SignificanceCalcium influx during action potentials triggers neurotransmitter release at presynaptic active zones. Calcium buffers limit the spread of calcium and restrict neurotransmitter release to the vicinity of calcium channels. To sustain synchronous release during repetitive activity, rapid removal of calcium from the active zone is essential, but the underlying mechanisms are unclear. Therefore, we focused on cerebellar mossy fiber synapses, which are among the fastest synapses in the mammalian brain and found very weak presynaptic calcium buffering. One might assume that strong calcium buffering has the potential to efficiently remove calcium from active zones. In contrast, our results show that weak calcium buffering speeds active zone calcium clearance. Thus, the strength of presynaptic buffering limits the rate of synaptic transmission.

When hearing fails, electrical cochlear implants (eCIs) provide the brain with auditory information. One important bottleneck of CIs is the poor spectral selectivity that results from the wide current spread from each of the electrode contacts. Optical CIs (oCIs) promise to make better use of the tonotopic order of spiral ganglion neurons (SGNs) inside the cochlea by spatially confined stimulation. Here, we established multichannel oCIs based on light-emitting diode (LED) arrays and used them for optical stimulation of channelrhodopsin (ChR)−expressing SGNs in rodents. Power-efficient blue LED chips were integrated onto microfabricated 15-μm-thin polyimide-based carriers comprising interconnecting lines to address individual LEDs by a stationary or mobile driver circuitry. We extensively characterized the optoelectronic, thermal, and mechanical properties of the oCIs and demonstrated stability over weeks in vitro. We then implanted the oCIs into ChR-expressing rats and gerbils, and characterized multichannel optogenetic SGN stimulation by electrophysiological and behavioral experiments. Improved spectral selectivity was directly demonstrated by recordings from the auditory midbrain. Long-term experiments in deafened ChR-expressing rats and in nontreated control animals demonstrated specificity of optogenetic stimulation. Behavioral studies on animals carrying a wireless oCI sound processor revealed auditory percepts. This study demonstrates hearing restoration with improved spectral selectivity by an LED-based multichannel oCI system.

Reconstitution of membrane proteins in artificial membranes is an essential prerequisite for functional studies that depend on the context of an intact membrane. While straight-forward protocols for reconstituting proteins in small unilamellar vesicles were developed many years ago, it is much more difficult to prepare large membranes containing membrane proteins at biologically relevant concentrations. Giant unilamellar vesicles (GUVs) represent a model system that is characterised by low curvature, controllable tension, and large surface that can be easily visualised with microscopy, but protein insertion is notoriously difficult. Here we describe a convenient method for efficient generation of GUVs containing functionally active SNARE proteins that govern exocytosis of synaptic vesicles. Preparation of proteo-GUVs requires a simple, in-house-built device, standard and inexpensive electronic equipment, and employs a straight-forward protocol that largely avoids damage of the proteins. The procedure allows upscaling and multiplexing, thus providing a platform for establishing and optimizing preparation of GUVs containing membrane proteins for a diverse array of applications.