Phorbol esters modulate spontaneous and Ca2+-evoked transmitter release via acting on both munc13 and protein kinase C

Diacylglycerol (DAG) and phorbol esters strongly potentiate transmitter release at synapses by activating protein kinase C (PKC) and members of the Munc13 family of presynaptic vesicle priming proteins. This PKC/Munc13 pathway has emerged as a crucial regulator of release probability during various forms of activity-dependent enhancement of release. Here, we investigated the relative roles of PKC and Munc13-1 in the phorbol ester potentiation of evoked and spontaneous transmitter release at the calyx of Held synapse. The phorbol ester phorbol 12,13-dibutyrate (1 mu M) potentiated the frequency of miniature EPSCs, and the amplitudes of evoked EPSCs with a similar time course. Preincubating slices with the PKC blocker Ro31-82200 reduced the potentiation, mainly by affecting a late phase of the phorbol ester potentiation. The Ro31-8220-insensitive potentiation was most likely mediated by Munc13-1, because in organotypic slices of Munc13-1H567K knock-in mice, in which DAG binding to Munc13-1 is abolished, the potentiation of spontaneous release by phorbol ester was strongly suppressed. Using direct presynaptic depolarizations in paired recordings, we show that the phorbol ester potentiation does not go along with an increase in the number of readily releasable vesicles, despite an increase in the cumulative EPSC amplitude during 100 Hz stimulation trains. Our data indicate that activation of Munc13 and PKC both contribute to an enhancement of the fusion probability of readily releasable vesicles. Thus, docked and readily releasable vesicles are a substrate for modulation via intracellular second-messenger pathways that act via Munc13 and PKC.

Structural basis and biophysical properties of synaptic plasticity studied by atomic force microscopy

Alexandre Yersin

In this thesis, we studied two systems important for synaptic plasticity, one presynaptic and another postsynaptic. The protein complex composed of VAMP 2, SNAP-25 and syntaxin 1 (SNARE complex) is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. In order to better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope (AFM) and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces, dissociation kinetics of the SNAREs and led us to propose a sequence of formation for the complex. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein nSec1 injected into the AFM chamber prevented the SNARE complex formation. Finally we measured the effect of tetanus toxin protease (TeTx) on the SNARE complex and its activity by on-line registration during TeTx injection. These experiments reveal that AFM is a valuable tool to investigate complicate interactions among a protein complex containing up to four components. Trafficking of glutamate receptors AMPA is closely related to postsynaptic induction of long-term potentiation. In a second part of this thesis, we used AFM to explore the distribution of AMPA receptors on-line at the surface of living cells in correlation with cellular viscoelastic properties. First, we showed that AFM permits to detect specifically AMPA receptors at the surface of living neurons and we proved that the specificity of detection is stable during time. Moreover, we were able to visualize variations in AMPA receptor density on surface in response to different stimulations. Finally, AFM also allowed us to study local viscoelastic properties of the cell and their modifications occurring during AMPA receptor trafficking. Our measurements reveal that AMPA receptors are situated at the cell surface on local stiffness maxima. We have noticed that an excitatory stimulation removes these stiffness maxima before producing internalization of receptors. Our results suggest also that AMPA receptors may be constituted of two subpopulations depending on the cell stiffness. A "hard" subpopulation may be preferentially internalized after an excitatory stimulation whereas a "soft" one seems to remain at the cell surface.

EPFL2004

Structural basis and biophysical properties of synaptic plasticity studied by atomic force microscopy

Alexandre Yersin

EPFL2004