SummarySustained rate-coded signals encode many types of sensory modalities. Some sensory synapses possess specialized ribbon structures, which tether vesicles, to enable high-frequency signaling. However, central synapses lack these structures, yet some can maintain signaling over a wide bandwidth. To analyze the underlying molecular mechanisms, we investigated the function of the active zone core component Bassoon in cerebellar mossy fiber to granule cell synapses. We show that short-term synaptic depression is enhanced in Bassoon knockout mice during sustained high-frequency trains but basal synaptic transmission is unaffected. Fluctuation and quantal analysis as well as quantification with constrained short-term plasticity models revealed that the vesicle reloading rate was halved in the absence of Bassoon. Thus, our data show that the cytomatrix protein Bassoon speeds the reloading of vesicles to release sites at a central excitatory synapse.

The coupling distance between presynaptic Ca(2+) influx and the sensor for vesicular transmitter release determines speed and reliability of synaptic transmission. Nanodomain coupling (<100 nm) favors fidelity and is employed by synapses specialized for escape reflexes and by inhibitory synapses involved in synchronizing fast network oscillations. Cortical glutamatergic synapses seem to forgo the benefits of tight coupling, yet quantitative detail is lacking. The reduced transmission fidelity of loose coupling, however, raises the question whether it is indeed a general characteristic of cortical synapses. Here we analyzed excitatory parallel fiber to Purkinje cell synapses, major processing sites for sensory information and well suited for analysis because they typically harbor only a single active zone. We quantified the coupling distance by combining multiprobability fluctuation analyses, presynaptic Ca(2+) imaging, and reaction-diffusion simulations in wild-type and calretinin-deficient mice. We found a coupling distance of <30 nm at these synapses, much shorter than at any other glutamatergic cortical synapse investigated to date. Our results suggest that nanodomain coupling is a general characteristic of conventional cortical synapses involved in high-frequency transmission, allowing for dense gray matter packing and cost-effective neurotransmission.

In Drosophila, central nervous system (CNS) formation starts with the delamination from the neuroectoderm of about 30 neuroblasts (NBs) per hemisegment. They give rise to approximately 350 neurons and 30 glial cells during embryonic development. Understanding the mechanisms leading to cell fate specification and differentiation in the CNS requires the identification of the NB lineages. The embryonic lineages derived from 17 NBs of the ventral part of the neuroectoderm have previously been described (Bossing et al., 1996). Here we present 13 lineages derived from the dorsal part of the neuroectoderm and we assign 12 of them to identified NBs. Together, the 13 lineages comprise approximately 120 neurons and 22 to 27 glial cells which we include in a systematic terminology. Therefore, NBs from the dorsal neuroectoderm produce about 90% of the glial cells in the embryonic ventral ganglion. Two of the NBs give rise to glial progeny exclusively (NB 6-4A, GP) and five to glia as well as neurons (NBs 1-3, 2-5, 5-6, 6-4T, 7-4). These seven NBs are arranged as a group in the most lateral region of the NB layer. The other lineages (NBs 2-4, 3-3, 3-5, 4-3, 4-4, 5-4, clone y) are composed exclusively of neurons (interneurons, motoneurons, or both). Additionally, it has been possible to link the lateral cluster of even-skipped expressing cells (EL) to the lineage of NB 3-3. Along with the previously described clones, the vast majority (more than 90%) of cell lineages in the embryonic ventral nerve cord (thorax, abdomen) are now known. Moreover, previously identified neurons and most glial cells are now linked to certain lineages and, thus, to particular NBs. This complete set of data provides a foundation for the interpretation of mutant phenotypes and for future investigations on cell fate specification and differentiation.

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.

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 moodstabilizing 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-trisphosphatedependent 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 membrane͞cytoskeleton-bound IMPase in central neurons.calcium ͉ inositol-1,4,5-trisphosphate ͉ two-photon microscopy ͉ diffusion ͉ mobility

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Key points• Endogenous Ca 2+ binding proteins such as calbinding-D28k (CB) and parvalbumin (PV) are considered important regulators of short-term synaptic plasticity.• Cerebellar Purkinje neurons express large amounts of CB and PV and are laterally connected by inhibitory synapses that show paired-pulse facilitation (PPF) during high-frequency activation.• We report quantal synaptic release parameters of these synapses in wild-type and in CB and PV knock-out mice; evidence is provided that these synapses operate at nanodomain influx-release coupling.• We find that PPF is independent of CB and PV, using a combination of paired electrophysiological recordings, synaptic Ca 2+ imaging and numerical computer simulations.• Our results suggest that PPF during high-frequency activation results from slow Ca 2+ unbinding from the sensor for transmitter release, which is reminiscent of the 'active Ca 2+ ' mechanism of PPF suggested by Katz and Miledi in 1968.Abstract Paired-pulse facilitation (PPF) is a dynamic enhancement of transmitter release considered crucial in CNS information processing. The mechanisms of PPF remain controversial and may differ between synapses. Endogenous Ca 2+ buffers such as parvalbumin (PV) and calbindin-D28k (CB) are regarded as important modulators of PPF, with PV acting as an anti-facilitating buffer while saturation of CB can promote PPF. We analysed transmitter release and PPF at intracortical, recurrent Purkinje neuron (PN) to PN synapses, which show PPF during high-frequency activation (200 Hz) and strongly express both PV and CB. We quantified presynaptic Ca 2+ dynamics and quantal release parameters in wild-type (WT), and CB and PV deficient mice. Lack of CB resulted in increased volume averaged presynaptic Ca 2+ amplitudes and in increased release probability, while loss of PV had no significant effect on these parameters. Unexpectedly, none of the buffers significantly influenced PPF, indicating that neither CB saturation nor residual free Ca 2+ ([Ca 2+ ] res ) was the main determinant of PPF. Experimentally constrained, numerical simulations of Ca 2+ -dependent release were used to estimate the contributions of [Ca 2+ ] res , CB, PV, calmodulin (CaM), immobile buffer fractions and Ca 2+ remaining bound to the release sensor after the first of two action potentials ('active

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.