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Upon trauma, the adult murine PNS displays a remarkable degree of spontaneous anatomical and functional regeneration. To explore extrinsic mechanisms of neural repair, we carried out single cell analysis of naïve mouse sciatic nerve, peripheral blood mononuclear cells, and crushed sciatic nerves at 1-day, 3-days, and 7-days following injury. During the first week, monocytes and macrophages (Mo/Mac) rapidly accumulate in the injured nerve and undergo extensive metabolic reprogramming. Proinflammatory Mo/Mac in the injured nerve show high glycolytic flux compared to Mo/Mac in blood and dominate the early injury response. They subsequently give way to inflammation resolving Mac, programmed toward oxidative phosphorylation. Nerve crush injury causes partial leakiness of the blood-nerve-barrier, proliferation of endoneurial and perineurial stromal cells, and accumulation of select serum proteins. Micro-dissection of the nerve injury site and distal nerve, followed by single-cell RNA-sequencing, identified distinct immune compartments, triggered by mechanical nerve wounding and Wallerian degeneration, respectively. This finding was independently confirmed with Sarm1-/- mice, where Wallerian degeneration is greatly delayed. Experiments with chimeric mice showed that wildtype immune cells readily enter the injury site in Sarm1-/- mice, but are sparse in the distal nerve, except for Mo. We used CellChat to explore intercellular communications in the naïve and injured PNS and report on hundreds of ligand-receptor interactions. Our longitudinal analysis represents a new resource for nerve regeneration, reveals location specific immune microenvironments, and reports on large intercellular communication networks. To facilitate mining of scRNAseq datasets, we generated the injured sciatic nerve atlas (iSNAT): https://cdb-rshiny.med.umich.edu/Giger_iSNAT/

For a long time, myelin was thought to be restricted to excitatory neurons, and studies on dysmyelination focused primarily on excitatory cells. Recent evidence showed that axons of inhibitory neurons in the neocortex are also myelinated, but the role of myelin on inhibitory circuits remains unknown. Here we studied the impact of mild hypomyelination on both excitatory and inhibitory connectivity in the primary auditory cortex (A1) with well-characterized mouse models of hypomyelination due to loss of oligodendrocyte ErbB receptor signaling. Using laser-scanning photostimulation, we found that mice with mild hypomyelination have reduced functional inhibitory connections to A1 L2/3 neurons without changes in excitatory connections, resulting in altered excitatory/inhibitory balance. These effects are not associated with altered expression of GABAergic and glutamatergic synaptic components, but with reduced density of parvalbumin-positive (PV

Age-related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly. This progressive pathology often has psychological and medical comorbidities, including social isolation, depression, and cognitive decline. Despite ARHL's enormous societal and economic impact, no therapies to prevent or slow its progression exist. Loss of synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs), a.k.a. IHC synaptopathy, is an early event in cochlear aging, preceding neuronal and hair cell loss. To determine if age-related IHC synaptopathy can be prevented, and if this impacts the time-course of ARHL, we tested the effects of cochlear overexpression of neurotrophin-3 (Ntf3) starting at middle age. We chose Ntf3 because this neurotrophin regulates the formation of IHC-SGN synapses in the neonatal period. We now show that triggering Ntf3 overexpression by IHC supporting cells starting in middle age rapidly increases the amplitude of sound-evoked neural potentials compared with age-matched controls, indicating that Ntf3 produces a positive effect on cochlear function when the pathology is minimal. Furthermore, near the end of their lifespan, Ntf3-overexpressing mice have milder ARHL, with larger sound-evoked potentials along the ascending auditory pathway and reduced IHC synaptopathy compared with age-matched controls. Our results also provide evidence that an age-related decrease in cochlear Ntf3 expression contributes to ARHL and that Ntf3 supplementation could serve as a therapeutic for this prevalent disorder. Furthermore, these findings suggest that factors that regulate synaptogenesis during development could prevent age-related synaptopathy in the brain, a process involved in several central nervous system degenerative disorders.

The auditory system detects and encodes sound information with high precision to provide a high-fidelity representation of the environment and communication. In mammals, detection occurs in the peripheral sensory organ (the cochlea) containing specialized mechanosensory cells (hair cells) that initiate the conversion of sound-generated vibrations into action potentials in the auditory nerve. Neural activity in the auditory nerve encodes information regarding the intensity and frequency of sound stimuli, which is transmitted to the auditory cortex through the ascending neural pathways. Glial cells are critical for precise control of neural conduction and synaptic transmission throughout the pathway, allowing for the precise detection of the timing, frequency, and intensity of sound signals, including the sub-millisecond temporal fidelity is necessary for tasks such as sound localization, and in humans, for processing complex sounds including speech and music. In this review, we focus on glia and glia-like cells that interact with hair cells and neurons in the ascending auditory pathway and contribute to the development, maintenance, and modulation of neural circuits and transmission in the auditory system. We also discuss the molecular mechanisms of these interactions, their impact on hearing and on auditory dysfunction associated with pathologies of each cell type.

Neurotrophin-3 growth factor can improve cochlear neuron survival, and localized delivery of this protein to the round window membrane in the middle ear may be able to reverse sensorineural hearing loss. Thus, the goal of this work was to develop an injectable hydrogel delivery system that can allow localized release of neurotrophin-3 in a controlled and sustained manner. We identified a PEG hydrogel formulation that uses thiol-vinyl sulfone Michael addition for crosslinking. This injectable formulation provides elastic hydrogels with higher mechanical rigidity, better bio-adhesion and longer residence time than Poloxamer hydrogels currently being investigated clinically for hearing loss. In vivo, PEG hydrogels induce local immune responses comparable to biocompatible Poloxamer hydrogels, yet they released payloads at a ~5-fold slower rate in the subcutaneous area. Based on this injectable hydrogel formulation, we designed an affinity-based protein release system by modifying PEG hydrogels with affinity peptides specific to neurotrophin-3 proteins. We verified the sustained release of neurotrophin-3 from peptide-conjugated PEG hydrogels resulting from the reversible interaction between peptides and proteins. The rate of affinity-controlled release depends on the polymer concentrations, the affinity of peptides and the peptide-to-protein ratios. Collectively, we developed an injectable hydrogel formulation for localized delivery of neurotrophin-3, which provides affinity-controlled release and longer delivery time compared to Poloxamer hydrogels.

Hidden hearing loss (HHL) is an auditory neuropathy characterized by normal hearing thresholds but reduced amplitude of the sound-evoked auditory nerve compound action potential (CAP). It has been proposed that in humans HHL leads to speech discrimination and intelligibility deficits, particularly in noisy environments. Animal models originally indicated that HHL can be caused by moderate noise exposures or aging, and that loss of inner hair cell (IHC) synapses could be its cause. A recent study provided evidence that transient loss of cochlear Schwann cells also causes permanent auditory deficits in mice which have characteristics of HHL. Histological analysis of the cochlea after auditory nerve remyelination showed a permanent disruption of the myelination patterns at the heminode of type I spiral ganglion neuron (SGN) peripheral terminals, suggesting that this defect could be contributing to HHL. To shed light on the mechanisms of different HHL scenarios and to test their impact on type I SGN activity, we constructed a reduced biophysical model for a population of SGN peripheral axons. We found that the amplitudes of simulated sound-evoked SGN CAPs are lower and have greater latencies when the heminodes are disorganized, i.e. they are placed at different distances from the hair cell rather than at the same distance as seen in the normal cochlea. Thus, our model confirms that disruption of the position of the heminode causes desynchronization of SGN spikes leading to a loss of temporal resolution and reduction of the sound-evoked SGN CAP. We also simulated synaptopathy by removing high threshold IHC-SGN synapses and found that the amplitude of simulated sound-evoked SGN CAPs decreases while latencies remain unchanged, corresponding to what has been observed in noise exposed animals. This model can be used to further study the effects of synaptopathy or demyelination on auditory function.Author summaryHidden hearing loss is an auditory disorder caused by noise exposure, aging or peripheral neuropathy which is estimated to affect 12-15% of the world’s population. It is a ‘hidden’ disorder because subjects have normal hearing thresholds, i.e., the condition cannot be revealed by standard audiological tests, but they report difficulties in understanding speech in noisy environments. Studies on animal models suggest two possible pathogenic mechanisms for hidden hearing loss: (1) loss of synapses between inner hair cells and auditory nerve fibers, and (2) disruption of auditory-nerve myelin. In this study, we constructed a computational model of sound-evoked auditory neuron fiber activity and auditory nerve compound action potential to understand how each one of these mechanisms affects nerve transmission. We show that disruption of auditory-nerve myelin desynchronizes sound-evoked auditory neuron spiking, decreasing the amplitude and increasing the latency of the compound action potential. In addition, elongation of the initial axon segment may cause spike generation failure leading to decreased spiking probability. In contrast, the effect of synapse loss is only to decrease the probability of firing, thus reducing the compound action potential amplitude without disturbing its latency. This model, which accurately represents the in vivo findings, could be useful to make further predictions on the consequences of HHL and extend it to explore the impact of synaptopathy and myelinopathy on hearing.

For a long time, myelin was thought to be restricted to excitatory neurons, and studies on dysmyelination focused primarily on excitatory cells. Recent evidence showed that axons of inhibitory neurons in the neocortex are also myelinated, but the role of myelin on inhibitory circuits remains unknown. Here we studied the impact of mild hypomyelination on both excitatory and inhibitory connectivity in the primary auditory cortex (A1) with well-characterized mouse models of hypomyelination due to loss of oligodendrocyte ErbB receptor signaling. Using laser-scanning photostimulation, we found that mice with mild hypomyelination have reduced functional inhibitory connections to A1 L2/3 neurons without changes in excitatory connections, resulting in altered excitatory/inhibitory balance. These effects are not associated with altered expression of GABAergic and glutamatergic synaptic components, but with reduced density of parvalbumin-positive (PV+) neurons, which reflects reduced PV expression by interneurons rather than PV+ neuronal loss. While immunostaining shows that hypomyelination occurs in both PV+ and PV- axons, there is a strong correlation between MBP and PV expression suggesting that myelination influences PV expression. Together, the results demonstrate that mild hypomyelination impacts A1 neuronal networks, reducing inhibitory activity, and shifting networks towards excitation.

SUMMARY Dentate gyrus (DG) development requires specification of granule cell (GC) progenitors in the hippocampal neuroepithelium, as well as their proliferation and migration into the primordial DG. We identify the Plexin family members Plxna2 and Plxna4 as important regulators of DG development. Distribution of immature GCs is regulated by Sema5A signaling through PlxnA2 and requires a functional PlxnA2 GTPase-activating protein (GAP) domain and Rap1 small GTPases. In adult Plxna2−/− but not Plxna2-GAP-deficient mice, the dentate GC layer is severely malformed, neurogenesis is compromised, and mossy fibers form aberrant synaptic boutons within CA3. Behavioral studies with Plxna2−/− mice revealed deficits in associative learning, sociability, and sensorimotor gating—traits commonly observed in neuropsychiatric disorder. Remarkably, while morphological defects are minimal in Plxna2-GAP-deficient brains, defects in fear memory and sensorimotor gating persist. Since allelic variants of human PLXNA2 and RAP1 associate with schizophrenia, our studies identify a biochemical pathway important for brain development and mental health., In Brief Zhao et al. find that Sema5A-PlexinA2 forward signaling through Rap1 GTPases is required for progenitor distribution in the developing mouse dentate gyrus. Adult Plxna2−/−, but not Plxna2-GAP-deficient, mice show defects in dentate morphology, neurogenesis, and mossy fiber connectivity. Plxna2−/− and Plxna2-GAP mice exhibit behavioral defects suggestive of neuropsychiatric illness.

Myelin allows for the rapid and precise timing of action potential propagation along neuronal circuits and is essential for healthy auditory system function. In this article, we discuss what is currently known about myelin in the auditory system with a focus on the timing of myelination during auditory system development, the role of myelin in supporting peripheral and central auditory circuit function, and how various myelin pathologies compromise auditory information processing. Additionally, in keeping with the increasing recognition that myelin is dynamic and is influenced by experience throughout life, we review the growing evidence that auditory sensory deprivation alters myelin along specific segments of the brain's auditory circuit. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 80-92, 2018.

Juvenile social experience is crucial for the functional development of forebrain regions, especially the prefrontal cortex (PFC). We previously reported that social isolation for 2 weeks after weaning induces prefrontal cortex dysfunction and hypomyelination. However, the effect of social isolation on physiological properties of PFC neuronal circuit remained unknown. Since hypomyelination due to isolation is prominent in deep-layer of medial PFC (mPFC), we focused on 2 types of Layer-5 pyramidal cells in the mPFC: prominent h-current (PH) cells and nonprominent h-current (non-PH) cells. We found that a 2-week social isolation after weaning leads to a specific deterioration in action potential properties and reduction in excitatory synaptic inputs in PH cells. The effects of social isolation on PH cells, which involve reduction in functional glutamatergic synapses and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl-d-aspartate charge ratio, are specific to the 2 weeks after weaning and to the mPFC. We conclude that juvenile social experience plays crucial roles in the functional development in a subtype of Layer-5 pyramidal cells in the mPFC. Since these neurons project to subcortical structures, a deficit in social experience during the critical period may result in immature neural circuitry between mPFC and subcortical targets.

Experience with procurement of research funding and grantsmanship is an essential skill and one that is rarely taught in a manner that adequately prepares trainees for the magnitude of this professional requirement. The aims of the program described in this article are (1) to provide a mentored experience in grantsmanship through designing and concisely outlining an individual research study and (2) to supplement extramural funding mechanisms for clinical trainees to produce meaningful and substantive clinical and/or basic science research. A total of $10,000 of departmental chair discretionary funds is allocated for resident research annually. The first 2 cycles have successfully allocated the allotted funding through a competitive, scored grant evaluation process. Awardees have already produced meaningful data that have been nationally presented, submitted for publication, and integrated into an National Institutes of Health grant submission. The feasibility of implementing an intramural competitive resident research grant may have broad application within varied training environments.

Noise exposures causing only transient threshold shifts can destroy auditory-nerve synapses without damaging hair cells. Here, we asked whether virally mediated neurotrophin3 (NT3) overexpression can repair this damage. CBA/CaJ mice at 6 wks were injected unilaterally with adeno-associated virus (AAV) containing either NT3 or GFP genes, via the posterior semicircular canal, 3 wks prior to, or 5 hrs after, noise exposure. Controls included exposed animals receiving vehicle only, and unexposed animals receiving virus. Thresholds were measured 2 wks post-exposure, just before cochleas were harvested for histological analysis. In separate virus-injected animals, unexposed cochleas were extracted for qRT-PCR. The GFP reporter showed that inner hair cells (IHCs) were transfected throughout the cochlea, and outer hair cells mainly in the apex. qRT-PCR showed 4- to 10-fold overexpression of NT3 from 1–21 days post-injection, and 1.7-fold overexpression at 40 days. AAV-NT3 delivered prior to noise exposure produced a dose-dependent reduction of synaptopathy, with nearly complete rescue at some cochlear locations. In unexposed ears, NT3 overexpression did not affect thresholds, however GFP overexpression caused IHC loss. In exposed ears, NT3 overexpression increased permanent threshold shifts. Thus, although NT3 overexpression can minimize noise-induced synaptic damage, the forced overexpression may be harmful to hair cells themselves during cochlear overstimulation.

Animal-based studies have provided important insights into the structural and functional consequences of noise exposure on the cochlea. Yet, less is known about the molecular mechanisms by which noise induces cochlear damage, particularly at relatively low exposure levels. While there is ample evidence that noise exposure leads to changes in inner ear metabolism, the specific effects of noise exposure on the cochlear metabolome are poorly understood. In this study we applied liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based metabolomics to analyze the effects of noise on the mouse inner ear. Mice were exposed to noise that induces temporary threshold shifts, synaptopathy and permanent hidden hearing loss. Inner ears were harvested immediately after exposure and analyzed by targeted metabolomics for the relative abundance of 220 metabolites across the major metabolic pathways in central carbon metabolism. We identified 40 metabolites differentially affected by noise. Our approach detected novel noise-modulated metabolites and pathways, as well as some already linked to noise exposure or cochlear function such as neurotransmission and oxidative stress. Furthermore, it showed that metabolic effects of noise on the inner ear depend on the intensity and duration of exposure. Collectively, our results illustrate that metabolomics provides a powerful approach for the characterization of inner ear metabolites affected by auditory trauma. This type of information could lead to the identification of drug targets and novel therapies for noise-induced hearing loss.

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.

Background: Neosaxitoxin (NeoSTX) is a site-1 sodium channel blocker undergoing clinical trials as a prolonged-duration local anesthetic. Rat sciatic block and intravenous infusion models were used to assess efficacy and local and systemic toxicities for NeoSTX in saline (NeoSTX-Saline), bupivacaine (Bup), and their combination (NeoSTX-Bup). Exploratory studies evaluated the effects of addition of epinephrine to NeoSTX-Bup (NeoSTX-Bup-Epi). Methods: Rats received percutaneous sciatic blocks with escalating doses of NeoSTX-Saline or NeoSTX-Bup. Sensory-nocifensive block was assessed using modified hotplate and Von Frey filaments. Motor-proprioceptive function was assessed by extensor postural thrust. Nerves were examined histologically after 7 days and scored on the Estebe–Myers scale. Median lethal dose was estimated for NeoSTX-Saline and in combinations. Accidental intravenous overdose was simulated in isoflurane-anesthetized, spontaneously breathing rats receiving NeoSTX-Saline (n = 6), Bup (n = 7), or NeoSTX-Bup (n = 13), with respiratory, hemodynamic, and electrocardiographic endpoints. Additional groups received blocks with NeoSTX-Bup-Epi (n = 80). Investigators were blinded for behavioral and histologic studies. Results: NeoSTX-Bup produced more prolonged sensory and motor block compared with NeoSTX-Saline or Bup. NeoSTX-Bup-Epi further prolonged median time to near-complete recovery for 3 μg/kg NeoSTX-Bup (hotplate: 48 vs. 6 h, P < 0.001). With sciatic injections, addition of Bup did not worsen the systemic toxicity (median lethal dose) compared with NeoSTX-Saline. Intravenous NeoSTX-Saline infusion had significantly longer times to apnea, first arrhythmia, and asystole compared with Bup (P < 0.001 for each). Histologic injury scores overall were low for all groups, with median scores of 0 (interquartile range, 0 to 0) on a 5-point scale. Conclusion: NeoSTX-Bup and NeoSTX-Bup-Epi hold promise for prolonged-duration local anesthesia.

In the enteric nervous system (ENS), glia outnumber neurons by 4-fold and form an extensive network throughout the gastrointestinal tract. Growing evidence for the essential role of enteric glia in bowel function makes it imperative to understand better their molecular marker expression and how they relate to glia in the rest of the nervous system. We analyzed expression of markers of astrocytes and oligodendrocytes in the ENS and found, unexpectedly, that proteolipid protein 1 (PLP1) is specifically expressed by glia in adult mouse intestine. PLP1 and S100β are the markers most widely expressed by enteric glia, while glial fibrillary acidic protein expression is more restricted. Marker expression in addition to cellular location and morphology distinguishes a specific subpopulation of intramuscular enteric glia, suggesting that a combinatorial code of molecular markers can be used to identify distinct subtypes. To assess the similarity between enteric and extraenteric glia, we performed RNA sequencing analysis on PLP1-expressing cells in the mouse intestine and compared their gene expression pattern to that of other types of glia. This analysis shows that enteric glia are transcriptionally unique and distinct from other cell types in the nervous system. Enteric glia express many genes characteristic of the myelinating glia, Schwann cells and oligodendrocytes, although there is no evidence of myelination in the murine ENS. GLIA 2015;63:2040-2057.

Myelin allows for the rapid and precise timing of action potential propagation along neuronal circuits and is essential for healthy auditory system function. In this article, we discuss what is currently known about myelin in the auditory system with a focus on the timing of myelination during auditory system development, the role of myelin in supporting peripheral and central auditory circuit function, and how various myelin pathologies compromise auditory information processing. Additionally, in keeping with the increasing recognition that myelin is dynamic and is influenced by experience throughout life, we review the growing evidence that auditory sensory deprivation alters myelin along specific segments of the brain's auditory circuit. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 80-92, 2018.

Hidden hearing loss (HHL) is a recently described auditory neuropathy believed to contribute to speech discrimination and intelligibility deficits in people with normal audiological tests. Animals and humans with HHL have normal auditory thresholds but defective cochlear neurotransmission, that is, reduced suprathreshold amplitude of the sound-evoked auditory nerve compound action potential. Currently, the only cellular mechanism known for HHL is loss of inner hair cell synapses (synaptopathy). Here we report that transient loss of cochlear Schwann cells results in permanent auditory deficits characteristic of HHL. This auditory neuropathy is not associated with synaptic loss, but rather with disruption of the first heminodes at the auditory nerve peripheral terminal. Thus, this study identifies a new mechanism for HHL, highlights the long-term consequences of transient Schwann cell loss on hearing and might provide insights into the causes of the auditory deficits reported in patients that recover from acute demyelinating diseases such as Guillain–Barré syndrome., Hidden hearing loss (HHL) is an auditory neuropathy that impairs one's ability to hear, particularly in a noisy environment. Here the authors show that in mice, transient loss of cochlear Schwann cells results in permanent disruption of the cochlear heminodal structure, leading to auditory deficits characteristic of HHL.

Small-fiber neuropathy (SFN) is a disorder of peripheral nerves commonly found in patients with diabetes mellitus, HIV infection, and those receiving chemotherapy. The complexity of disease etiology has led to a scarcity of effective treatments. Using two models of progressive SFN, we show that overexpression of glial cell line-derived neurotrophic factor (GDNF) in skin keratinocytes or topical application of XIB4035, a reported nonpeptidyl agonist of GDNF receptor α1 (GFRα1), are effective treatments for SFN. We also demonstrate that XIB4035 is not a GFRα1 agonist, but rather it enhances GFRα family receptor signaling in conjunction with ligand stimulation. Taken together, our results indicate that topical application of GFRα/RET receptor signaling modulators may be a unique therapy for SFN, and we have identified XIB4035 as a candidate therapeutic agent.

Hidden hearing loss (HHL), a recently described auditory disorder, has been proposed to affect auditory neural processing and hearing acuity in subjects with normal audiometric thresholds, particularly in noisy environments. In contrast to central auditory processing disorders, HHL is caused by defects in the cochlea, the peripheral auditory organ. Noise exposure, aging, ototoxic drugs, and peripheral neuropathies are some of the known risk factors for HHL. Our knowledge of the causes and mechanisms of HHL are based primarily on animal models. However, recent clinical studies have also shed light on the etiology and prevalence of this cochlear disorder and how it may affect auditory perception in humans. Here, we review the current knowledge regarding the causes and cellular mechanisms of HHL, summarize information on available noninvasive tests for differential diagnosis, and discuss potential therapeutic approaches for treatment of HHL.

Sensory epithelia of the inner ear contain two major cell types: hair cells and supporting cells. It has been clear for a long time that hair cells play critical roles in mechanoreception and synaptic transmission. In contrast, until recently the more abundant supporting cells were viewed primarily as serving primarily structural and homeostatic functions. In this review we discuss the growing information about the roles that supporting cells play in the development, function and maintenance of the inner ear, their activities in pathological states, their potential for hair cell regeneration, and the mechanisms underlying these processes.

Upon peripheral nerve injury, specific molecular events, including increases in the expression of selected neurotrophic factors, are initiated to prepare the tissue for regeneration. However, the mechanisms underlying these events and the nature of the cells involved are poorly understood. We used the injury-induced upregulation of glial cell-derived neurotrophic factor (GDNF) expression as a tool to gain insights into these processes. We found that both myelinating and nonmyelinating Schwann cells are responsible for the dramatic increase in GDNF expression after injury. We also demonstrate that the GDNF upregulation is mediated by a signaling cascade involving activation of Schwann cell purinergic receptors, followed by protein kinase C signaling which activates protein kinase D (PKD), which leads to increased GDNF transcription. Given the potent effects of GDNF on survival and repair of injured peripheral neurons, we propose that targeting these pathways may yield therapeutic tools to treat peripheral nerve injury and neuropathies.

In acquired sensorineural hearing loss, such as that produced by noise or aging, there can be massive loss of the synaptic connections between cochlear sensory cells and primary sensory neurons, without loss of the sensory cells themselves. Because the cell bodies and central projections of these cochlear neurons survive for months to years, there is a long therapeutic window in which to re-establish functional connections and improve hearing ability. Here we show in noise-exposed mice that local delivery of neurotrophin-3 (NT-3) to the round window niche, 24 hours after an exposure that causes an immediate loss of up to 50% loss of synapses in the cochlear basal region, can regenerate pre- and post-synaptic elements at the hair cell / cochlear nerve interface. This synaptic regeneration, as documented by confocal microscopy of immunostained cochlear sensory epithelia, was coupled with a corresponding functional recovery, as seen in the suprathreshold amplitude of auditory brainstem response Wave 1. Cochlear delivery of neurotrophins in humans is likely achievable as an office procedure via transtympanic injection, making our results highly significant in a translational context.

Altres ajuts: Fundació La Marato-TV3(TV3201428-10), AFM-Telethon (Nrg14ALS) Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1G93A mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemistry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation.

A Critical Period for Glia The brain develops in fits and starts—while one system is completed, another system may still be under construction. Such transient states are known as critical periods, and during these specific aspects of brain development may become more sensitive to outside agents than they would be later. Makinodan et al. (p. 1357 ) observed the effect of environmental conditions on the brains of mice bioengineered to develop fluorescent oligodendrocytes. The mice were exposed to a variety of social conditions during rearing, ranging from isolation to a normal laboratory cage setting, or to settings enriched with extra buddies and a steady rotation of new play toys. The results show that social isolation leaves a developmental trace that persists into adulthood. Specifically, they found that oligodendrocytes, which produce the myelin that insulates neurons, were underdeveloped, suggesting that there may be a critical period that governs development of these glial oligodendrocyte cells.

Transient receptor potential (TRP) channels have been implicated as sensors of diverse stimuli in mature neurons. However, developmental roles for TRP channels in the establishment of neuronal connectivity remain largely unexplored. Here, we identify an essential function for TRPC5, a member of the canonical TRP subfamily, in the regulation of dendrite patterning in the mammalian brain. Strikingly, TRPC5 knockout mice harbor long, highly branched granule neuron dendrites with impaired dendritic claw differentiation in the cerebellar cortex. In vivo RNAi analyses suggest that TRPC5 regulates dendrite morphogenesis in the cerebellar cortex in a cell-autonomous manner. Correlating with impaired dendrite patterning in the cerebellar cortex, behavioral analyses reveal that TRPC5 knockout mice have deficits in gait and motor coordination. Finally, we uncover the molecular basis of TRPC5's function in dendrite patterning. We identify the major protein kinase calcium/calmodulin-dependent kinase II β (CaMKIIβ) as a critical effector of TRPC5 function in neurons. Remarkably, TRPC5 forms a complex specifically with CaMKIIβ, but not the closely related kinase CaMKIIα, and thereby induces the CaMKIIβ-dependent phosphorylation of the ubiquitin ligase Cdc20-APC at the centrosome. Accordingly, centrosomal CaMKIIβ signaling mediates the ability of TRPC5 to regulate dendrite morphogenesis in neurons. Our findings define a novel function for TRPC5 that couples calcium signaling to a ubiquitin ligase pathway at the centrosome and thereby orchestrates dendrite patterning and connectivity in the brain.

Astrocytes in the hypothalamus release prostaglandin E 2 (PGE 2 ) in response to cell–cell signaling initiated by neurons and glial cells. Upon release, PGE 2 stimulates the secretion of gonadotropin-releasing hormone (GnRH), the neuropeptide that controls reproduction, from hypothalamic neuroendocrine neurons. Whether this effect on GnRH secretion is accompanied by changes in the firing behavior of these neurons is unknown. Using patch-clamp recording we demonstrate that PGE 2 exerts a dose-dependent postsynaptic excitatory effect on GnRH neurons. These effects are mimicked by an EP2 receptor agonist and attenuated by protein kinase A (PKA) inhibitors. The acute blockade of prostaglandin synthesis by indomethacin (INDO) or the selective inhibition of astrocyte metabolism by fluoroacetate (FA) suppresses the spontaneous firing activity of GnRH neurons in brain slices. Similarly, GnRH neuronal activity is reduced in mice with impaired astrocytic PGE 2 release due to defective erbB signaling in astrocytes. These results indicate that astrocyte-to-neuron communication in the hypothalamus is essential for the activity of GnRH neurons and suggest that PGE 2 acts as a gliotransmitter within the GnRH neurosecretory system.

The potential for harmful effects of general anesthesia on the developing brain has been described as pediatric anesthesiology’s greatest current challenge.1 Anesthetic agents appear to have the greatest neurodegenerative impact when exposure occurs during the period of rapid synaptogenesis.2-15 Among species, peak synaptogenesis occurs at different rates and at different parts of the life cycle. This critical period of synaptogenesis in the rat lasts from roughly day of birth to day 10, peaking in cortex at day 7. In humans, this corresponds roughly to a period from the third trimester of gestation and the initial 3 yr of postnatal life. In rats and mice, brain growth is most rapid during the first 2 weeks of postnatal life. The brain’s maturational state in postnatal day 7 mice is comparable to 7-day-old rats and corresponds to premature human neonates.16,17 Studies in infant rat models have shown that exposure to a number of classes of sedative-hypnotic medications, including benzodiazepines, n-methyl-d-aspartate receptor antagonists, and inhalation anesthetics produce striking degrees of apoptotic neurodegeneration, particularly at 7 days postnatal age (P7) but not at postnatal days 21 (P21). Similar concerns have been raised by studies in other species, including infant mice young primates and in brain slice preparations in vitro.2-15 Conversely, other investigators, including one of us (S.S.), have found increased brain apoptosis in sections of parietal and temporal cortex and thalamus in P7 rats with prolonged (6-h) ketamine exposure, but not with brief (1-h) exposure.4,18 During programmed cell death, also termed apoptosis, a cascade of autodigestive enzymes known as caspases are activated. Cleavage of the final member of this cascade, caspase-3, will leads to activation of endonucleases, resulting in apoptotic cell death.19 Case-control studies have shown that infants undergoing surgery under general anesthesia, especially with more than one procedure20 may have a higher frequency of neurodevelopmental difficulties than infants who did not require anesthesia in infancy.20 Conversely, a recent twin study did not find evidence for harmful sequelae of anesthesia in infancy.21 Case-control studies do not fully answer the question of whether the anesthesia per se is associated with increased risk, due to the confounding effect of surgery, postsurgical stress, and other comorbidities that may be more common among infants requiring surgery. To address these concerns, a large international randomized controlled trial is in progress examining neurodevelopmental outcomes at 2 and 5 years of age in a group of infants undergoing inguinal hernia repairs under spinal or general anesthesia.22 The rationale for this trial is based in part on the assumption that spinal anesthesia would not be expected to produce harmful effects on neurodevelopment. Nevertheless, this assumption has not previously been examined in infant animal surrogate models. The present study describes a technique for spinal anesthesia in the young rat. Our general hypothesis is that spinal anesthesia is neurologically benign in young rats. In particular, we hypothesized that (1): a spinal bupivacaine dose could be identified that produced signs of lower body blockade without signs of high spinal blockade, severe physiologic instability, or high mortality, (2) these doses would not increase apoptotic neurodegeneration in the brain or spinal cord (as assayed by cleaved caspase-3 staining), as seen with prolonged exposure to a volatile anesthetic, (3) spinal anesthesia would not produce long-term deficits in motor performance in those rats when tested as adults, and (4) histologic effects of spinal anesthesia on lumbar spinal cord would appear benign.

Recent studies indicate that molecules released by glia can induce synapse formation. However, what induces glia to produce such signals, their identity, and their in vivo relevance remain poorly understood. Here we demonstrate that supporting cells of the vestibular organ—cells that have many characteristics of glia—promote synapse formation only when induced by neuron-derived signals. Furthermore, we identify BDNF as the synaptogenic signal produced by these nonneuronal cells. Mice in which erbB signaling has been eliminated in supporting cells have vestibular dysfunction caused by failure of synapse formation between hair cells and sensory neurons. This phenotype correlates with reduced BDNF expression in supporting cells and is rescued by reexpression of BDNF in these cells. Furthermore, knockdown of BDNF expression in supporting cells postnatally phenocopies the loss of erbB signaling. These results indicate that vestibular supporting cells contribute in vivo to vestibular synapse formation and that this is mediated by reciprocal signals between sensory neurons and supporting cells involving erbB receptors and BDNF.