RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses.

The tight spatial coupling of synaptic vesicles and voltage-gated Ca2+ channels (CaVs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca2+ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2-deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in CaV2.1 clustering at AZs, which likely alters Ca2+ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.

Serotonin Attenuates Feedback Excitation onto O-LM Interneurons.

The serotonergic system is a subcortical neuromodulatory center that controls cortical information processing in a state-dependent manner. In the hippocampus, serotonin (5-HT) is released by ascending serotonergic fibers from the midbrain raphe nuclei, thereby mediating numerous modulatory functions on various neuronal subtypes. Here, we focus on the neuromodulatory effects of 5-HT on GABAergic inhibitory oriens lacunosum-moleculare (O-LM) cells in the hippocampal area CA1 of the rat. These interneurons are thought to receive primarily local excitatory input and are, via their axonal projections to stratum lacunosum-moleculare, ideally suited to control entorhinal cortex input. We show that 5-HT reduces excitatory glutamatergic transmission onto O-LM interneurons. By means of paired recordings from synaptically connected CA1 pyramidal cells and O-LM interneurons we reveal that this synapse is modulated by 5-HT. Furthermore, we demonstrate that the reduction of glutamatergic transmission by serotonin is likely to be mediated via a decrease of calcium influx into presynaptic terminals of CA1 pyramidal cells. This modulation of excitatory synaptic transmission onto O-LM interneurons by 5-HT might be a mechanism to vary the activation of O-LM interneurons during ongoing network activity and serve as a brain state-dependent switch gating the efficiency of entorhinal cortex input to CA1 pyramidal neurons.

Recruitment of oriens-lacunosum-moleculare interneurons during hippocampal ripples.

Sharp wave-associated ∼200-Hz ripple oscillations in the hippocampus have been implicated in the consolidation of memories. However, knowledge on mechanisms underlying ripples is still scarce, in particular with respect to synaptic involvement of specific cell types. Here, we used cell-attached and whole-cell recordings in vitro to study activity of pyramidal cells and oriens-lacunosum-moleculare (O-LM) interneurons during ripples. O-LM cells received ripple-associated synaptic input that arrived delayed (3.3 ± 0.3 ms) with respect to the maximum amplitude of field ripples and was locked to the ascending phase of field oscillations (mean phase: 209 ± 6°). In line, O-LM cells episodically discharged late during ripples (∼6.5 ms after the ripple maximum), and firing was phase-locked to field oscillations (mean phase: 219 ± 9°). Our data unveil recruitment of O-LM neurons during ripples, suggesting a previously uncharacterized role of this cell type during sharp wave-associated activity.

From TER to trans- and paracellular resistance: lessons from impedance spectroscopy.

In epithelia and endothelia, overall resistance (TER) is determined by all ion-conductive structures, such as membrane channels, tight junctions, and the intercellular space, whereas the epithelial capacitance is due to the hydrophobic phase of the plasma membrane. Impedance means alternating current resistance and, in contrast to ohmic resistance, takes into account that, e.g., capacitors become increasingly conductive with increasing frequency. Impedance spectroscopy uses the association of the capacitance with the transcellular pathway to distinguish between this capacitive pathway and purely conductive components (tight junctions, subepithelium). In detail, one-path impedance spectroscopy distinguishes the resistance of the epithelium from the resistance of subepithelial tissues. Beyond that, two-path impedance spectroscopy allows for the separation of paracellular resistance (governed by tight junctional properties) from transcellular resistance (determined by conductive structures residing in the cell membranes). The present paper reviews the basic principles of these techniques, some historic milestones, as well as recent developments in epithelial physiology.

Purine receptors and Ca(2+) signalling in the human blood-brain barrier endothelial cell line hCMEC/D3.

The expression and physiology of purine receptors of the human blood-brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca(2+)-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y(2)-, P2Y(6)-, P2Y(11)- as well as the ionotropic P2X(4)-, P2X(5)- and P2X(7)-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca(2+) concentration ([Ca(2+)](i)) from 150 to 300 nM in single cells. The change in [Ca(2+)](i) corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, αß-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP(3) cascade-mediated Ca(2+) release, indicating that the nucleotide-induced Ca(2+) signal was mainly related to P2Y(2, 6 and 11) receptors. The gene silencing of the P2Y(2) receptor reduced the ATP- or UTP-induced Ca(2+) signal and suppressed the Ca(2+) signal mediated by P2Y(6) and P2Y(11) more specific agonists like UDP (P2Y(6)), BzATP (P2Y(11)) and ATPγS (P2Y(11)). This report identifies the P2Y(2) receptor subtype as the main purine receptor involved in Ca(2+) signalling of the hCMEC/D3 cells.

Action potentials in primary osteoblasts and in the MG-63 osteoblast-like cell line.

Whole-cell patch-clamp analysis revealed a resting membrane potential of -60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of -20 mV and a repolarization between the spikes to -45 mV. Expressed channels were characterized by application of voltage pulses between -150 mV and 90 mV in 10 mV steps, from a holding potential of -40 mV. Voltages below -60 mV induced an inward current. Depolarizing voltages above -30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between -30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above -30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K(+) (K(ir)) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na(+) (Na(v)) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K(+) (K(v)) channels. In addition, RT-PCR showed expression of Na(v)1.3, Na(v)1.4, Na(v)1.5, Na(v)1.6, Na(v)1.7, and K(ir)2.1, K(ir)2.3, and K(ir)2.4 as well as K(v)2.1. We conclude that osteoblasts express channels that allow firing of action potentials.