Max Planck Institute for Dynamics and Self-Organization -- Department for Nonlinear Dynamics and Network Dynamics Group
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The Synaptic Nanomachine Underlying Auditory Encoding

The temporal structure of sound impinging on our ears is used by the brain with an astonishingly high degree of temporal resolution on the order of a dozen microseconds. Examples of such high temporal precision of neuronal sound processing range from for binaural sound source localisation to speech recognition [1]. The temporal precision of sound encoding is preserved over many orders of magnitude in sound intensity [1]. Accumulating evidence indicates that the decision to generate an action potential in the auditory nerve relies on the behaviour of a surprisingly small number of molecules. In our current research, we are investigating the dynamics of intracellular signaling that underlies this intriguing example of temporal fidelity using an approach integrating high resolution electrophysiological measurements and biophysical and mathematical modelling. Sound information is conveyed to the brain by action potential patterns of spiral ganglion neurons in the inner ear and auditory nerve (Fig 1B). Individual action potentials of auditory nerve fibers are triggered by vesicle release at specialized synapses (Fig 1D) of the sensory cells, the inner hair cells. These cells perform several signal transduction steps from liquid movement over membrane voltage transients to ion channel gating, Ca2+- influx and finally release of transmitter filled vesicles. Recently, strong evidence was found that at the hair cell synapse several vesicles can be released in a coordinated, synchronized fashion [2,3]. Furthermore, control of vesicle release by Ca2+-channel gating seems to follow a nano-domain regime i.e. the fusion of a certain vesicle is typically triggered by the Ca2+ entering through only one or very few Ca2+-channels adjacent to the vesicle (Fig. 1E;[4,5]). The transmitter content of one vesicle is presumably sufficient to trigger postsynaptic action potential generation. As a consequence, the spike timing in an auditory nerve fiber is determined by the stochastic opening of one or very few Ca2+-channels. Thus the molecular and biophysical organization of the inner hair cell synapse at first sight appears rather vulnerable to molecular noise. In order to understand the mechanistic basis of the temporal fidelity in sound encoding, it is thus important to characterize this intriguing cellular signaling architecture with quantitative precision and to systematically assess how temporal fidelity is achieved by a molecular nanomachine operating in a regime where perceptual decisions are apparently left to single molecule thermal fluctuations.

figure_1_nano_machine.gif

Coordinated vesicle release

Electrophysiological recordings from auditory nerve fibers in rat and bullfrog detected excitatory postsynaptic currents with widely varying amplitude [2,5,6]. This suggests that a presynaptic release event can involve multiple vesicles – either released in a highly synchronous fashion, or fused with each other before release. To clarify whether the observed postsynaptic variability indeed has a presynaptic cause, we used presynaptic whole cell measurements. Model studies demonstrated that non-stationary fluctuation analysis using
non parametric statistical methods of error estimation would be able to estimate the capacitance of an average release event (in the range of 100 aF) on a fluctuating background capacitance of a whole cell (7 fF) and despite the considerable measurement noise (tens of fF peak-to-peak)[3]. Analyzing whole cell capacitance measurements obtained in the perforated patch configuration using these methods, we found the average release event at the ribbon synapse of mouse inner hair cells indeed comprises more than one vesicle (Fig.2A;[3]). However the extend of coordination in our experiments was smaller than expected from postsynaptic recordings. We conclude that only about half of the fusion events involve more than one vesicle [3]. Currently, we are using a detailed biophysical model of local Ca2+-signals to investigate if such synchronous release events might be a consequence of the nano-domain coupling and how the coordination of release might be computationally exploited by postsynaptic mechanisms to encode sound intensity information.

 

Achieving single channel-single Vesicle resolution

To directly observe Ca2+-influx – vesicle secretion coupling at an individual synapse we modified existing methods to reach very low noise levels in simultaneous current and capacitance recordings [7]. It is now possible to simultaneously detect Ca2+-channel openings and the fusion of single vesicles. Initial results showed exocytic events (vesicle fusion) ranging from 50 to 600 aF in size (Fig. 2B), supporting the idea of coordinated multivesicular release. Furthermore these measurements can further scrutinize the hypothesis of nano-domain control. However, some experimental problems remain to be solved as the frequency of fusion events observed is extremely low and the time course of individual events is much slower than expected. In the near future, we are starting to combine single channel/single vesicle detection with physiologically motivated i.e. smooth and periodic stimuli rather than square pulses that are usually used to study synaptic transmission. This will allow us to clarify how the temporal structure of auditory stimuli is encoded at the hair cell synapse. Figure1: A Section through a human ear. The VIII nerve, comprising the auditory fibres is drawn in green. B Diagram of single auditory fibres and respective sound evoked spike trains. C Schematic of a hair cell with 5 ribbon synapses and initial segments of the auditory nerve fibres shown. D Enlargement of a single hair cell synapse, showing the ribbon in dark brown, synaptic vesicles in light brown and Ca2+-channels in blue. E Diagram of Ca2+ channels (diamond symbols) and vesicles (light brown, fusing vesicles crossed out). The range of intracellular [Ca2+] that is sufficient to trigger fusion is schematically represented by the light blue area. Left panel: In a case with high channel open probability but of brief openings the Ca2+-domains around multiple channels merge into a microdomain. Fusion of any vesicle is triggered by Ca2+ entering through a number of channels. Right panel: Longer lasting but rare channel openings create strong Ca2+-nano-domains that can trigger fusion, if a vesicle is sufficiently close to the channel.


figure_2_nano_machine.gif

Towards a biophysical model of the ribbon synapse


The complexity of the sound encoding nanomachine makes it desirable to simulate its function to aid understanding and constrain hypotheses. Fortunately, the large amount of electrophysiological and morphological data makes it possible to create a detailed biophysical model of this system. A detailed biophysical model of the ribbon synapse is currently being implemented with a event driven Monte-Carlo simulation system for single molecule based diffusion-reaction systems (Jentsch et al. in preparation). This model comprises stochastic channel gating and Ca2+-influx, buffered Ca2+ diffusion and binding to the Ca2+ sensors of vesicles that lead to vesicle release.

We plan to extend it to the postsynaptic element. Previous simulations with reduced models demonstrated that exocytosis driven by whole cell calcium currents and using experimentally constrained model of the hair cell Ca2+-sensor failed to reproduce the experimentally observed kinetics. This finding is in line with previous evidence indicating that exocytosis operates under nano-domain control and thus presumably is driven by single channel currents. As a consequence the behavior of any realistic biophysical model of the inner hair cell ribbon synapse is expected to sensitively depend on the precise gating kinetics of individual Ca2+ channels. As currently available data do not sufficiently constrain the dynamics of nano-domain [Ca2+] signals we are currently systematically scanning the impact of different hypothetical single channel kinetics. In the future, our own in situ single Ca2+ channel recordings will yield important tests of and constraints on this key determinant of auditory encoding.
 


Contact:  Fred Wolf 

Members working within this Project:

 Andreas Neef 
 Fred Wolf 

Former Members:

 Nikolai M. Chapochnikov 
 Min Huang 

Selected Publications:

S. Jung, T. Maritzen, C. Wichmann, Z. Jing, A. Neef, N.H. Revelo, H. Al-Moyed, S. Meese, S.M. Wojcik, I.P. , H. Bulut, P. Schu, R. Ficner, E. Reisinger, S.O. Rizzoli, J. Neef, N. Strenzke, V. Haucke, and T. Moser (2015).
Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing
EMBO.

B.N. Buran, N. Strenzke, A. Neef, E.D. Gundelfinger, T. Moser, and M.C. Liberman (2010).
Onset coding is degraded in auditory nerve fibers from mutant mice lacking synaptic ribbons.
Journal of Neuroscience 30(22):7587-97. download file

T. Frank, M.A. Rutherford, N. Strenzke, A. Neef, T. Pangršič, D. Khimich, A. Fejtova, E.D. Gundelfinger, M.C. Liberman, B. Harke, K.E. Bryan, A. Lee, A. Egner, D. Riedel, and T. Moser (2010).
Bassoon and the Synaptic Ribbon Organize Ca2+ Channels and Vesicles to Add Release Sites and Promote Refilling
Neuron 68(4):724-38. download file

N. Strenzke, S. Chanda, C. Kopp-Scheinpflug, D. Khimich, K. Reim, A.V. Bulankina, A. Neef, F. Wolf, N. Brose, M. Xu-Friedman, and M. Tobias (2009).
Complexin-I is required for high-fidelity transmission at the endbulb of Held auditory synapse.
Journal of Neuroscience 29(25):7991-8004. download file

M.G. Holt, A. Cooke, A. Neef, and L. Lagnado (2004).
High mobility of vesicles supports continuous exocytosis at a ribbon synapse
Curr Biol 14(3):173-83. download file