BetaIVSigma1 spectrin stabilizes the nodes of Ranvier and axon initial segments.

Saltatory electric conduction requires clustered voltage-gated sodium channels (VGSCs) at axon initial segments (AIS) and nodes of Ranvier (NR). A dense membrane undercoat is present at these sites, which is thought to be key for the focal accumulation of channels. Here, we prove that betaIVSigma1 spectrin, the only betaIV spectrin with an actin-binding domain, is an essential component of this coat. Specifically, betaIVSigma1 coexists with betaIVSigma6 at both AIS and NR, being the predominant spectrin at AIS. Removal of betaIVSigma1 alone causes the disappearance of the nodal coat, an increased diameter of the NR, and the presence of dilations filled with organelles. Moreover, in myelinated cochlear afferent fibers, VGSC and ankyrin G clusters appear fragmented. These ultrastructural changes can explain the motor and auditory neuropathies present in betaIVSigma1 -/- mice and point to the betaIVSigma1 spectrin isoform as a master-stabilizing factor of AIS/NR membranes.

Survival, synaptogenesis, and regeneration of adult mouse spiral ganglion neurons in vitro.

The inner ear spiral ganglion is populated by bipolar neurons connecting the peripheral sensory receptors, the hair cells, with central neurons in auditory brain stem nuclei. Hearing impairment is often a consequence of hair cell death, e.g., from acoustic trauma. When deprived of their peripheral targets, the spiral ganglion neurons (SGNs) progressively degenerate. For effective clinical treatment using cochlear prostheses, it is essential to maintain the SGN population. To investigate their survival dependence, synaptogenesis, and regenerative capacity, adult mouse SGNs were separated from hair cells and studied in vitro in the presence of various neurotrophins and growth factors. Coadministration of fibroblast growth factor 2 (FGF-2) and glial cell line-derived neurotrophic factor (GDNF) provided support for long-term survival, while FGF-2 alone could strongly promote neurite regeneration. Fibroblast growth factor receptor FGFR-3-IIIc was found to upregulate and translocate to the nucleus in surviving SGNs. Surviving SGNs formed contacts with other SGNs after they were deprived of the signals from the hair cells. In coculture experiments, neurites extending from SGNs projected toward hair cells. Interestingly, adult mouse spiral ganglion cells could carry out both symmetric and asymmetric cell division and give rise to new neurons. The authors propose that a combination of FGF-2 and GDNF could be an efficient route for clinical intervention of secondary degeneration of SGNs. The authors also demonstrate that the adult mammalian inner ear retains progenitor cells, which could commit neurogenesis.

Cross talk between vestibular neurons and Schwann cells mediates BDNF release and neuronal regeneration.

It is now well-established that an active cross-talk occurs between neurons and glial cells, in the adult as well as in the developing and regenerating nervous systems. These functional interactions not only actively modulate synaptic transmission, but also support neuronal growth and differentiation. We have investigated the possible existence of a reciprocal interaction between inner ear vestibular neurons and Schwann cells maintained in primary cultures. We show that ATP released by the extending vestibular axons elevates intracellular calcium levels within Schwann cells. Purinergic activation of the Schwann P2X(7) receptor induces the release of neurotrophin BDNF, which occurs via a regulated, tetanus-toxin sensitive, vesicular pathway. BDNF, in turn, is required by the vestibular neuron to support its own survival and growth. Given the massive release of ATP during tissue damage, cross-talk between vestibular neurons and Schwann cells could play a primary role during regeneration.

Image-adaptive deconvolution for three-dimensional deep biological imaging.

Deconvolution algorithms are widely used in conventional fluorescence microscopy, but they remain difficult to apply to deep imaging systems such as confocal and two-photon microscopy, due to the practical difficulty of measuring the system's point spread function (PSF), especially in biological experiments. Since a separate PSF measurement performed under the design optical conditions of the microscope cannot reproduce the true experimental conditions prevailing in situ, the most natural approach to solve the problem is to extract the PSF from the images themselves. We investigate here the approach of cropping an approximate PSF directly from the images, by exploiting the presence of small structures within the samples under study. This approach turns out to be practical in many cases, allowing significantly better restorations than with a design PSF obtained by imaging fluorescent beads in gel. We demonstrate the advantages of this approach with a number of deconvolution experiments performed both on artificially blurred and noisy test images, and on real confocal images taken within an in vitro preparation of the mouse hearing organ.

BDNF gene replacement reveals multiple mechanisms for establishing neurotrophin specificity during sensory nervous system development.

Neurotrophins have multiple functions during peripheral nervous system development such as controlling neuronal survival, target innervation and synaptogenesis. Neurotrophin specificity has been attributed to the selective expression of the Trk tyrosine kinase receptors in different neuronal subpopulations. However, despite overlapping expression of TrkB and TrkC in many sensory ganglia, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) null mutant mice display selective losses in neuronal subpopulations. In the present study we have replaced the coding part of the BDNF gene in mice with that of NT3 (BDNF(NT3/NT3)) to analyse the specificity and selective roles of BDNF and NT3 during development. Analysis of BDNF(NT3/NT3) mice showed striking differences in the ability of NT3 to promote survival, short-range innervation and synaptogenesis in different sensory systems. In the cochlea, specificity is achieved by a tightly controlled spatial and temporal ligand expression. In the vestibular system TrkB or TrkC activation is sufficient to promote vestibular ganglion neuron survival, while TrkB activation is required to promote proper innervation and synaptogenesis. In the gustatory system, NT3 is unable to replace the actions of BDNF possibly because of a temporally selective expression of TrkB in taste neurons. We conclude that there is no general mechanism by which neurotrophin specificity is attained and that specificity is achieved by (i) a tightly controlled spatial and temporal expression of ligands, (ii) different Trk receptors playing distinct roles within the same neuronal subpopulation, or (iii) selective receptor expression in sensory neuron subpopulations.