Outer Hair Cell Glutamate Signaling through Type II Spiral Ganglion Afferents Activates Neurons in the Cochlear Nucleus in Response to Nondamaging Sounds.

Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells, are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons: in this case, onto the sparse Type II SG neurons. However, unlike glutamate signaling at the inner hair cell-Type I SG neuron synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-Type II SG neuron synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-Type II SG pathway to signal to the brain in response to moderate, nondamaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the VGluTs associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to Type II SG neurons in KO mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus in response to sound. From these analyses, we show, for the first time, that glutamatergic signaling at the OHC-Type II SG neuron synapse is capable of activating cochlear nucleus neurons, even at moderate sound levels.SIGNIFICANCE STATEMENT Evidence suggests that cochlear outer hair cells (OHCs) release glutamate onto Type II spiral ganglion neurons only when exposed to loud sound, and that Type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGluT3 for synaptic glutamate release. We then used a genetically modified mouse line in which OHCs, but not inner hair cells, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC-Type II spiral ganglion pathway. Together, these data indicate that glutamate signaling at the OHC-Type II afferent synapse participates in auditory function at moderate sound levels.

Wnt9a Can Influence Cell Fates and Neural Connectivity across the Radial Axis of the Developing Cochlea.

Vertebrate hearing organs manifest cellular asymmetries across the radial axis that underlie afferent versus efferent circuits between the inner ear and the brain. Therefore, understanding the molecular control of patterning across this axis has important functional implications. Radial axis patterning begins before the cells become postmitotic and is likely linked to the onset of asymmetric expression of secreted factors adjacent to the sensory primordium. This study explores one such asymmetrically expressed gene, Wnt9a, which becomes restricted to the neural edge of the avian auditory organ, the basilar papilla, by embryonic day 5 (E5). Radial patterning is disrupted when Wnt9a is overexpressed throughout the prosensory domain beginning on E3. Sexes were pooled for analysis and sex differences were not studied. Analysis of gene expression and afferent innervation on E6 suggests that ectopic Wnt9a expands the neural-side fate, possibly by re-specifying the abneural fate. RNA sequencing reveals quantitative changes, not only in Wnt-pathway genes, but also in genes involved in axon guidance and cytoskeletal remodeling. By E18, these early patterning effects are manifest as profound changes in cell fates [short hair cells (HCs) are missing], ribbon synapse numbers, outward ionic currents, and efferent innervation. These observations suggest that Wnt9a may be one of the molecules responsible for breaking symmetry across the radial axis of the avian auditory organ. Indirectly, Wnt9a can regulate the mature phenotype whereby afferent axons predominantly innervate neural-side tall HCs, resulting in more ribbon synapses per HC compared with abneural-side short HCs with few ribbons and large efferent synapses.SIGNIFICANCE STATEMENT Wnts are a class of secreted factors that are best known for stimulating cell division in development and cancer. However, in certain contexts during development, Wnt-expressing cells can direct neighboring cells to take on specific fates. This study suggests that the Wnt9a ligand may play such a role in the developing hearing organ of the bird cochlea. This was shown through patterning defects that occur in response to the overexpression of Wnt9a. This manipulation increased one type of sensory hair cell (tall HCs) at the expense of another (short HCs) that is usually located furthest from the Wnt9a source. The extraneous tall HCs that replaced short HCs showed some physiological properties and neuronal connections consistent with a fate switch.

Interferon gamma protects neonatal neural stem/progenitor cells during measles virus infection of the brain.

BACKGROUND: In the developing brain, self-renewing neural stem/progenitor cells (NSPC) give rise to neuronal and glial lineages. NSPC survival and differentiation can be altered by neurotropic viruses and by the anti-viral immune response. Several neurotropic viruses specifically target and infect NSPCs, in addition to inducing neuronal loss, which makes it difficult to distinguish between effects on NSPCs that are due to direct viral infection or due to the anti-viral immune response. METHODS: We have investigated the impact of anti-viral immunity on NSPCs in measles virus (MV)-infected neonates. A neuron-restricted viral infection model was used, where NSPCs remain uninfected. Thus, an anti-viral immune response was induced without the confounding issue of NSPC infection. Two-transgenic mouse lines were used: CD46+ mice express the human isoform of CD46, the MV entry receptor, under the control of the neuron-specific enolase promoter; CD46+/IFNγ-KO mice lack the key anti-viral cytokine IFNγ. Multi-color flow cytometry and Western Blot analysis were used to quantify effects on NSPC, neuronal, and glial cell number, and quantify effects on IFNγ-mediated signaling and cell markers, respectively. RESULTS: Flow cytometric analysis revealed that NSPCs were reduced in CD46+/IFNγ-KO mice at 3, 7, and 10 days post-infection (dpi), but were unaffected in CD46+ mice. Early neurons showed the greatest cell loss at 7 dpi in both genotypes, with no effect on mature neurons and glial cells. Thus, IFNγ protected against NSPC loss, but did not protect young neurons. Western Blot analyses on hippocampal explants showed reduced nestin expression in the absence of IFNγ, and reduced doublecortin and ßIII-tubulin in both genotypes. Phosphorylation of STAT1 and STAT2 occurred independently of IFNγ in the hippocampus, albeit with distinct regulation of activation. CONCLUSIONS: This is the first study to demonstrate bystander effects of anti-viral immunity on NSPC function. Our results show IFNγ protects the NSPC population during a neonatal viral CNS infection. Significant loss of NSPCs in CD46+/IFNγ-KO neonates suggests that the adaptive immune response is detrimental to NSPCs in the absence of IFNγ. These results reveal the importance and contribution of the anti-viral immune response to neuropathology and may be relevant to other neuroinflammatory conditions.

A subset of chicken statoacoustic ganglion neurites are repelled by Slit1 and Slit2.

Mechanosensory hair cells in the chicken inner ear are innervated by bipolar afferent neurons of the statoacoustic ganglion (SAG). During development, individual SAG neurons project their peripheral process to only one of eight distinct sensory organs. These neuronal subtypes may respond differently to guidance cues as they explore the periphery in search of their target. Previous gene expression data suggested that Slit repellants might channel SAG neurites into the sensory primordia, based on the presence of robo transcripts in the neurons and the confinement of slit transcripts to the flanks of the prosensory domains. This led to the prediction that excess Slit proteins would impede the outgrowth of SAG neurites. As predicted, axonal projections to the primordium of the anterior crista were reduced 2-3 days after electroporation of either slit1 or slit2 expression plasmids into the anterior pole of the otocyst on embryonic day 3 (E3). The posterior crista afferents, which normally grow through and adjacent to slit expression domains as they are navigating towards the posterior pole of the otocyst, did not show Slit responsiveness when similarly challenged by ectopic delivery of slit to their targets. The sensitivity to ectopic Slits shown by the anterior crista afferents was more the exception than the rule: responsiveness to Slits was not observed when the entire E4 SAG was challenged with Slits for 40 h in vitro. The corona of neurites emanating from SAG explants was unaffected by the presence of purified human Slit1 and Slit2 in the culture medium. Reduced axon outgrowth from E8 olfactory bulbs cultured under similar conditions for 24 h confirmed bioactivity of purified human Slits on chicken neurons. In summary, differential sensitivity to Slit repellents may influence the directional outgrowth of otic axons toward either the anterior or posterior otocyst.

Members of the BMP, Shh, and FGF morphogen families promote chicken statoacoustic ganglion neurite outgrowth and neuron survival in vitro.

Mechanosensory hair cells of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Members of several morphogen families are expressed within and surrounding the chick inner ear during stages of SAG axon outgrowth and pathfinding. On the basis of their localized expression patterns, we hypothesized that bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and sonic hedgehog (Shh) may function as guidance cues for growing axons and/or may function as trophic factors once axons have reached their targets. To test this hypothesis, three-dimensional collagen cultures were used to grow Embryonic Day 4 (E4) chick SAG explants for 24 h in the presence of purified proteins or beads soaked in proteins. The density of neurite outgrowth was quantified to determine effects on neurite outgrowth. Explants displayed enhanced neurite outgrowth when cultured in the presence of purified BMP4, BMP7, a low concentration of Shh, FGF8, FGF10, or FGF19. In contrast, SAG neurons appeared unresponsive to FGF2. Collagen gel cultures were labeled with terminal dUTP nick-end labeling and immunostained with anti-phosphohistone H3 to determine effects on neuron survival and proliferation, respectively. Treatments that increased neurite outgrowth also yielded significantly fewer apoptotic cells, with no effect on cell proliferation. When presented as focal sources, BMP4, Shh, and FGFs -8, -10, and -19 promoted asymmetric outgrowth from the ganglion in the direction of the beads. BMP7-soaked beads did not induce this response. These results suggest that a subset of morphogens enhance both survival and axon outgrowth of otic neurons.

Wnts and Wnt inhibitors do not influence axon outgrowth from chicken statoacoustic ganglion neurons.

The peripheral growth cones of statoacoustic ganglion (SAG) neurons are presumed to sense molecular cues to navigate to their sensory targets during development. Based on previously reported expression data for Frizzled receptors, Wnt ligands, and Wnt inhibitors, we hypothesized that some members of the Wnt morphogen family may function as repulsive cues for SAG neurites. The responses of SAG neurons to mammalian Wnts -1, -4, -5a, -6, and -7b, and the Wnt inhibitors sFRP -1, -2, and -3, were tested in vitro by growing SAG explants from embryonic day 4 (E4) chicken embryos for two days in 3D collagen gels. Average neurite length and density were quantified to determine effects on neurite outgrowth. SAG neurites were strongly repelled by human Sema3E, demonstrating SAG neurons are responsive under these assay conditions. In contrast, SAG neurons showed no changes in neurite outgrowth when cultured in the presence of Wnts and Wnt inhibitors. As an alternative approach, Wnt4 and Wnt5a were also tested in vivo by injecting retroviruses encoding these genes into the chicken otocyst on E3. On E6, no differences were evident in the peripheral projections of SAG axons terminating in infected sensory organs as compared to uninfected organs on the contralateral side of the same embryo. For all Wnt sources, bioactivity was confirmed on E6 chick spinal cord explants by observing enhanced axon outgrowth, as reported previously in the mouse. These results suggest that the tested Wnts do not play a role in guiding peripheral axons in the chicken inner ear.

Non-cell-autonomous planar cell polarity propagation in the auditory sensory epithelium of vertebrates.

Sensory epithelia of the inner ear require a coordinated alignment of hair cell stereociliary bundles as an essential element of mechanoreceptive function. Hair cell bundle alignment is mediated by core planar cell polarity (PCP) proteins, such as Vangl2, that localize asymmetrically to the circumference of the cell near its apical surface. During early phases of cell orientation in the chicken basilar papilla (BP), Vangl2 is present at supporting cell junctions that lie orthogonal to the polarity axis. Several days later, there is a striking shift in the Vangl2 pattern associated with hair cells that reorient towards the distal (apical) end of the organ. How the localization of PCP proteins transmits planar polarity information across the developing sensory epithelium remains unclear. To address this question, the normal asymmetric localization of Vangl2 was disrupted by overexpressing Vangl2 in clusters of cells. The BP was infected with replication-competent retrovirus encoding Vangl2 prior to hair cell differentiation. Virus-infected cells showed normal development of individual stereociliary bundles, indicating that asymmetry was established at the cellular level. Yet, bundles were misoriented in ears infected with Vangl2 virus but not Wnt5a virus. Notably, Vangl2 misexpression did not randomize bundle orientations but rather generated larger variations around a normal mean angle. Cell clusters with excess Vangl2 could induce non-autonomous polarity disruptions in wild-type neighboring cells. Furthermore, there appears to be a directional bias in the propagation of bundle misorientation that is towards the abneural edge of the epithelium. Finally, regional bundle reorientation was inhibited by Vangl2 overexpression. In conclusion, ectopic Vangl2 protein causes inaccurate local propagation of polarity information, and Vangl2 acts in a non-cell-autonomous fashion in the sensory system of vertebrates.

Dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays.

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues and survival factors located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro. Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival.