FM1-43 reveals membrane recycling in adult inner hair cells of the mammalian cochlea

Neural transmission of complex sounds demands fast and sustained rates of synaptic release from the primary cochlear receptors, the inner hair cells (IHCs). The cells therefore require efficient membrane recycling. Using two-photon imaging of the membrane marker FM1-43 in the intact sensory epithelium within the cochlear bone of the adult guinea pig, we show that IHCs possess fast calcium-dependent membrane uptake at their apical pole. FM1-43 did not permeate through the stereocilial mechanotransducer channel because uptake kinetics were neither changed by the blockers dihydrostreptomycin and D-tubocurarine nor by treatment of the apical membrane with BAPTA, known to disrupt mechanotransduction. Moreover, the fluid phase marker Lucifer Yellow produced a similar labeling pattern to FM1-43, consistent with FM1-43 uptake via endocytosis. We estimate the membrane retrieval rate at similar to0.5% of the surface area of the cell per second. Labeled membrane was rapidly transported to the base of IHCs by kinesin-dependent trafficking and accumulated in structures that resembled synaptic release sites. Using confocal imaging of FM1-43 in excised strips of the organ of Corti, we show that the time constants of fluorescence decay at the basolateral pole of IHCs and apical endocytosis were increased after depolarization of IHCs with 40 mM potassium, a stimulus that triggers calcium influx and increases synaptic release. Blocking calcium channels with either cadmium or nimodipine during depolarization abolished the rate increase of apical endocytosis. We suggest that IHCs use fast calcium-dependent apical endocytosis for activity-associated replenishment of synaptic membrane

synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses

A Compact Multiphoton 3D Imaging System for Recording Fast Neuronal Activity

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at http://neurospy.org/ under an open source license

A Missense Mutation in a Highly Conserved Alternate Exon of Dynamin-1 Causes Epilepsy in Fitful Mice

Dynamin-1 (Dnm1) encodes a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Mice heterozygous for a novel spontaneous Dnm1 mutation—fitful—experience recurrent seizures, and homozygotes have more debilitating, often lethal seizures in addition to severe ataxia and neurosensory deficits. Fitful is a missense mutation in an exon that defines the DNM1a isoform, leaving intact the alternatively spliced exon that encodes DNM1b. The expression of the corresponding alternate transcripts is developmentally regulated, with DNM1b expression highest during early neuronal development and DNM1a expression increasing postnatally with synaptic maturation. Mutant DNM1a does not efficiently self-assemble into higher order complexes known to be necessary for proper dynamin function, and it also interferes with endocytic recycling in cell culture. In mice, the mutation results in defective synaptic transmission characterized by a slower recovery from depression after trains of stimulation. The DNM1a and DNM1b isoform pair is highly conserved in vertebrate evolution, whereas invertebrates have only one isoform. We speculate that the emergence of more specialized forms of DNM1 may be important in organisms with complex neuronal function

Ectopic synaptic ribbons in dendrites of mouse retinal ON- and OFF-bipolar cells

The ectopic distribution of synaptic ribbons in dendrites of mouse retinal bipolar cells was examined by using genetic ablation of metabotropic glutamate receptor subtype 6 (mGluR6), electron microscopy, and immunocytochemistry. Ectopic ribbons were observed in dendrites of rod and ON-cone bipolar cells in the mGluR6-deficient mouse but not in those of wild-type mice. The number of rod spherules facing the ectopic ribbons in mGluR6-deficient rod bipolar dendrites increased gradually during early growth and reached a plateau level of about 20% at 12 weeks. These ectopic ribbons were immunopositive for RIBEYE, a ribbon-specific protein, but the associated vesicles were immunonegative for synaptophysin, a synaptic-vesicle-specific protein. The presence of ectopic ribbons was correlated with an increase in the roundness of the invaginating dendrites of the rod bipolar cells. We further confirmed ectopic ribbons in dendrites of OFF-cone bipolar cells in wild-type retinas. Of the four types of OFF-cone bipolar cells (T1–T4), only the T2-type, which had a greater number of synaptic ribbons at the axon terminal and a thicker axon cylinder than the other types, had ectopic ribbons. Light-adapted experiments revealed that, in wild-type mice under enhanced-light adaptation (considered similar to the mGluR6-deficient state), the roundness in the invaginating dendrites and axon terminals of rod bipolar cells increased, but no ectopic ribbons were detected. Based on these findings and known mechanisms for neurotransmitter release and protein trafficking, the possible mechanisms underlying the ectopic ribbons are discussed on the basis of intracellular transport for the replenishment of synaptic proteins

FM Dye Photo-Oxidation as a Tool for Monitoring Membrane Recycling in Inner Hair Cells

Styryl (FM) dyes have been used for more than two decades to investigate exo- and endocytosis in conventional synapses. However, they are difficult to use in the inner hair cells of the auditory pathway (IHCs), as FM dyes appear to penetrate through mechanotransducer channels into the cytosol of IHCs, masking endocytotic uptake. To solve this problem we applied to IHCs the FM dye photo-oxidation technique, which renders the dyes into electron microscopy markers. Photo-oxidation allowed the unambiguous identification of labeled organelles, despite the presence of FM dye in the cytosol. This enabled us to describe the morphologies of several organelles that take up membrane in IHCs, both at rest and during stimulation. At rest, endosome-like organelles were detected in the region of the cuticular plate. Larger tubulo-cisternal organelles dominated the top and nuclear regions. Finally, the basal region, where the IHC active zones are located, contained few labeled organelles. Stimulation increased significantly membrane trafficking in the basal region, inducing the appearance of labeled vesicles and cistern-like organelles. The latter were replaced by small, synaptic-like vesicles during recovery after stimulation. In contrast, no changes in membrane trafficking were induced by stimulation in the cuticular plate region or in the top and nuclear regions. We conclude that synaptic vesicle recycling takes place mostly in the basal region of the IHCs. Other organelles participate in abundant constitutive membrane trafficking throughout the rest of the IHC volume.Open-Access-Publikationsfonds 2014peerReviewe

Altered Traveling Wave Propagation and Reduced Endocochlear Potential Associated with Cochlear Dysplasia in the BETA2/NeuroD1 Null Mouse

The BETA2/NeuroD1 null mouse has cochlear dysplasia. Its cochlear duct is shorter than normal, there is a lack of spiral ganglion neurons, and there is hair cell disorganization. We measured vertical movements of the tectorial membrane at acoustic frequencies in excised cochleae in response to mechanical stimulation of the stapes using laser doppler vibrometry. While tuning curve sharpness was similar between wild-type, heterozygotes, and null mice in the base, null mutants had broader tuning in the apex. At both the base and the apex, null mice had less phase lag accumulation with increasing stimulus frequency than wild-type or heterozygote mice. In vivo studies demonstrated that the null mouse lacked distortion product otoacoustic emissions, and the cochlear microphonic and endocochlear potential were found to be severely reduced. Electrically evoked otoacoustic emissions could be elicited, although the amplitudes were lower than those of wild-type mice. Cochlear cross-sections revealed an incomplete partition malformation, with fenestrations within the modiolus that connected the cochlear turns. Outer hair cells from null mice demonstrated the normal pattern of prestin expression within their lateral walls and normal FM 1-43 dye entry. Overall, these data demonstrate that while tonotopicity can exist with cochlear dysplasia, traveling wave propagation is abnormally fast. Additionally, the presence of electrically evoked otoacoustic emissions suggests that outer hair cell reverse transduction is present, although the acoustic response is shaped by the alterations in cochlear mechanics