouse telencephala. Using the McCarthy and de Vellis mechanical separation method OPCs were separated from mixed culture of glial cells. When the OPCs were plated onto fibronectin coated tissue culture plates in a biochemically defined medium that contained fibroblast growth factor-2 (FGF-2) and platelet derived growth factor AA (PDGFAA), and they were maintained in a standard tissue culture incubator, they proliferated very slowly. By contrast, mouse OPCs doubled approximately every 7 days when maintained in a 2% oxygen, nitrogen buffered environment. After 3 passages, greater than 99% of these OPCs were NG2+/PDGFR+. In medium containing only FGF-2, mouse OPCs progressed to late stage OPCs whereupon A2B5 expression decreased and O4 expression increased. When these cells were differentiated between passages 1 and 3, the majority of the OPCs differentiated into MBP+ mature oligodendrocytes However, cells that were repeatedly passaged beyond 4 passages progressed to a late O4+ OPC (even with mitogens present) and when differentiated by mitogen removal a minority of the OPCs differentiated into MBP+ cells. These studies reveal significant differences between mouse and rat OPCs and an inhibitory role for oxygen in mouse OPC proliferation.

Neurodegenerative diseases such as Alzheimers and Parkinsons currently affect [~]25 million people worldwide (EO_SCPLOWRKKINENC_SCPLOW et al. 2018). The global incidence of traumatic brain injury (TBI) is estimated at [~]70 million/year (DO_SCPLOWEWANC_SCPLOW et al. 2018). Both neurodegenerative diseases and TBI remain without effective treatments. We are utilizing adult Drosophila melanogaster to investigate the mechanisms of brain regeneration with the long term goal of identifying targets for neural regenerative therapies. Like mammals, Drosophila have few proliferating cells in the adult brain. Nonetheless, within 24 hours of a Penetrating Traumatic Brain Injury (PTBI) to the central brain, there is a significant increase in the number of proliferating cells. We subsequently detect both new glia and new neurons and the formation of new axon tracts that target appropriate brain regions. Glial cells divide rapidly upon injury to give rise to new glial cells. Other cells near the injury site upregulate neural progenitor genes including asense and deadpan and later give rise to the new neurons. Locomotor abnormalities observed after PTBI are reversed within two weeks of injury, supporting the idea that there is functional recovery. Together, these data indicate that adult Drosophila brains are capable of neuronal repair. We anticipate that this paradigm will facilitate the dissection of the mechanisms of neural regeneration and that these processes will be relevant to human brain repair.

In the central nervous system (CNS), axons and its surrounding myelin sheaths, generated by oligodendrocytes, greatly depend on each other, where oligodendrocytes provide axons with both trophic and metabolic support. Across spices, assessment of the axon-myelin ultrastructure is the key-approach to visualize de- and re-myelination of axons. However, this assessment omits to provide information on axonal homeostasis or how axon-myelin influence one another. Since mitochondria may adjust in size thus mirroring the intracellular physiological and metabolic status we applied this to myelinated axons in the CNS. We herein show that a large axonal mitochondria diameter correlates with thinner surrounding myelin sheaths across different CNS tracts and species, including human. We also show that the relation between axonal mitochondria diameter and surrounding myelin thickness is a valuable measurement to verify advanced remyelination in two commonly used experimental demyelinating models, namely the cuprizone and the lysolecithin (LPC) model. Lastly, we show that axonal mitochondria adjust in diameter in response to the thickness of the axonal surrounding myelin whereas the opposite adaption was absent. In summary, the link between axonal mitochondria diameter and surrounding myelin thickness provide insight on the axon-myelin relation both during homeostasis and pathological conditions. This link is also translational applicable and can thus contribute to a better understanding on how to study remyelination using experimental models.

The perineuronal net (PNN) is a condensed form of extracellular matrix (ECM) that enwraps specific populations of neurons and regulates plasticity. To create a PNN, only three classes of components are needed: membrane bound hyaluronan by its synthetic enzyme hyaluronan synthases (HASs), a link protein and a CSPG. However, there is redundancy within the classes as multiple HAS isoforms, link proteins and CSPGs have been found in the PNN in vivo. The effect of this heterogeneity has on PNN function is unresolved. Currently, the most common way to address this question is through the creation and study of PNN component in knockout animals. Here, we reported the development of a primary neuronal culture model which reproduces the in vivo maturation and heterogeneity of PNNs. This model accurately replicated mature cortical PNNs, both in terms of the heterogeneity in PNN composition and its maturation. PNNs transitioned from an immature punctate morphology to the reticular morphology as observed in the mature CNS. We also observed a small population of PNNs that were mature at an earlier time point and a distinct composition, highlighting further heterogeneity. This model will provide a valuable tool for the study of PNN biology, their roles in diseases and the development of PNN focused plasticity treatment.

Interactions between neurons, astrocytes and microglia critically influence neuroinflammatory responses to insult in the central nervous system. Studying neuroinflammation in vitro has been difficult because most primary culture models do not include all three critical cell types. We describe an in vitro model of neuroinflammation comprised of neurons, astrocytes and microglia. Primary rat cortical cells were cultured in a serum-free medium used to co-culture neurons and astrocytes that is supplemented with three factors (IL-34, TGF-{beta} and cholesterol) used to support isolated microglia. This "tri-culture" can be maintained for at least 14 days in vitro while retaining a physiologically-relevant representation of all three cell types. Additionally, we demonstrate that the tri-culture system responds to lipopolysaccharide, mechanical trauma and excitotoxicity with both neurotoxic and neuroprotective aspects of the neuroinflammatory response observed in vivo. We expect the tri-culture model will enable mechanistic studies of neuroinflammation in vitro with enhanced physiological relevance.

Recent work shows that certain immunological assays for the neurofilament light chain NF-L detect informative signals in the CSF and blood of human and animals affected by a variety of CNS injury and disease states. Much of this work has been performed using two mouse monoclonal antibodies to NF-L, UD1 and UD2, also known as 2.1 and 47.3 respectively. These are the essential components of the Uman Diagnostics NF-Light ELISA kit, the Quanterix Simoa bead based NF-L assay and others. We show here that the antibodies bind to neighboring epitopes in a short, conserved and unusual peptide in the NF-L "rod" Coil 2 region. We also describe a surprising and useful feature of Uman and similar reagents. While other well characterized NF-L antibodies show robust staining of countless cells and processes in CNS sections from healthy rats, both Uman antibodies reveal only a minor subset of presumably spontaneously degenerating or degenerated neurons and their processes. However following experimental mid-cervical injuries to rat spinal cord both Uman antibodies recognize numerous profiles in tissue sections. The Uman positive material was associated with fiber tracts expected to be damaged by the injury administered and the profiles had the swollen, beaded, discontinuous and sinusoidal morphology expected for degenerating and degenerated processes. We also found that several antibodies to the C terminal "tail" region of NF-L stain undamaged axonal profiles but fail to recognize the Uman positive material. The unmasking of the Uman epitopes and the loss of the NF-L tail epitopes can be mimicked by treating sections from healthy animals with proteases suggesting that the immunological changes we have discovered are due to neurodegeneration induced proteolysis. We have also generated a novel panel of monoclonal and polyclonal antibody reagents directed against the region of NF-L including the Uman epitopes which have staining properties identical to the Uman reagents. Using these we show that the NF-L region to which the Uman reagents bind contains further hidden epitopes distinct from those recognized by the two Uman reagents. We speculate that the Uman type epitopes are part of a binding region important for higher order neurofilament assembly. The work provides important insights into the properties of the NF-L biomarker, describes novel and useful properties of Uman type and NF-L tail binding antibodies and provides a hypothesis relevant to further understanding of neurofilament assembly.

The Aii glycinergic amacrine cell (Aii) plays a central role in bridging rod pathways with cone pathways, enabling an increased dynamic range of vision from scotopic to photopic ranges. The Aii integrates scotopic signals via chemical synapses from rod bipolar cells (RodBCs) onto the arboreal processes of Aii ACs, injecting signals into ON-cone bipolar cells (CBbs) via gap junctions with Aiis on the arboreal processes and the waist of the Aii ACs. The CBbs then carry this information to ON and OFF ganglion cell classes. In addition, the Aii is involved in the surround inhibition of OFF cone bipolar cells (CBas) through glycinergic chemical synapses from Aii ACs onto CBas. We have previously shown changes in RodBC connectivity as a consequence of rod photoreceptor degeneration in a pathoconnectome of early retinal degeneration: RPC1. Here, we evaluated the impact of rod photoreceptor degeneration on the connectivity of the Aii to determine the impacts of photoreceptor degeneration on the downstream network of the neural retina and its suitability for integrating therapeutic interventions as rod photoreceptors are lost. Previously, we reported that in early retinal degeneration, prior to photoreceptor cell loss, Rod BCs make pathological gap junctions with Aiis. Here, we further characterize this altered connectivity and additional shifts in both the excitatory drive and gap junctional coupling of Aiis in retinal degeneration, along with discussion of the broader impact of altered connectivity networks. New findings reported here demonstrate that Aiis make additional gap junctions with CBas increasing the number of BC classes that make pathological gap junctional connectivity with Aiis in degenerating retina. In this study, we also report that the Aii, a tertiary retinal neuron alters their synaptic contacts early in photoreceptor degeneration, indicating that rewiring occurs in more distant members of the retinal network earlier in degeneration than was previously predicted. This rewiring impacts retinal processing, presumably acuity, and ultimately its ability to support therapeutics designed to restore image-forming vision. Finally, these Aii alterations may be the cellular network level finding that explains one of the first clinical complaints from human patients with retinal degenerative disease, an inability to adapt back and forth from photopic to scotopic conditions.

Multiple Sclerosis is a chronic inflammatory disease in which disability results from the disruption of myelin and axons. During the initial stages of the disease, injured myelin is replaced by mature myelinating oligodendrocytes that differentiate from oligodendrocyte precursor cells. However, myelin repair fails in secondary and chronic progressive stages of the disease and with aging, as the environment becomes progressively more hostile. This may be attributable to inhibitory molecules in the multiple sclerosis environment including activation of the p38MAPK family of kinases. We explored oligodendrocyte precursor cell differentiation and myelin repair using animals with conditional ablation of p38MAPK{gamma} from oligodendrocyte precursors. We found that p38{gamma}MAPK ablation accelerated oligodendrocyte precursor cell differentiation and myelination. This resulted in an increase in both the total number of oligodendrocytes and the migration of progenitors ex vivo and faster remyelination in the cuprizone model of demyelination/remyelination. Consistent with its role as an inhibitor of myelination, p38{gamma}MAPK was significantly downregulated as oligodendrocyte precursor cells matured into oligodendrocytes. Notably, p38{gamma}MAPK was enriched in samples of leukocortical multiple sclerosis lesions from patients, which represent areas of failed remyelination. Our data suggest that p38{gamma} could be targeted to improve myelin repair in multiple sclerosis.

During demyelination, lipid-rich myelin debris is released in the central nervous system (CNS) and must be phagocytosed and processed before new myelin can form. Although myelin comprises over 70% lipids, relatively little is known about how the CNS lipidome changes during demyelination and remyelination. In this study, we obtained a longitudinal lipidomic profile of the brain, spinal cord, and serum using a genetic mouse model of demyelination, known as Plp1-iCKO-Myrf mice. This model has distinct phases of demyelination and remyelination over the course of 24 weeks, in which loss of motor function peaks during demyelination. Using principal component analysis (PCA) and volcano plots, we have demonstrated that the brain and spinal cord have different remyelination capabilities and that this is reflected in different lipidomic profiles over time. We observed that plasmalogens (ether-linked phosphatidylserine and ether-linked phosphatidylcholine) were elevated specifically during the early stages of active demyelination. In addition, we identified lipids in the brain that were altered when mice were treated with a remyelinating drug, which may be CNS biomarkers of remyelination. The results of this study provide new insights into how the lipidome changes in response to demyelination, which will enable future studies to elucidate mechanisms of lipid regulation during demyelination and remyelination.

The cuprizone model is a well-characterized model to study processes of demyelination and remyelination, which are known features of multiple sclerosis. Cuprizone induces oligodendrocyte loss and severe demyelination in the brain, including the corpus callosum, hippocampus, and cortex. Loss of oligodendrocytes and myelin is accompanied by microgliosis and astrogliosis, wherein microglia and astrocytes partially lose their homeostatic functions and acquire a reactive/activated state. Cuprizone-induced demyelination peaks later in grey matter (GM) than in white matter (WM), and remyelination is more efficient in WM areas. Here, we aim to better understand regional diversity in microglia, astrocytes, and oligodendrocytes and their respective role in remyelination efficiency, by characterizing their response to cuprizone across brain regions. We applied spatial transcriptomics (ST) for unbiased gene activity profiling of multiple brain regions in a single tissue section, to identify region-associated changes in gene activity following cuprizone treatment. Gene activity changes were detected in highly abundant cell types, like neurons, oligodendrocytes, and astrocytes, but challenging to detect in low-abundant cell types such as microglia and oligodendrocyte precursor cells. ST revealed a significant increase in the expression of astrocyte markers Clu, Slc1a3, and Gfap during the demyelination phase in the WM fiber tract. In the cortex, the changes in GFAP expression were less prominent, both at the transcriptional and protein level. By mapping genes obtained from scRNAseq of FACS-sorted ACSA2-positive astrocytes onto the ST data, we observed astrocyte heterogeneity beyond the simple classification of WM- and GM-astrocytes in both control and cuprizone-treated mice. In the future, the characterization of these regional astrocyte populations could aid the development of novel strategies to halt the progression of demyelination and support remyelination. Highlights Astrocyte markers Clu, Slc1a3, and Gfap are increased in WM fiber tracts during demyelination Expression dynamics of astrogliosis markers Gfap and Vim during de-and remyelination depend on the brain region Combining scRNAseq with ST data revealed astrocyte heterogeneity beyond WM- and GM-differences scRNAseq-identified gene sets were differently affected by cuprizone treatment across brain regions

Piezo channels are associated with neuropathology in diseases like traumatic brain injury and glaucoma, but pathways linking tissue stretch to aberrant neural signaling remain unclear. The present study demonstrates that Piezo1 activation increases action potential frequency in response to light and the spontaneous dark signal from mouse retinal explants. Piezo1 stimulation was sufficient to increase cytoplasmic Ca2+ in soma and neurites, while stretch increased spiking activity in current clamp recordings from of isolated retinal ganglion cells (RGCs). Axon-marker beta-tubulin III colocalized with both Piezo1 and Piezo2 protein in the mouse optic nerve head, while RGC nuclear marker BRN3A colocalized with Piezo channels in the soma. Piezo1 was also present on GFAP-positive regions in the optic nerve head and colocalized with glutamine synthetase in the nerve fiber layer, suggesting expression in optic nerve head astrocytes and Muller glia end feet, respectively. Human RGCs from induced pluripotent stem cells also expressed Piezo1 and Piezo2 in soma and axons, while staining patterns in rats resembled those in mice. mRNA message for Piezo1 was greatest in the RPE/choroid tissue, while Piezo2 levels were highest in the optic nerve, with both channels also expressed in the retina. Increased expression of Piezo1 and Piezo2 occurred both 1 and 10 days after a single stretch in vivo; this increase suggests a potential role in rising sensitivity to repeated nerve stretch. In summary, Piezo1 and Piezo2 were detected in the soma and axons of RGCs, and stimulation affected the light-dependent output of RGCs. The rise in RGCs excitability induced by Piezo stimulation may have parallels to the early disease progression in models of glaucoma and other retinal degenerations. HighlightsO_LIActivation of Piezo1 excites retinal ganglion cells, paralleling the early neurodegenerative progression in glaucoma mouse models and retinal degeneration. C_LIO_LIPiezo1 and Piezo2 were expressed in axons and soma of retinal ganglion cells in mice, rats, and human iPSC-RGCs. C_LIO_LIFunctional assays confirmed Piezo1 in soma and neurites of neurons. C_LIO_LISustained elevation of Piezo1 and Piezo2 occurred after a single transient stretch may enhance damage from repeated traumatic nerve injury. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/599602v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@7bb1a0org.highwire.dtl.DTLVardef@cab0dcorg.highwire.dtl.DTLVardef@121690eorg.highwire.dtl.DTLVardef@7848fa_HPS_FORMAT_FIGEXP M_FIG Graphical abstract Piezo1 and Piezo2 channels in retinal ganglion cells and the impact of Piezo1 stimulation on light-dependent neural activity. Puttipong Sripinun, Lily P. See, Sergei Nikonov, Venkata Ramana Murthy Chavali, Vrathasha Vrathasha, Jie He, Joan M. OBrien, Jingsheng Xia, Wennan Lu, Claire H. Mitchell*. Activation of Piezo channels through mechanical or pharmacological stimulation leads to an influx of Ca2+ and other cations into RGCs, depolarizing the membrane and increasing the action potential frequency to modulate the visual signal. Created with Biorender.com C_FIG

In adults, peripheral nerves comprise bundles of disseminated motor, sensory and autonomic axons that are considered stable neuroanatomical units. The only exceptions to this established wiring are very distal terminal branches in target organs, such as skin. Here we provide a remarkable deviation from this state of affairs in the peripheral nerves of mice with a conditional knockout of sensory neuron PTEN (phosphatase and tensin homolog deleted on chromosome ten). PTEN is normally expressed in adult sensory neurons, particularly small IB4 nonpeptidergic subtypes and its knockdown after injury or during experimental diabetes improves axon regrowth. We studied Advillin Cre;PTEN null mice lacking PTEN in their sensory neurons. As might be expected, their harvested and cultured DRG neurons displayed enhanced neurite outgrowth in vitro. In vivo, these mice were healthy and had a normal sensory behavioural phenotype. However, the nerves of mice lacking sensory neuron PTEN were highly abnormal, with augmented clusters of small myelinated and unmyelinated axons populating endoneurial fascicles of their peripheral nerve trunks. The axon clusters did not disrupt normal fascicular anatomy but invested the epidermis with greater axon numbers. Within endoneurial fascicles, supernumerary axons formed regenerative units and expressed ongoing growth markers, unlike normal adult axons. This was not accompanied by rises in dorsal root ganglia (DRG) neuron numbers, indicating enhanced distal sprouting from parent neurons. Additionally, sprouting axons were electrophysiologically intact, generating rises in the amplitudes of sensory nerve action potentials. Despite this extensive regenerative activity of intact nerves, regeneration indices after superimposed injury were only modestly enhanced or unchanged. This unusual behaviour of adult sensory axons lacking a single growth-suppressive molecule may identify insights into what molecular constraints the nervous system normally utilizes to suppress inappropriate plasticity.

Improving models to investigate neurodegenerative disease, neurodevelopmental disease, neuro-toxicology and neuropharmacology is critical to improve our basic understanding of the human nervous system, as well as to accelerate discovery of interventions and drugs. Improved models of the human central nervous system could enable critical discoveries related to functional changes induced by sensory stimulation or toxic exposures. Neural organoids, complex three-dimensional cell cultures derived from adult human stem cells, have been grown with complex connectivity and neuroanatomy. Moreover, these cultures have been interfaced with bi-directional electrical stimulation and recording, as well as chemical stimulation. This effort sought to develop new computational techniques which could be applied to comparative studies using neural organoids. In particular, we adapted CEBRA, a state of the art model from the in vivo modeling literature, to generate 2D and 3D embeddings (projections into structured low-dimensional spaces) of high dimensional neural organoid electrophysiology data. This can be done in an unsupervised or semi-supervised manner. Results indicate these embeddings can be quickly and reliably generated and serve as a low-dimensional, interpretable embeddings for characterizing changes in neural organoid activity over time, as well as clustering results around known bursting phenomena. Moreover, we demonstrate that mixtures of von Mises-Fisher distributions can be used as a parametric model for these embedding spaces to enable statistics hypothesis testing. This technique may enable new types of comparative studies using neural organoids, and may be critical for creating a representation for quantitative comparison and validation of neural organoid models against human and animal data. Looking ahead, this work could allow the formulation of a new class of experiments investigating the functional impact of toxins, genetic manipulations, or pharmacological interventions on human neurons.

Myelination is the terminal step in a complex and precisely timed program that orchestrates the proliferation, migration and differentiation of oligodendroglial cells. It is thought that Sonic Hedgehog (Shh) acting on Smoothened (Smo) participates in regulating this process, but that these effects are highly context dependent. Here, we investigate oligodendroglial development and remyelination from three specific transgenic lines: NG2-CreERT2 (control), Smofl/fl/NG2-CreERT2 (loss of function) and SmoM2/NG2-CreERT2 (gain of function), as well as pharmacological manipulation that enhance or inhibit the Smo pathway (SAG or cyclopamine treatment respectively). To explore the effects of Shh/Smo on differentiation and myelination in vivo, we developed a highly quantifiable model by transplanting OPCs in the retina. We find that myelination is greatly enhanced upon cyclopamine treatment and hypothesize that Shh/Smo could promote OPC proliferation to subsequently inhibit differentiation. Consistent with this hypothesis, we find that the genetic activation of Smo significantly increased numbers of OPCs and decreased oligodendrocyte differentiation when we examined the corpus callosum during development and after cuprizone demyelination and remyelination. However, upon loss of function with the conditional ablation of Smo, myelination in the same scenarios are unchanged. Taken together, our present findings suggest that the Shh pathway is sufficient to maintain OPCs in an undifferentiated state, but is not necessary for myelination and remyelination.

AO_SCPLOWBSTRACTC_SCPLOWSpiral ganglion neurons (SGNs) relay auditory information from cochlear hair cells to the central nervous system. After hair cells are destroyed by aminoglycoside antibiotics, SGNs gradually die. However, the reasons for this cochlear neurodegeneration are unclear. We used microarray gene expression profiling to assess transcriptomic changes in the spiral ganglia of kanamycin-deafened and age-matched control rats and found that many of the genes upregulated after deafening are associated with immune/inflammatory responses. In support of this, we observed increased numbers of macrophages in the spiral ganglion of deafened rats. We also found, via CD68 immunoreactivity, an increase in activated macrophages after deafening. An increase in CD68-associated nuclei was observed by postnatal day 23, a time before significant SGN degeneration is observed. Finally, we show that the immunosuppressive drugs dexamethasone and ibuprofen, as well as the NAD salvage pathway activator P7C3, provide at least some neuroprotection post-deafening. Ibuprofen and dexamethasone also decreased the degree of macrophage activation. These results suggest that activated macrophages specifically, and perhaps a more general neuroinflammatory response, are actively contributing to SGN degeneration after hair cell loss.