Hartmut Schmidt

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 PVs 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. Calbindins 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.

Bassoon Speeds Vesicle Reloading at a Central Excitatory Synapse

Summary Sustained 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.

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.

Nanodomain coupling at an excitatory cortical 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.

Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

Background Voltage-gated potassium (Kv) channels are among the earliest ion channels to appear during brain development, suggesting a functional requirement for progenitor cell proliferation and/or differentiation. We tested this hypothesis, using human neural progenitor cells (hNPCs) as a model system. Methodology/Principal Findings In proliferating hNPCs a broad spectrum of Kv channel subtypes was identified using quantitative real-time PCR with a predominant expression of the A-type channel Kv4.2. In whole-cell patch-clamp recordings Kv currents were separated into a large transient component characteristic for fast-inactivating A-type potassium channels (IA) and a small, sustained component produced by delayed-rectifying channels (IK). During differentiation the expression of IA as well as A-type channel transcripts dramatically decreased, while IK producing delayed-rectifiers were upregulated. Both Kv currents were differentially inhibited by selective neurotoxins like phrixotoxin-1 and α-dendrotoxin as well as by antagonists like 4-aminopyridine, ammoniumchloride, tetraethylammonium chloride and quinidine. In viability and proliferation assays chronic inhibition of the A-type currents severely disturbed the cell cycle and precluded proper hNPC proliferation, while the blockade of delayed-rectifiers by α-dendrotoxin increased proliferation. Conclusions/Significance These findings suggest that A-type potassium currents are essential for proper proliferation of immature multipotent hNPCs.

Parvalbumin is freely mobile in axons, somata and nuclei of cerebellar Purkinje neurones

The Ca2+‐binding protein (CaBP) parvalbumin (PV) is strongly expressed in cerebellar Purkinje neurones (PNs). It is considered a pure Ca2+ buffer, lacking any Ca2+ 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 two‐photon 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 one‐dimensional diffusion equation and a diffusion coefficient (D) of 12 (IQR 6–20) µm2/s. For the soma and nucleus a three‐dimensional 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 Ca2+ buffer and will aid simulations of neuronal Ca2+ signalling.

Three functional facets of calbindin D-28k

Many neurons of the vertebrate central nervous system (CNS) express the Ca2+ binding protein calbindin D-28k (CB), including important projection neurons like cerebellar Purkinje cells but also neocortical interneurons. CB has moderate cytoplasmic mobility and comprises at least four EF-hands that function in Ca2+ binding with rapid to intermediate kinetics and affinity. Classically it was viewed as a pure Ca2+ buffer important for neuronal survival. This view was extended by showing that CB is a critical determinant in the control of synaptic Ca2+ dynamics, presumably with strong impact on plasticity and information processing. Already 30 years ago, in vitro studies suggested that CB could have an additional Ca2+ sensor function, like its prominent acquaintance calmodulin (CaM). More recent work substantiated this hypothesis, revealing direct CB interactions with several target proteins. Different from a classical sensor, however, CB appears to interact with its targets both, in its Ca2+-loaded and Ca2+-free forms. Finally, CB has been shown to be involved in buffered transport of Ca2+, in neurons but also in kidney. Thus, CB serves a threefold function as buffer, transporter and likely as a non-canonical sensor.

P2Y1 receptors inhibit long-term depression in the prefrontal cortex.

Long-term depression (LTD) is a form of synaptic plasticity that may contribute to information storage in the central nervous system. Here we report that LTD can be elicited in layer 5 pyramidal neurons of the rat prefrontal cortex by pairing low frequency stimulation with a modest postsynaptic depolarization. The induction of LTD required the activation of both metabotropic glutamate receptors of the mGlu1 subtype and voltage-sensitive Ca(2+) channels (VSCCs) of the T/R, P/Q and N types, leading to the stimulation of intracellular inositol trisphosphate (IP3) receptors by IP3 and Ca(2+). The subsequent release of Ca(2+) from intracellular stores activated the protein phosphatase cascade involving calcineurin and protein phosphatase 1. The activation of purinergic P2Y(1) receptors blocked LTD. This effect was prevented by P2Y(1) receptor antagonists and was absent in mice lacking P2Y(1) but not P2Y(2) receptors. We also found that activation of P2Y(1) receptors inhibits Ca(2+) transients via VSCCs in the apical dendrites and spines of pyramidal neurons. In addition, we show that the release of ATP under hypoxia is able to inhibit LTD by acting on postsynaptic P2Y(1) receptors. In conclusion, these data suggest that the reduction of Ca(2+) influx via VSCCs caused by the activation of P2Y(1) receptors by ATP is the possible mechanism for the inhibition of LTD in prefrontal cortex.

KV10.1 opposes activity-dependent increase in Ca2+ influx into the presynaptic terminal of the parallel fibre - Purkinje cell synapse.

Voltage‐gated KV10.1 potassium channels are widely expressed in the mammalian brain but their function remains poorly understood. We report that KV10.1 is enriched in the presynaptic terminals and does not take part in somatic action potentials. In parallel fibre synapses in the cerebellar cortex, we find that KV10.1 regulates Ca2+ influx and neurotransmitter release during repetitive high‐frequency activity. Our results describe the physiological role of mammalian KV10.1 for the first time and help understand the fine‐tuning of synaptic transmission.

Paired-pulse facilitation at recurrent Purkinje neuron synapses is independent of calbindin and parvalbumin during high-frequency activation

•  Endogenous Ca2+ 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 Ca2+ imaging and numerical computer simulations. •  Our results suggest that PPF during high‐frequency activation results from slow Ca2+ unbinding from the sensor for transmitter release, which is reminiscent of the ‘active Ca2+’ mechanism of PPF suggested by Katz and Miledi in 1968.