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.

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

The Ca 2+ -binding protein (CaBP) parvalbumin (PV) is strongly expressed in cerebellar Purkinje neurones (PNs). It is considered a pure Ca 2+ buffer, lacking any Ca 2+ sensor function. Consistent with this notion, no PV ligand was found in dendrites of PNs. Recently, however, we observed for a related CaBP that ligand-targeting differs substantially between dendrites and axons. Thus, here we quantified the diffusion of dye-labelled PV in axons, somata and nuclei of PNs by twophoton fluorescence recovery after photobleaching (FRAP). In all three compartments the fluorescence rapidly returned to baseline, indicating that no large or immobile PV ligand was present. In the axon, FRAP was well described by a onedimensional diffusion equation and a diffusion coefficient (D) of 12 (IQR 6-20) lm 2 /s. For the soma and nucleus a threedimensional model yielded similar D values. The diffusional mobility in these compartments was $3 times smaller than in dendrites. Based on control experiments with fluorescein dextrans, we attributed this reduced mobility of PV to different cytoplasmic properties rather than to specific PV interactions in these compartments. Our findings support the notion that PV functions as a pure Ca 2+ buffer and will aid simulations of neuronal Ca 2+ signalling.

Synaptically induced calcium transients in dendrites of Purkinje neurons (PNs) play a key role in the induction of plasticity in the cerebellar cortex (Ito, Physiol Rev 81:1143-1195, 2001). Long-term depression at parallel fiber-PN synapses can be induced by stimulation paradigms that are associated with long-lasting (>1 min) calcium signals. These signals remain strictly localized (Eilers et al., Learn Mem 3:159-168, 1997), an observation that was rather unexpected, given the high concentration of the mobile endogenous calcium-binding proteins parvalbumin and calbindin in PNs (Fierro and Llano, J Physiol (Lond) 496:617-625, 1996; Kosaka et al., Exp Brain Res 93:483-491, 1993). By combining two-photon calcium imaging experiments in acute slices with numerical computer simulations, we found that significant calcium diffusion out of active branches indeed takes places. It is outweighed, however, by rapid and powerful calcium extrusion along the dendritic shaft. The close interplay of diffusion and extrusion defines the spread of calcium between active and inactive dendritic branches, forming a steep gradient in calcium with drop ranges of ~13 μm (interquartile range, 10-18 μm).

Key points• The dynamics of the second messenger Ca 2+ are tightly controlled by Ca 2+ -binding proteins (CaBPs).• The diffusional mobility of a given CaBP, such as calretinin (CR), defines how it affects the range-of-action of Ca 2+ but, if unexpectedly low, may also indicate that the CaBP acts as a Ca 2+ sensor, undergoing specific protein interactions.• Here we quantified the diffusional mobility of CR in dendrites of cerebellar granule cells using microscopic methods.• We find that CR diffuses unexpectedly slow, that its mobility is further reduced when Ca 2+ levels are elevated and that a distinct region of CR interacts with specific targets in a Ca 2+ -dependent manner.• Our findings indicate a new 'sensor' role for CR, which may allow for Ca 2+ -dependent feedback control of neuronal excitability.Abstract Ca 2+ -binding proteins (CaBPs) are important regulators of neuronal Ca 2+ signalling, acting either as buffers that shape Ca 2+ transients and Ca 2+ diffusion and/or as Ca 2+ sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range-of-action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye-labelled CR in mouse granule cells using two-photon fluorescence recovery after photobleaching. We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca 2+ transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF-hand 5 of CR. Our results suggest long-lasting, Ca 2+ -dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca 2+ buffer and point towards a Ca 2+ sensor function of CR.