Current and calcium responses to local activation of axonal NMDA receptors in developing cerebellar molecular layer interneurons

In developing cerebellar molecular layer interneurons (MLIs), NMDA increases spontaneous GABA release. This effect had been attributed to either direct activation of presynaptic NMDA receptors (preNMDARs) or an indirect pathway involving activation of somato-dendritic NMDARs followed by passive spread of somatic depolarization along the axon and activation of axonal voltage dependent Ca(2+) channels (VDCCs). Using Ca(2+) imaging and electrophysiology, we searched for preNMDARs by uncaging NMDAR agonists either broadly throughout the whole field or locally at specific axonal locations. Releasing either NMDA or glutamate in the presence of NBQX using short laser pulses elicited current transients that were highly sensitive to the location of the spot and restricted to a small number of varicosities. The signal was abolished in the presence of high Mg(2+) or by the addition of APV. Similar paradigms yielded restricted Ca(2+) transients in interneurons loaded with a Ca(2+) indicator. We found that the synaptic effects of NMDA were not inhibited by blocking VDCCs but were impaired in the presence of the ryanodine receptor antagonist dantrolene. Furthermore, in voltage clamped cells, bath applied NMDA triggers Ca(2+) elevations and induces neurotransmitter release in the axonal compartment. Our results suggest the existence of preNMDARs in developing MLIs and propose their involvement in the NMDA-evoked increase in GABA release by triggering a Ca(2+)-induced Ca(2+) release process mediated by presynaptic Ca(2+) stores. Such a mechanism is likely to exert a crucial role in various forms of Ca(2+)-mediated synaptic plasticity.     

Dendritic NMDA receptors activate axonal calcium channels

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.     

Extracellular mild acidosis decreases the Ca2+ permeability of the human NMDA receptors

NMDA receptors (NMDARs) are glutamate-gated ion channels involved in excitatory synaptic transmission and in others physiological processes such as synaptic plasticity and development. The overload of Ca²⁺ ions through NMDARs, caused by an excessive activation of receptors, leads to excitotoxic neuronal cell death. For this reason, the reduction of Ca²⁺ flux through NMDARs has been a central focus in finding therapeutic strategies to prevent neuronal cell damage.Extracellular H⁺ are allosteric modulators of NMDARs. Starting from previous studies showing that extracellular mild acidosis reduces NMDA-evoked whole cell currents, we analyzed the effects of this condition on the NMDARs Ca²⁺ permeability, measured as “fractional calcium current” (P_f, i.e. the percentage of the total current carried by Ca²⁺ ions), of human NMDARs NR1/NR2A and NR1/NR2B transiently transfected in HeLa cells. Extracellular mild acidosis significantly reduces P_f of both human NR1/NR2A and NR1/NR2B NMDARs, also decreasing single channel conductance in outside out patches for NR1/NR2A receptor. Reduction of Ca²⁺ flux through NMDARs was also confirmed in cortical neurons in culture. A comparative analysis of both NMDA evoked Ca²⁺ transients and whole cell currents showed that extracellular H⁺ differentially modulate the permeation of Na⁺ and Ca²⁺ through NMDARs.Our data highlight the synergy of two distinct neuroprotective mechanisms during acidosis: Ca²⁺ entry through NMDARs is lowered due to the modulation of both open probability and Ca²⁺ permeability. Furthermore, this study provides the proof of concept that it is possible to reduce Ca²⁺ overload in neurons modulating the NMDAR Ca²⁺ permeability.     

Synaptic NMDA Receptor-Dependent Ca2+ Entry Drives Membrane Potential and Ca2+ Oscillations in Spinal Ventral Horn Neurons

During vertebrate locomotion, spinal neurons act as oscillators when initiated by glutamate release from descending systems. Activation of NMDA receptors initiates Ca²⁺-mediated intrinsic membrane potential oscillations in central pattern generator (CPG) neurons. NMDA receptor-dependent intrinsic oscillations require Ca²⁺-dependent K⁺ (K_Ca2) channels for burst termination. However, the location of Ca²⁺ entry mediating K_Ca2 channel activation, and type of Ca²⁺ channel – which includes NMDA receptors and voltage-gated Ca²⁺ channels (VGCCs) – remains elusive. NMDA receptor-dependent Ca²⁺ entry necessitates presynaptic release of glutamate, implying a location at active synapses within dendrites, whereas VGCC-dependent Ca²⁺ entry is not similarly constrained. Where Ca²⁺ enters relative to K_Ca2 channels is crucial to information processing of synaptic inputs necessary to coordinate locomotion. We demonstrate that Ca²⁺ permeating NMDA receptors is the dominant source of Ca²⁺ during NMDA-dependent oscillations in lamprey spinal neurons. This Ca²⁺ entry is synaptically located, NMDA receptor-dependent, and sufficient to activate K_Ca2 channels at excitatory interneuron synapses onto other CPG neurons. Selective blockade of VGCCs reduces whole-cell Ca²⁺ entry but leaves membrane potential and Ca²⁺ oscillations unaffected. Furthermore, repetitive oscillations are prevented by fast, but not slow, Ca²⁺ chelation. Taken together, these results demonstrate that K_Ca2 channels are closely located to NMDA receptor-dependent Ca²⁺ entry. The close spatial relationship between NMDA receptors and K_Ca2 channels provides an intrinsic mechanism whereby synaptic excitation both excites and subsequently inhibits ventral horn neurons of the spinal motor system. This places the components necessary for oscillation generation, and hence locomotion, at glutamatergic synapses.     

Spike‐triggered dendritic calcium transients depend on synaptic activity in the cricket giant interneurons

The relationship between electrical activity and spike‐induced Ca²⁺ increases in dendrites was investigated in the identified wind‐sensitive giant interneurons in the cricket. We applied a high‐speed Ca²⁺ imaging technique to the giant interneurons, and succeeded in recording the transient Ca²⁺ increases (Ca²⁺ transients) induced by a single action potential, which was evoked by presynaptic stimulus to the sensory neurons. The dendritic Ca²⁺ transients evoked by a pair of action potentials accumulated when spike intervals were shorter than 100 ms. The amplitude of the Ca²⁺ transients induced by a train of spikes depended on the number of action potentials. When stimulation pulses evoking the same numbers of action potentials were separately applied to the ipsi‐ or contra‐lateral cercal sensory nerves, the dendritic Ca²⁺ transients induced by these presynaptic stimuli were different in their amplitude. Furthermore, the side of presynaptic stimulation that evoked larger Ca²⁺ transients depended on the location of the recorded dendritic regions. This result means that the spike‐triggered Ca²⁺ transients in dendrites depend on postsynaptic activity. It is proposed that Ca²⁺ entry through voltage‐dependent Ca²⁺ channels activated by the action potentials will be enhanced by excitatory synaptic inputs at the dendrites in the cricket giant interneurons. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 234–244, 2002; DOI 10.1002/neu.10032     

Extracellular Ca2+ ions reduce NMDA receptor conductance and gating

Brief intracellular Ca²⁺ transients initiate signaling routines that direct cellular activities. Consequently, activation of Ca²⁺-permeable neurotransmitter-gated channels can both depolarize and initiate remodeling of the postsynaptic cell. In particular, the Ca²⁺ transient produced by NMDA receptors is essential to normal synaptic physiology, drives the development and plasticity of excitatory central synapses, and also mediates glutamate excitotoxicity. The amplitude and time course of the Ca²⁺ signal depends on the receptor’s conductance and gating kinetics; these properties are themselves influenced both directly and indirectly by fluctuations in the extracellular Ca²⁺ concentration. Here, we used electrophysiology and kinetic modeling to delineate the direct effects of extracellular Ca²⁺ on recombinant GluN1/GluN2A receptor conductance and gating. We report that, in addition to decreasing unitary conductance, Ca²⁺ also decreased channel open probability primarily by lengthening closed-channel periods. Using one-channel current recordings, we derive a kinetic model for GluN1/GluN2A receptors in physiological Ca²⁺ concentrations that accurately describes macroscopic channel behaviors. This model represents a practical instrument to probe the mechanisms that control the Ca²⁺ transients produced by NMDA receptors during both normal and aberrant synaptic signaling.     

Presynaptic kainate and NMDA receptors are implicated in the modulation of GABA release from cortical and hippocampal nerve terminals

One of the pathways implicated in a fine-tuning control of synaptic transmission is activation of the receptors located at the presynaptic terminal. Here we investigated the intracellular events in rat brain cortical and hippocampal nerve terminals occurring under the activation of presynaptic glutamate receptors by exogenous glutamate and specific agonists of ionotropic receptors, NMDA and kainate. Involvement of synaptic vesicles in exocytotic process was assessed using [³H]GABA and pH-sensitive fluorescent dye acridine orange (AO). Glutamate as well as NMDA and kainate were revealed to induce [³H]GABA release that was not blocked by NO-711, a selective blocker of GABA transporters. AO-loaded nerve terminals responded to glutamate application by the development of a two-phase process. The first phase, a fluorescence transient completed in ∼1 min, was similar to the response to high K⁺. It was highly sensitive to extracellular Ca²⁺ and was decreased in the presence of the NMDA receptor antagonist, MK-801. The second phase, a long-lasting process, was absolutely dependent on extracellular Na⁺ and attenuated in the presence of CNQX, the kainate receptor antagonist. NMDA as well as kainate per se caused a rapid and abrupt neurosecretory process confirming that both glutamate receptors, NMDA and kainate, are involved in the control of neurotransmitter release. It could be suggested that at least two types ionotropic receptor are attributed to glutamate-induced two-phase process, which appears to reflect a rapid synchronous and a more prolonged asynchronous vesicle fusion.     

Translocation of CaMKII to dendritic microtubules supports the plasticity of local synapses

The processing of excitatory synaptic inputs involves compartmentalized dendritic Ca²⁺ oscillations. The downstream signaling evoked by these local Ca²⁺ transients and their impact on local synaptic development and remodeling are unknown. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an important decoder of Ca²⁺ signals and mediator of synaptic plasticity. In addition to its known accumulation at spines, we observed with live imaging the dynamic recruitment of CaMKII to dendritic subdomains adjacent to activated synapses in cultured hippocampal neurons. This localized and transient enrichment of CaMKII to dendritic sites coincided spatially and temporally with dendritic Ca²⁺ transients. We show that it involved an interaction with microtubular elements, required activation of the kinase, and led to localized dendritic CaMKII autophosphorylation. This process was accompanied by the adjacent remodeling of spines and synaptic AMPA receptor insertion. Replacement of endogenous CaMKII with a mutant that cannot translocate within dendrites lessened this activity-dependent synaptic plasticity. Thus, CaMKII could decode compartmental dendritic Ca²⁺ transients to support remodeling of local synapses.     

Target-Specific Expression of Presynaptic NMDA Receptors in Neocortical Microcircuits

Traditionally, NMDA receptors are located postsynaptically; yet, putatively presynaptic NMDA receptors (preNMDARs) have been reported. Although implicated in controlling synaptic plasticity, their function is not well understood and their expression patterns are debated. We demonstrate that, in layer 5 of developing mouse visual cortex, preNMDARs specifically control synaptic transmission at pyramidal cell inputs to other pyramidal cells and to Martinotti cells, while leaving those to basket cells unaffected. We also reveal a type of interneuron that mediates ascending inhibition. In agreement with synapse-specific expression, we find preNMDAR-mediated calcium signals in a subset of pyramidal cell terminals. A tuned network model predicts that preNMDARs specifically reroute information flow in local circuits during high-frequency firing, in particular by impacting frequency-dependent disynaptic inhibition mediated by Martinotti cells, a finding that we experimentally verify. We conclude that postsynaptic cell type determines presynaptic terminal molecular identity and that preNMDARs govern information processing in neocortical columns.     

Zinc reverses glycine-dependent inactivation of NMDARs in cultured rat hippocampal neurons

In the presence of glutamate and co-agonists, e.g., glycine, the N-methyl-D-aspartate receptor (NMDAR) plays an important role in physiological and pathophysiological brain processes. Previous studies indicate glycine could inhibit NMDAR responses induced by high concentration of NMDA in hippocampal neurons. The mechanism underlying this inhibitory impact, however, has been unclear. In this study, the whole-cell patch-clamp recording and Ca²⁺ imaging with Fluo-3/AM under laser scanning confocal microscope were used to analyze the possible involvement of NMDAR subunits in this effect. We found that the peak current of NMDARs and Ca²⁺ influx induced by high concentration of NMDA were reduced by treatment of glycine (0.03?C10 ??mol L^?1) in a dose-dependent manner, and that the glycine-dependent inhibition of NMDAR responses, which were induced at 300 ??mol L^?1 NMDA, was reversed by ZnCl₂ through the blocking of the NR2A subunit of NMDARs, but was less influenced by ifenprodil, a NR2B inhibitor. Our results suggest that the glycine-dependent inactivation of NMDARs is potentially modulated by the regulatory subunit NR2A.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

Mechanisms for localising calcineurin and CaMKII in dendritic spines

Calcineurin and calmodulin-dependent protein kinase II (CaMKII) are both highly abundant in neurons, and both are activated by calmodulin at similar Ca²⁺ concentrations in the test tube. However, they fulfill opposite functions in dendritic spines, with CaMKII activity driving long-term synaptic potentiation following large influxes of Ca²⁺ through NMDA-type glutamate receptors (NMDARs), and calcineurin responding to smaller influxes of Ca²⁺ through the same receptors to induce long-term depression. In this review, we explore the notion that precise dynamic localisation of the two enzymes at different sites within dendritic spines is fundamental to this behaviour. We describe the structural basis of calcineurin and CaMKII localisation by their interaction with proteins including AKAP79, densin-180, α-actinin, and NMDARs. We then consider how interactions with these proteins likely position calcineurin and CaMKII at different distances from Ca²⁺ microdomains emanating from the mouths of NMDARs in order to drive the divergent responses. We also highlight shortcomings in our current understanding of synaptic localisation of these two important signalling enzymes.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Effects of blockers of voltage-operated potassium channels on an NMDA component of excitatory synaptic transmission in theCA1 subfield of the rat hippocampus

The effects of voltage-operated potassium channel blockers on evoked excitatory synaptic transmission were studied in theCA1 subfield of rat hippocampal slices. Incubation with 50 μM 4-aminopyridine (n=27), 300 nM α-dendrotoxin (n=3), or 5 to 25 mM tetraethylammonium (n=7) resulted in an enhancement of the peak amplitude of excitatory postsynaptic currents (EPSC) and significant prolongation of their decay at strong stimuli, due to an increased contribution of NMDA receptors into EPSC. In five experiments, the presence of an AMPA receptor antagonist, 4-aminopyridine, led to the appearance of NMDA receptor-mediated field excitatory postsynaptic potentials (fEPSP). It is suggested that various modulations increasing presynaptic Ca²⁺ entry and, consequently, glutamate release may increase an NMDA component of synaptic transmission via excitation of polysynaptic excitatory pathways and/or due to glutamate spillover to distant extrasynaptic NMDA receptors.     

An Atypical Role for Collapsin Response Mediator Protein 2 (CRMP-2) in Neurotransmitter Release via Interaction with Presynaptic Voltage-gated Calcium Channels

Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate and axonal growth of neurons through protein-protein interactions. Their functions in presynaptic biology remain unknown. Here, we identify the presynaptic N-type Ca²⁺ channel (CaV2.2) as a CRMP-2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus. Both proteins co-localize within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to enhanced green fluorescent protein caused a significant increase in Ca²⁺ channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca²⁺ current density was not due to a change in channel gating. Rather, cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2-overexpressing neurons. These neurons also exhibited a significant increase in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-enhanced green fluorescent protein-overexpressing neurons elicited a significant increase in release of glutamate compared with control neurons. Toxin block of Ca²⁺ entry via CaV2.2 abolished this stimulated release. Thus, the CRMP-2-Ca²⁺ channel interaction represents a novel mechanism for modulation of Ca²⁺ influx into nerve terminals and, hence, of synaptic strength.     

Functional organization of dendritic Ca2+ signals in midbrain dopamine neurons

Dendritic Ca²⁺ plays an important role not only in synaptic integration and synaptic plasticity, but also in dendritic excitability in midbrain dopamine neurons. However, the functional organization of dendritic Ca²⁺ signals in the dopamine neurons remains largely unknown. We therefore investigated dendritic Ca²⁺ signals by measuring glutamate-induced Ca²⁺ increases along the dendrites of acutely isolated midbrain dopamine neurons.Maximal doses of glutamate induced a [Ca²⁺]_c rise with similar amplitudes in proximal and distal dendritic regions of a dopamine neuron. Glutamate receptors contributed incrementally to the [Ca²⁺]_c rise according to their distance from the soma, with a reciprocal decrement in the contribution of voltage-operated Ca²⁺ channels (VOCCs). The contribution of AMPA and NMDA receptors increased with dendritic length, but that of metabotropic glutamate receptors decreased. At low doses of glutamate at which spontaneous firing was sustained, the [Ca²⁺]_c rise was higher in the distal than the proximal regions of a dendrite, possibly due to the increased spontaneous firing rate.These results indicate that functional organization of Ca²⁺ signals in the dendrites of dopamine neurons requires different combination of VOCCs and glutamate receptors according to dendritic length, and that regional Ca²⁺ rises in dendrites respond differently to applied glutamate concentration.     

Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA_ARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA_A autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca²⁺ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl⁻]_i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABA_A channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na_v-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA_A autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.     

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice

SK2- and K_V4.2-containing K⁺ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca²⁺ source that provides Ca²⁺-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca²⁺ entry through NMDARs, whereas K_V4.2-containing channel availability is increased by Ca²⁺ entry through SNX-482 (SNX) sensitive Ca_V2.3 R-type Ca²⁺ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca²⁺ entry through R-type Ca²⁺ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca²⁺ channels and K_V4.2-containing K⁺ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca²⁺ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from Ca_V2.3 null mice. The results show that in the absence of Ca_V2.3 channels, apamin application still boosted EPSPs. The boosting effect of Ca_V2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from Ca_V2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and K_V4.2-containing channels to Ca_V2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Structural and physiological properties of leech giant glial cells as studied by confocal microscopy

We have studied the architecture of giant neuropile glial cells of the medicinal leech Hirudo medicinalis L. using confocal laser scanning microscopy. We also measured changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by activation of glutamate receptors or voltage-gated Ca²⁺ channels in different glial cell compartments. Glial cells of isolated segmental ganglia were filled iontophoretically with the Ca²⁺ indicator dye Fluo-3. The three-dimensional structure, calculated from serial sections, showed that numerous fine glial branches extend within the whole neuropile, where most of the synapses between neurones are established. Activation of glial glutamate receptors by glutamate or kainate, or depolarizing the cell membrane by elevating the external K⁺ concentration resulted in a transient increase in [Ca²⁺]_i, as measured by Fluo-3 fluorescence. The comparison of [Ca²⁺]_i changes in glial cell branches with changes in the cell body demonstrated that transients in the branches were 2–3 times larger than those in the cell body. The results suggest that glutamate receptors and voltage-gated Ca²⁺ channels are located in the membrane not only of the glial cell body but also of the cellular branches, which may extend close to synaptic domains.