Biomedical subjects

Jens Eilers

Publications and source records attributed to Jens Eilers.

4 recordsLinked to original sources

PubMed2006

Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis.

Two-photon microscopy (TPM) revolutionized Ca2+ imaging by allowing recordings in the depth of intact tissue and live organisms. A serious limitation in TPM, however, is the lack of an accurate and straightforward approach for the quantification of Ca2+ signals, an ability that became an invaluable tool in fluorescence microscopy. Here, we present time-correlated fluorescence lifetime imaging (tcFLIM) as a ratiometric method for the quantification of Ca2+ signals in TPM. The fluorescence lifetime of the Ca2+-indicator dye Oregon Green BAPTA-1 (OGB-1) can be recorded using the approximately 80 MHz excitation pulses utilized in TPM. It shows a Ca2+ dependence that can be explained by the Ca2+-affinity, spectral properties and purity of the dye. Pixel-wise lifetime recordings, controlled by a laser-scanning microscope, allowed quantitative Ca2+ imaging in full-frame and linescan mode. Although we focused on the high-affinity Ca2+ indicator OGB-1, our tcFLIM-based quantification is applicable to other Ca2+ dyes and to fluorescence indicators in general.

Calcium↗

PubMed2005

Calbindin D28k targets myo-inositol monophosphatase in spines and dendrites of cerebellar Purkinje neurons.

The Ca(2+)-binding protein calbindin D28k (CB) is vital for the normal function of the central nervous system but its specific functional role is largely unclear. CB is typically described as a mobile Ca(2+)buffer that shapes the spatiotemporal extent of cellular Ca(2+)signals. Recent biochemical data, however, indicate that CB also has characteristics of a Ca(2+) sensor and activates myo-inositol monophosphatase (IMPase), a key enzyme of the inositol-1,4,5-trisphosphate signaling cascade and an assumed target of mood-stabilizing drugs in the treatment of bipolar disorder. Here, we show that CB interacts with IMPase in cerebellar Purkinje neurons, a cell type well known to rely on inositol-1,4,5-trisphosphate-dependent synaptic integration. Quantification of the mobility of dye-labeled CB with two-photon fluorescence recovery after photobleaching revealed that a substantial fraction of CB is immobilized in spines and dendrites, but not in axons. Immobilization occurs over several seconds, is increased by suprathreshold synaptic activity, and can be relieved by a synthetic peptide that resembles the putative CB-binding site of IMPase, indicating that CB binds to immobilized IMPase. Measurements of the apparent diffusion coefficients of CB imply that CB does not interact with cytosolic IMPase or that the latter is present only in minute amounts in the spiny dendrites of Purkinje neurons. Our results suggest that CB acts as an activity-dependent sensor that targets membrane/cytoskeleton-bound IMPase in central neurons.

Animals↗

PubMed2003

Mutational analysis of dendritic Ca2+ kinetics in rodent Purkinje cells: role of parvalbumin and calbindin D28k.

The mechanisms governing the kinetics of climbing fibre-mediated Ca2+ transients in spiny dendrites of cerebellar Purkinje cells (PCs) were quantified with high-resolution confocal Ca2+ imaging. Ca2+ dynamics in parvalbumin (PV-/-) and parvalbumin/calbindin D28k null-mutant (PV/CB-/-) mice were compared with responses in wild-type (WT) animals. In the WT, Ca2+ transients in dendritic shafts were characterised by double exponential decay kinetics that were not due to buffered Ca2+ diffusion or saturation of the indicator dye. Ca2+ transients in PV-/- PCs reached the same peak amplitude as in the WT but the biphasic nature of the decay was less pronounced, an effect that could be attributed to PV's slow binding kinetics. In contrast, peak amplitudes in PV/CB-/- PCs were about two times higher than in the WT and the decay became nearly monophasic. Numerical simulations indicate that the residual deviation from a single exponential decay in PV/CB-/- is due to saturation of the Ca2+ indicator dye. Furthermore, the simulations imply that the effect of uncharacterised endogenous Ca2+ binding proteins is negligible, that buffered diffusion and dye saturation significantly affects spineous Ca2+ transients but not those in the dendritic shafts, and that neither CB nor PV undergoes saturation in spines or dendrites during climbing fibre-evoked Ca2+ transients. Calbindin's medium-affinity binding sites are fast enough to reduce the peak amplitude of the Ca2+ signal. However, similar to PV, delayed binding by CB leads to biphasic Ca2+ decay kinetics. Our results suggest that the distinct kinetics of PV and CB underlie the biphasic kinetics of synaptically evoked Ca2+ transients in dendritic shafts of PCs.

Animals↗

PubMed2003

Diffusional mobility of parvalbumin in spiny dendrites of cerebellar Purkinje neurons quantified by fluorescence recovery after photobleaching.

Ca(2+)-binding proteins (CaBPs) represent key factors for the modulation of cellular Ca(2+) dynamics. Especially in thin extensions of nerve cells, Ca(2+) binding and buffered diffusion of Ca(2+) by CaBPs is assumed to effectively control the spatio-temporal extend of Ca(2+) signals. However, no quantitative data about the mobility of specific CaBPs in the neuronal cytosol are available. We quantified the diffusion of the endogenous CaPB parvalbumin (PV) in spiny dendrites of cerebellar Purkinje neurons with two-photon fluorescence recovery after photobleaching. Fluorescently labeled PV diffused readily between spines and dendrites with a median time constant of 49 ms (37-61 ms, interquartile range). Based on published data on spine geometry, this value corresponds to an apparent diffusion coefficient of 43 microm(2) s(-1) (34-56 microm(2) s(-1)). The absence of large or immobile binding partners for PV was confirmed in PV null-mutant mice. Our data validate the common but so far unproven assumption that PV is highly mobile in neurons and will facilitate simulations of neuronal Ca(2+) buffering. Our experimental approach represents a versatile tool for quantifying the mobility of proteins in neuronal dendrites.

Animals↗