Biomedical subjects

Andreas Neef

Publications and source records attributed to Andreas Neef.

3 recordsLinked to original sources

PubMed2007

Measurements of membrane patch capacitance using a software-based lock-in system.

On-cell patch-clamp capacitance measurements can resolve the fusion of individual vesicles to a membrane patch and the accompanying dilation of the fusion pore. So far, these measurements have used a patch-clamp amplifier in combination with a hardware lock-in amplifier. Usually, solely the capacitance and conductance outputs of hardware lock-in amplifiers were recorded, which needed to be filtered rather heavily to suppress spectral components at the stimulus frequency. Therefore, the temporal resolution was limited, and information carried in the patch current was not utilized. In this paper, we describe an alternative and more versatile approach for measuring patch capacitance and conductance, using a digitally controlled patch-clamp amplifier. The software lock-in system showed better bandwidth and identical signal-to-noise performance needing less instrumentation. High temporal resolution measurements on patches of chromaffin cells showed that vesicle fission can be completed in only tens of microseconds. Capacitance calculation based on the patch current allows for straightforward offline phase correction. Moreover, the close inspection of direct current for the first time revealed small current changes accompanying the fusion and fission of large secretory vesicles, promising new insights into the vesicles' membrane properties. A practical guide to high-resolution on-cell patch-clamp capacitance measurements using the software lock-in is provided.

Algorithms↗

PubMed2006

Mechanisms underlying the temporal precision of sound coding at the inner hair cell ribbon synapse.

Our auditory system is capable of perceiving the azimuthal location of a low frequency sound source with a precision of a few degrees. This requires the auditory system to detect time differences in sound arrival between the two ears down to tens of microseconds. The detection of these interaural time differences relies on network computation by auditory brainstem neurons sharpening the temporal precision of the afferent signals. Nevertheless, the system requires the hair cell synapse to encode sound with the highest possible temporal acuity. In mammals, each auditory nerve fibre receives input from only one inner hair cell (IHC) synapse. Hence, this single synapse determines the temporal precision of the fibre. As if this was not enough of a challenge, the auditory system is also capable of maintaining such high temporal fidelity with acoustic signals that vary greatly in their intensity. Recent research has started to uncover the cellular basis of sound coding. Functional and structural descriptions of synaptic vesicle pools and estimates for the number of Ca(2+) channels at the ribbon synapse have been obtained, as have insights into how the receptor potential couples to the release of synaptic vesicles. Here, we review current concepts about the mechanisms that control the timing of transmitter release in inner hair cells of the cochlea.

Afferent Pathways↗

PubMed2004

High mobility of vesicles supports continuous exocytosis at a ribbon synapse.

BACKGROUND: Most synapses release neurotransmitter as transient pulses, but ribbon synapses of sensory neurons support continuous exocytosis in response to maintained stimulation. We have investigated how the movement and retrieval of vesicles might contribute to continuous exocytosis at the ribbon synapse of retinal bipolar cells. RESULTS: Using a combination of total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching, we found that the great majority of vesicles within 50-120 nm of the plasma membrane move in a random fashion with an effective diffusion coefficient of approximately 1.5 x 10(-2) microm(2) s(-1). Using confocal microscopy, we found that vesicles are similarly mobile across the whole terminal and that this motion is not altered by calcium influx or the actin cytoskeleton. We calculated that the cytoplasmic reservoir of approximately 300,000 vesicles would generate about 900 vesicle collisions/s against ribbons and 28,000 collisions/s against the surface membrane. The efficient resupply of vesicles to ribbons was confirmed by electron microscopy. A 1 min depolarization, releasing 500-1000 vesicles/s, caused a 70% reduction in the number of vesicles docked at the active zone without reducing the number of vesicles attached to ribbons or remote areas of the plasma membrane. These sites were not repopulated by retrieved vesicles because 80-90% of the recycled membrane was taken up into cisternae that pinched off from the surface. CONCLUSIONS: These results indicate that the random motion of cytoplasmic vesicles provides an efficient supply to the ribbon and plasma membrane and allows the maintenance of high rates of exocytosis without an equally rapid recycling of vesicles. The selective depletion of vesicles docked under ribbons suggests that the transfer of vesicles to the active zone limits the rate of exocytosis during maintained stimulation.

Animals↗