Experience-dependent flexibility in a molecularly diverse central-to-peripheral auditory feedback system.

Brainstem olivocochlear neurons (OCNs) modulate the earliest stages of auditory processing through feedback projections to the cochlea and have been shown to influence hearing and protect the ear from sound-induced damage. Here, we used single-nucleus sequencing, anatomical reconstructions, and electrophysiology to characterize murine OCNs during postnatal development, in mature animals, and after sound exposure. We identified markers for known medial (MOC) and lateral (LOC) OCN subtypes, and show that they express distinct cohorts of physiologically relevant genes that change over development. In addition, we discovered a neuropeptide-enriched LOC subtype that produces Neuropeptide Y along with other neurotransmitters. Throughout the cochlea, both LOC subtypes extend arborizations over wide frequency domains. Moreover, LOC neuropeptide expression is strongly upregulated days after acoustic trauma, potentially providing a sustained protective signal to the cochlea. OCNs are therefore poised to have diffuse, dynamic effects on early auditory processing over timescales ranging from milliseconds to days.

Just as our pupils dilate or shrink depending on the amount of light available to our eyes, our ears adjust their sensitivity based on the sound environment we encounter. Evidence suggests that a group of cells known as olivocochlear neurons (OCNs for short) may be involved in this process. These cells are located in the brainstem but project into the cochlea, the inner ear structure that converts sound waves into the electrical impulses relayed to the brain. OCNs may mediate how sounds are detected and encoded "at the source." Historically, OCNs have been divided into two groups (medial or lateral OCNs) based on different morphologies and roles in hearing. For instance, medial OCNs are thought to protect our ears against loud sounds by sending molecular signals to the inner ear cells that amplify certain auditory signals. However, it remains difficult to disentangle the precise function of the different types of OCNs, in part because scientists still lack markers that would allow them to distinguish between medial and lateral cells simply based on genetic activity. Frank et al. aimed to eliminate this bottleneck by identifying which genes were switched on and to what degree in individual mouse medial and lateral OCNs; this was done throughout development and after exposure to loud noises. The experiments uncovered a range of genetic markers for medial and lateral OCNs, showing that these cells switch on different sets of genes relevant to their role over development. This gene expression data also revealed that two distinct groups of lateral OCNs exist, one of which is characterised by the production of large amounts of neuropeptides, a type of chemical messenger that can modulate neural circuit activity. Further work in both developing and adult mice showed that this production is shaped by the activity of the cells, with the neuropeptide levels increasing when the animals are exposed to damaging levels of noise. This change lasts for several days, suggesting that such an experience can have long-lasting effects on how the brain provides feedback to the ear. Overall, the results by Frank et al. will help to better identify and characterize the different types of OCNs and the role that they have in hearing. By uncovering the chemical messengers that mediate the response to loud noises, this research may contribute to a better understanding of how to prevent or reduce hearing loss.

Diversity of developing peripheral glia revealed by single-cell RNA sequencing.

The peripheral nervous system responds to a wide variety of sensory stimuli, a process that requires great neuronal diversity. These diverse neurons are closely associated with glial cells originating from the neural crest. However, the molecular nature and diversity among peripheral glia are not understood. Here, we used single-cell RNA sequencing to profile developing and mature glia from somatosensory dorsal root ganglia and auditory spiral ganglia. We found that glial precursors (GPs) in these two systems differ in their transcriptional profiles. Despite their unique features, somatosensory and auditory GPs undergo convergent differentiation to generate molecularly uniform myelinating and non-myelinating Schwann cells. By contrast, somatosensory and auditory satellite glial cells retain system-specific features. Lastly, we identified a glial signature gene set, providing new insights into commonalities among glia across the nervous system. This survey of gene expression in peripheral glia constitutes a resource for understanding functions of glia across different sensory modalities.

Spatiotemporal distribution of glia in and around the developing mouse optic tract.

In the developing mouse optic tract, retinal ganglion cell (RGC) axon position is organized by topography and laterality (i.e., eye-specific or ipsi- and contralateral segregation). Our lab previously showed that ipsilaterally projecting RGCs are segregated to the lateral aspect of the developing optic tract and found that ipsilateral axons self-fasciculate to a greater extent than contralaterally projecting RGC axons in vitro. However, the full complement of axon-intrinsic and -extrinsic factors mediating eye-specific segregation in the tract remain poorly understood. Glia, which are known to express several guidance cues in the visual system and regulate the navigation of ipsilateral and contralateral RGC axons at the optic chiasm, are natural candidates for contributing to eye-specific pre-target axon organization. Here, we investigate the spatiotemporal expression patterns of both putative astrocytes (Aldh1l1+ cells) and microglia (Iba1+ cells) in the embryonic and neonatal optic tract. We quantified the localization of ipsilateral RGC axons to the lateral two-thirds of the optic tract and analyzed glia position and distribution relative to eye-specific axon organization. While our results indicate that glial segregation patterns do not strictly align with eye-specific RGC axon segregation in the tract, we identify distinct spatiotemporal organization of both Aldh1l1+ cells and microglia in and around the developing optic tract. These findings inform future research into molecular mechanisms of glial involvement in RGC axon growth and organization in the developing retinogeniculate pathway.

Eye-specific segregation and differential fasciculation of developing retinal ganglion cell axons in the mouse visual pathway.

Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for axon navigation through intermediate choice points, whether and how axons are organized within tracts is less clear. Here we analyze the organization of retinal ganglion cell (RGC) axons in the developing mouse retinogeniculate pathway. RGC axons are organized by both eye-specificity and topography in the optic nerve and tract: ipsilateral RGC axons are segregated from contralateral axons and are offset laterally in the tract relative to contralateral axon topographic position. To identify potential cell-autonomous factors contributing to the segregation of ipsilateral and contralateral RGC axons in the visual pathway, we assessed their fasciculation behavior in a retinal explant assay. Ipsilateral RGC neurites self-fasciculate more than contralateral neurites in vitro and maintain this difference in the presence of extrinsic chiasm cues. To further probe the role of axon self-association in circuit formation in vivo, we examined RGC axon organization and fasciculation in an EphB1-/- mutant, in which a subset of ipsilateral RGC axons aberrantly crosses the midline but targets the ipsilateral zone in the dorsal lateral geniculate nucleus on the opposite side. Aberrantly crossing axons retain their association with ipsilateral axons in the contralateral tract, indicating that cohort-specific axon affinity is maintained independently of guidance signals present at the midline. Our results provide a comprehensive assessment of RGC axon organization in the retinogeniculate pathway and suggest that axon self-association contributes to pre-target axon organization.

Shh-ushing Midline Crossing through Remote Protein Transport.

Shh contributes to neural circuit formation with different mechanisms. In this issue, Peng and colleagues (2018) identify a novel trans-axonal mechanism by which Shh derived from contralateral projecting retinal ganglion cells prevents midline crossing of Boc-expressing ipsilateral axons at the optic chiasm.

SoxC Transcription Factors Promote Contralateral Retinal Ganglion Cell Differentiation and Axon Guidance in the Mouse Visual System.

Transcription factors control cell identity by regulating diverse developmental steps such as differentiation and axon guidance. The mammalian binocular visual circuit is comprised of projections of retinal ganglion cells (RGCs) to ipsilateral and contralateral targets in the brain. A transcriptional code for ipsilateral RGC identity has been identified, but less is known about the transcriptional regulation of contralateral RGC development. Here we demonstrate that SoxC genes (Sox4, 11, and 12) act on the progenitor-to-postmitotic transition to implement contralateral, but not ipsilateral, RGC differentiation, by binding to Hes5 and thus repressing Notch signaling. When SoxC genes are deleted in postmitotic RGCs, contralateral RGC axons grow poorly on chiasm cells in vitro and project ipsilaterally at the chiasm midline in vivo, and Plexin-A1 and Nr-CAM expression in RGCs is downregulated. These data implicate SoxC transcription factors in the regulation of contralateral RGC differentiation and axon guidance.

Enduring Poststroke Motor Functional Improvements by a Well-Timed Combination of Motor Rehabilitative Training and Cortical Stimulation in Rats.

BACKGROUND: In animal stroke models, peri-infarct cortical stimulation (CS) combined with rehabilitative reach training (RT) enhances motor functional outcome and cortical reorganization, compared with RT alone. It was unknown whether the effects of CS + RT (a) persist long after treatment, (b) can be enhanced by forcing greater use of the paretic limb, and (C) vary with treatment onset time. OBJECTIVE: To test the endurance, time sensitivity, and the potential for augmentation by forced forelimb use of CS + RT treatment effects following ischemic stroke. METHODS: Adult rats that were proficient in skilled reaching received unilateral ischemic motor cortical lesions. RT was delivered for 3 weeks alone or concurrently with 100-Hz cathodal epidural CS, delivered at 50% of movement thresholds. In study 1, this treatment was initiated at 14 days postinfarct, with some subgroups receiving an overlapping period of continuous constraint of the nonparetic forelimb to force use of the paretic limb. The function of the paretic limb was assessed weekly for 9 to 10 months posttreatment. In study 2, rats underwent CS, RT, and the combination during the chronic postinfarct period. RESULTS: Early onset CS + RT resulted in greater functional improvements than RT alone. The CS-related gains persisted for 9 to 10 months posttreatment and were not significantly influenced by forced use of the paretic limb. When treatment onset was delayed until 3 months post-infarct, RT alone improved function, but CS + RT was no more effective than RT alone. CONCLUSION: CS can enhance the persistence, as well as the magnitude of RT-driven functional improvements, but its effectiveness in doing so may vary with time postinfarct.

The LRR receptor Islr2 is required for retinal axon routing at the vertebrate optic chiasm.

BACKGROUND: In the visual system of most binocular vertebrates, the axons of retinal ganglion cells (RGCs) diverge at the diencephalic midline and extend to targets on both ipsi- and contralateral sides of the brain. While a molecular mechanism explaining ipsilateral guidance decisions has been characterized, less is known of how RGC axons cross the midline. RESULTS: Here, we took advantage of the zebrafish, in which all RGC axons project contralaterally at the optic chiasm, to characterize Islr2 as an RGC receptor required for complete retinal axon midline crossing. We used a systematic extracellular protein-protein interaction screening assay to identify two Vasorin paralogs, Vasna and Vasnb, as specific Islr2 ligands. Antibodies against Vasna and Vasnb reveal cellular populations surrounding the retinal axon pathway, suggesting the involvement of these proteins in guidance decisions made by axons of the optic nerve. Specifically, Vasnb marks the membranes of a cellular barricade located anteriorly to the optic chiasm, a structure termed the "glial knot" in higher vertebrates. Loss of function mutations in either vasorin paralog, individually or combined, however, do not exhibit an overt retinal axon projection phenotype, suggesting that additional midline factors, acting either independently or redundantly, compensate for their loss. Analysis of Islr2 knockout mice supports a scenario in which Islr2 controls the coherence of RGC axons through the ventral midline and optic tract. CONCLUSIONS: Although stereotypic guidance of RGC axons at the vertebrate optic chiasm is controlled by multiple, redundant mechanisms, and despite the differences in ventral diencephalic tissue architecture, we identify a novel role for the LRR receptor Islr2 in ensuring proper axon navigation at the optic chiasm of both zebrafish and mouse.

ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.

We describe a clearing method for enhanced visualization of cell morphology and connections in neuronal and non-neuronal tissue. Using Clear(T) or Clear(T2), which are composed of formamide or formamide/polyethylene glycol, respectively, embryos, whole mounts and thick brain sections can be rapidly cleared with minimal volume changes. Unlike other available clearing techniques, these methods do not use detergents or solvents, and thus preserve lipophilic dyes, fluorescent tracers and immunohistochemical labeling, as well as fluorescent-protein labeling.