Showing papers by "Erwin Neher published in 2007"

Quantitative analysis of calcium-dependent vesicle recruitment and its functional role at the calyx of held synapse

Abstract: Recruitment of release-ready vesicles at synapses is one of the important factors, which determine dynamic properties of signaling between neurons in the brain. It has been shown that the rate of vesicle recruitment is accelerated by strong synaptic activity. An elevated concentration of calcium ions in the presynaptic terminal ([Ca2+]i) has been proposed to be responsible for this effect. However, the precise relationship between [Ca2+]i and recruitment has not been established yet, and the functional consequences of accelerated recruitment during synaptic activity have not been quantified experimentally. To probe the intracellular Ca2+ dependence of vesicle recruitment and to examine its functional role during trains of action potential (AP)-like stimuli, we monitored [Ca2+]i and synaptic responses simultaneously with paired recordings at the calyx of Held synapse. We found that a distinct, rapidly releasing vesicle pool is replenished with a rate that increases linearly with [Ca2+]i, without any apparent cooperativity. The slope factor for this increase is approximately 1 pool/(microM x s). Blocking Ca2+-dependent recruitment specifically with a calmodulin binding peptide revealed that the steady-state EPSCs during 100 Hz AP-like trains were maintained through this Ca2+-dependent recruitment mechanism. Using a simple model of vesicle dynamics, we estimated that the recruitment rate accelerated 10-fold during the steady-state compared with the rate at resting [Ca2+]i. We could also demonstrate an approximate sixfold increase in release probability (facilitation) during the initial 5-15 AP-like stimuli of such trains in our experimental condition, regardless of EPSC depression.

Kinetics of both synchronous and asynchronous quantal release during trains of action potential-evoked EPSCs at the rat calyx of Held

Abstract: We studied the kinetics of transmitter release during trains of action potential (AP)-evoked excitatory postsynaptic currents (EPSCs) at the calyx of Held synapse of juvenile rats. Using a new quantitative method based on a combination of ensemble fluctuation analysis and deconvolution, we were able to analyse mean quantal size (q) and release rate (ξ) continuously in a time-resolved manner. Estimates derived this way agreed well with values of q and quantal content (M) calculated for each EPSC within the train from ensemble means of peak amplitudes and their variances. Separate analysis of synchronous and asynchronous quantal release during long stimulus trains (200 ms, 100 Hz) revealed that the latter component was highly variable among different synapses but it was unequivocally identified in 18 out of 37 synapses analysed. Peak rates of asynchronous release ranged from 0.2 to 15.2 vesicles ms−1 (ves ms−1) with a mean of 2.3 ± 0.6 ves ms−1. On average, asynchronous release accounted for less than 14% of the total number of about 3670 ± 350 vesicles released during 200 ms trains. Following such trains, asynchronous release decayed with several time constants, the fastest one being in the order of 15 ms. The short duration of asynchronous release at the calyx of Held synapse may aid in generating brief postsynaptic depolarizations, avoiding temporal summation and preserving action potential timing during high frequency bursts.

Short-term plasticity turns plastic. Focus on "synaptic transmission at the calyx of Held under in vivo-like activity levels".

Abstract: Short-term plasticity (STP) is a use-dependent change in synaptic strength on the time scale of millisecond to seconds and is observed in almost every synapse of the CNS. Each type of synapse has its own “personality” with respect to this property. When stimulated a few times within a second,

Spectral analysis of fret using two wavelengths excitation - ii: application

Abstract: In the following the applicability of a novel method [1] for spectral analysis of FRET-signals is explored. First, to confirm the validity of the proposed method, test measurements of a suspension of N1E-115 cells expressing a cytosolic tandem construct of donor (CFP) and acceptor (YFP) fluorophores were performed using a spectrofluorometer. The results obtained using the proposed method (i.e. apparent FRET efficiency and ratio of total donor to acceptor concentration) were compared to those obtained using classical 3-cube method. The resulting values were also in line with the FRET efficiency calculated from a time correlated single photon counting fluorescence lifetime method under two-photon excitation as well as single cell acceptor photobleaching experiments performed on a laser scanning microscope. Acceptor photobleaching was also performed on a cell suspension in a cuvette to examine the effect of progressive acceptor photobleaching on apparent FRET efficiency of the cytosolic tandem construct. This allowed us to explore the effect of correct labeling on measured donor related and acceptor related apparent FRET efficiencies, EfD and EfA. Secondly, we use the method to analyze the fluorescence from cells expressing mixtures of the CFP (donor) or YFP (acceptor) labeled serotonin 1A receptors, which are known to form oligomers. The results verify receptor-receptor interaction and accurately conform to the predictions of a high-affinity oligomerization reaction.