Analysis of relations between NMDA receptors and GABA release at olfactory bulb reciprocal synapses

In the mammalian olfactory bulb, signal processing is mediated by synaptic interactions between dendrites. Glutamate released from mitral cell dendrites excites dendritic spines of granule cells, which in turn release GABA back onto the mitral cell dendrites, forming a reciprocal synaptic pair. This feedback synaptic circuit was shown to be mediated predominantly by NMDA receptors. We further utilized caged Ca2+ compounds to obtain insight into the mechanism that couples NMDA receptor activation to GABA release. Feedback inhibition elicited by photo-release of caged Ca2+ in mitral cell secondary dendrites persisted when voltage-gated Ca2+ channels were blocked by cadmium (Cd2+) and nickel (Ni2+). These results indicate that Ca2+ influx through NMDA receptors can directly trigger presynaptic GABA release for local dendrodendritic feedback inhibition.     

Backpropagating action potentials enable detection of extrasynaptic glutamate by NMDA receptors

Synaptic NMDA receptors (NMDARs) are crucial for neural coding and plasticity. However, little is known about the adaptive function of extrasynaptic NMDARs occurring mainly on dendritic shafts. Here, we find that in CA1 pyramidal neurons, back-propagating action potentials (bAPs) recruit shaft NMDARs exposed to ambient glutamate. In contrast, spine NMDARs are "protected," under baseline conditions, from such glutamate influences by peri-synaptic transporters: we detect bAP-evoked Ca(2+) entry through these receptors upon local synaptic or photolytic glutamate release. During theta-burst firing, NMDAR-dependent Ca(2+) entry either downregulates or upregulates an h-channel conductance (G(h)) of the cell depending on whether synaptic glutamate release is intact or blocked. Thus, the balance between activation of synaptic and extrasynaptic NMDARs can determine the sign of G(h) plasticity. G(h) plasticity in turn regulates dendritic input probed by local glutamate uncaging. These results uncover a metaplasticity mechanism potentially important for neural coding and memory formation.     

Mg(2+) block of Drosophila NMDA receptors is required for long-term memory formation and CREB-dependent gene expression

NMDA receptor (NMDAR) channels allow Ca(2+) influx only during correlated activation of both pre- and postsynaptic cells; a Mg(2+) block mechanism suppresses NMDAR activity when the postsynaptic cell is inactive. Although the importance of NMDARs in associative learning and long-term memory (LTM) formation has been demonstrated, the role of Mg(2+) block in these processes remains unclear. Using transgenic flies expressing NMDARs defective for Mg(2+) block, we found that Mg(2+) block mutants are defective for LTM formation but not associative learning. We demonstrate that LTM-dependent increases in expression of synaptic genes, including homer, staufen, and activin, are abolished in flies expressing Mg(2+) block defective NMDARs. Furthermore, we show that genetic and pharmacological reduction of Mg(2+) block significantly increases expression of a CREB repressor isoform. Our results suggest that Mg(2+) block of NMDARs functions to suppress basal expression of a CREB repressor, thus permitting CREB-dependent gene expression upon LTM induction.     

CaMKII translocation requires local NMDA receptor-mediated Ca2+ signaling

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIalpha translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIalpha. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIalpha mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event.     

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.     

Reconstituted slow muscarinic inhibition of neuronal (Ca(v)1.2c) L-type Ca2+ channels

Ca(2+) influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca(2+) channels. We studied modulation of neuronal Ca(V)1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Galphaq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca(V)1.2c, whereas Galphai/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca(V)1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Galphaq/11. In contrast, inhibition was not reduced by coexpressing betaARK1ct or Galphat. These results indicate that slow inhibition required signaling by Galphaq/11, but not Gbetagamma, subunits. Slow inhibition did not require Ca(2+) transients or Ca(2+) influx through Ca(V)1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca(2+) chelation. The cardiac splice-variant of Ca(V)1.2 (Ca(V)1.2a) and a splice-variant of the neuronal/neuroendocrine Ca(V)1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.     

Aluminum as a specific inhibitor of plant TPC1 Ca2+ channels

In plant cells, Al ion plays dual roles as an inducer and an inhibitor of Ca(2+) influx depending on the concentration. Here, the effects of Al on Ca(2+) signaling were assessed in tobacco BY-2 cells expressing aequorin and a putative plant Ca(2+) channel from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). In wild-type cells (expressing only aequorin), Al treatment induced the generation of superoxide, and Ca(2+) influx was secondarily induced by superoxide. Higher Al concentrations inhibited the Al-stimulated and superoxide-mediated Ca(2+) influx, indicating that Ca(2+) channels responsive to reactive oxygen species (ROS) are blocked by high concentration of Al. H(2)O(2)-induced Ca(2+) influx was also inhibited by Al. Thus, inhibitory action of Al against ROS-induced Ca(2+) influx was confirmed. Similarly, known Ca(2+) channel blockers such as ions of La and Gd inhibited the H(2)O(2)-induced Ca(2+) influx. While La also inhibited the hypoosmotically induced Ca(2+) influx, Al showed no inhibitory effect against the hypoosmotic Ca(2+) influx. The effects of Al and La on Ca(2+) influx were also tested in the cell line overexpressing AtTPC1 and the cell line AtTPC1-dependently cosuppressing the endogenous TPC1 equivalents. Notably, responsiveness to H(2)O(2) was lost in the cosuppression cell line, thus TPC1 channels are required for ROS-responsive Ca(2+) influx. Data also suggested that hypoosmotic shock induces TPC1-independent Ca(2+) influx and Al shows no inhibitory action against the TPC1-independent event. In addition, AtTPC1 overexpression resulted in a marked increase in Al-sensitive Ca(2+) influx, indicating that TPC1 channels participate in osmotic Ca(2+) influx only when overexpressed. We concluded that members of TPC1 channel family are the only ROS-responsive Ca(2+) channels and are the possible targets of Al-dependent inhibition.     

Biphasic Synaptic Ca Influx Arising from Compartmentalized Electrical Signals in Dendritic Spines

Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca) imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively) that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK) channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.     

Activity-induced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing

The continued addition of new neurons to mature olfactory circuits represents a remarkable mode of cellular and structural brain plasticity. However, the anatomical configuration of newly established circuits, the types and numbers of neurons that form new synaptic connections, and the effect of sensory experience on synaptic connectivity in the olfactory bulb remain poorly understood. Using in vivo electroporation and monosynaptic tracing, we show that postnatal-born granule cells form synaptic connections with centrifugal inputs and mitral/tufted cells in the mouse olfactory bulb. In addition, newly born granule cells receive extensive input from local inhibitory short axon cells, a poorly understood cell population. The connectivity of short axon cells shows clustered organization, and their synaptic input onto newborn granule cells dramatically and selectively expands with odor stimulation. Our findings suggest that sensory experience promotes the synaptic integration of new neurons into cell type-specific olfactory circuits.     

Nongenomic mechanism of glucocorticoid inhibition of bradykinin-induced calcium influx in PC12 cells: possible involvement of protein kinase C

Many stimulants, including bradykinin (BK), can induce increase in [Ca(2+)](i) in PC12 cells. Bradykinin induces an increase in [Ca(2+)](i) via intracellular Ca(2+) release and extracellular Ca(2+) influx through the transduction of G protein, but not through voltage-sensitive calcium channels. In this experiment, We analyzed how corticosterone (Cort) influences BK-induced intracellular Ca(2+) release and extracellular Ca(2+) influx, and further studied the mechanism of glucocorticoid's action. To dissociate the intracellular Ca(2+) release and extracellular Ca(2+) influx induced by BK, the Ca(2+)-free/Ca(2+)- reintroduction protocol was used. The results were as follows: (1) The Ca(2+) influx induced by BK could be rapidly inhibited by Cort, but intracellular Ca(2+) release could not be affected significantly. (2) The inhibitory effect of Cort-BSA (BSA -conjugated Cort) on Ca(2+) influx induced by BK was the same as the effect of free Cort. (3) Protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) could mimic and PKC inhibitor G?6976 could reverse the inhibitory effect of Cort. (4) There was no inhibitory effect of Cort on Ca(2+) influx induced by BK when pretreated with pertussis toxin. The results suggested, for the first time, that Cort might act via a putative membrane receptor and inhibit the Ca(2+) influx induced by BK through the pertussis toxin -sensitive G protein-PKC pathway.     

Activity-dependent IGF-1 exocytosis is controlled by the Ca(2+)-sensor synaptotagmin-10

Synaptotagmins Syt1, Syt2, Syt7, and Syt9 act as Ca(2+)-sensors for synaptic and neuroendocrine exocytosis, but the function of other synaptotagmins remains unknown. Here, we show that olfactory bulb neurons secrete IGF-1 by an activity-dependent pathway of exocytosis, and that Syt10 functions as the Ca(2+)-sensor that triggers IGF-1 exocytosis in these neurons. Deletion of Syt10 impaired activity-dependent IGF-1 secretion in olfactory bulb neurons, resulting in smaller neurons and an overall decrease in synapse numbers. Exogenous IGF-1 completely reversed the Syt10 knockout phenotype. Syt10 colocalized with IGF-1 in somatodendritic vesicles of olfactory bulb neurons, and Ca(2+)-binding to Syt10 caused these vesicles to undergo exocytosis, thereby secreting IGF-1. Thus, Syt10 controls a previously unrecognized pathway of Ca(2+)-dependent exocytosis that is spatially and temporally distinct from Ca(2+)-dependent synaptic vesicle exocytosis controlled by Syt1. Our findings thereby reveal that two different synaptotagmins can regulate functionally distinct Ca(2+)-dependent membrane fusion reactions in the same neuron.     

A constitutive, transient receptor potential-like Ca2+ influx pathway in presynaptic nerve endings independent of voltage-gated Ca2+ channels and Na+/Ca2+ exchange

Calcium levels in the presynaptic nerve terminal are altered by several pathways, including voltage-gated Ca(2+) channels, the Na(+)/Ca(2+) exchanger, Ca(2+)-ATPase, and the mitochondria. The influx pathway for homeostatic control of [Ca(2+)](i) in the nerve terminal has been unclear. One approach to detecting the pathway that maintains internal Ca(2+) is to test for activation of Ca(2+) influx following Ca(2+) depletion. Here, we demonstrate that a constitutive influx pathway for Ca(2+) exists in presynaptic terminals to maintain internal Ca(2+) independent of voltage-gated Ca(2+) channels and Na(+)/Ca(2+) exchange, as measured in intact isolated nerve endings from mouse cortex and in intact varicosities in a neuronal cell line using fluorescence spectroscopy and confocal imaging. The Mg(2+) and lanthanide sensitivity of the influx pathway, in addition to its pharmacological and short hairpin RNA sensitivity, and the results of immunostaining for transient receptor potential (TRP) channels indicate the involvement of TRPC channels, possibly TRPC5 and TRPC1. This constitutive Ca(2+) influx pathway likely serves to maintain synaptic function under widely varying levels of synaptic activity.     

Ca2+ influx into identified leech neurons induced by 5-hydroxytryptamine

The role of 5-hydroxytryptamine (5-HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5-HT on the cytosolic free calcium concentration ([Ca(2+)](i)) in leech neurons. As [Ca(2+)](i) plays a pivotal role in numerous cellular processes, we investigated the effect of 5-HT on [Ca(2+)](i) (measured by Fura-2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5-HT induced a [Ca(2+)](i) increase which was predominantly due to Ca(2+) influx since it was abolished in Ca(2+)-free solution. The 5-HT-induced Ca(2+) influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage-dependent Ca(2+) channels. In P and N1 neurons, the membrane depolarization was due to Na(+) influx through cation channels coupled to 5-HT receptors, whereby the dose-dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5-HT receptor-coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage-dependent Ca(2+) channels was evoked by 5-HT-triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5-HT had no effect on [Ca(2+)](i).     

Cannabinoid receptor agonists inhibit depolarization-induced calcium influx in cerebellar granule neurons.

Neuronal cannabinoid receptors (CB(1)) are coupled to inhibition of voltage-sensitive Ca(2+) channels (VSCCs) in several cell types. The purpose of these studies was to characterize the interaction between endogenous CB(1) receptors and VSCCs in cerebellar granule neurons (CGN). Ca(2+) transients were evoked by KCl-induced depolarization and imaged using fura-2. The CB(1) receptor agonists CP55940, Win 55212-2 and N-arachidonylethanolamine (anandamide) produced concentration-related decreases in peak amplitude of the Ca(2+) response and total Ca(2+) influx. Pre-treatment of CGN with pertussis toxin abolished agonist-mediated inhibition. The inhibitory effect of Win 55212-2 on Ca(2+) influx was additive with inhibition produced by omega-agatoxin IVA and nifedipine but not with omega-conotoxin GVIA, indicating that N-type VSCCs are the primary effector. Paradoxically, the CB(1) receptor antagonist, SR141716, also inhibited KCl-induced Ca(2+) influx into CGN in a concentration-related manner. SR141716 inhibition was pertussis toxin-insensitive and was not additive with the inhibition produced by Win 55212-2. Confocal imaging of CGN in primary culture demonstrate a high density of CB(1) receptor expression on CGN plasma membranes, including the neuritic processes. These data demonstrate that the CB(1) receptor is highly expressed by CGN and agonists serve as potent and efficacious inhibitory modulators of Ca(2+) influx through N-type VSCC.     

Control of excitatory synaptic transmission by C-terminal Src kinase

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca(2+), followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca(2+) into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.     

Blanes cells mediate persistent feedforward inhibition onto granule cells in the olfactory bulb

Inhibitory local circuits in the olfactory bulb play a critical role in determining the firing patterns of output neurons. However, little is known about the circuitry in the major plexiform layers of the olfactory bulb that regulate this output. Here we report the first electrophysiological recordings from Blanes cells, large stellate-shaped interneurons located in the granule cell layer. We find that Blanes cells are GABAergic and generate large I(CAN)-mediated afterdepolarizations following bursts of action potentials. Using paired two-photon guided intracellular recordings, we show that Blanes cells have a presumptive axon and monosynaptically inhibit granule cells. Sensory axon stimulation evokes barrages of EPSPs in Blanes cells that trigger long epochs of persistent spiking; this firing mode was reset by hyperpolarizing membrane potential steps. Persistent firing in Blanes cells may represent a novel mechanism for encoding short-term olfactory information through modulation of tonic inhibitory synaptic input onto bulbar neurons.     

RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca(2+) channels

Ca(2+) influx through voltage-gated channels initiates the exocytotic fusion of synaptic vesicles to the plasma membrane. Here we show that RIM binding proteins (RBPs), which associate with Ca(2+) channels in hair cells, photoreceptors, and neurons, interact with alpha(1D) (L type) and alpha(1B) (N type) Ca(2+) channel subunits. RBPs contain three Src homology 3 domains that bind to proline-rich motifs in alpha(1) subunits and Rab3-interacting molecules (RIMs). Overexpression in PC12 cells of fusion proteins that suppress the interactions of RBPs with RIMs and alpha(1) augments the exocytosis triggered by depolarization. RBPs may regulate the strength of synaptic transmission by creating a functional link between the synaptic-vesicle tethering apparatus, which includes RIMs and Rab3, and the fusion machinery, which includes Ca(2+) channels and the SNARE complex.     

NMDA receptors turn to another channel for inhibition

Activation of glutamate receptors generally increases neuronal excitability. However, Isaacson and Murphy show in olfactory bulb granule cells that NMDA receptor-mediated calcium influx couples to large conductance (BK) calcium-activated potassium channels. The resulting inhibition is long lasting, which may be critical to the operation of the dynamic circuitry of the bulb.     

Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). The part played by Ca(2+) released from intracellular stores in the regulation of secretion is well established; however, the role of Ca(2+) influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (>or=10 microM) elicited an additional component of exocytosis despite reducing Ca(2+) influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (approximately 70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 microM) was potentiated (approximately 45%) by wortmannin. Exocytosis stimulated by 1 microM acetylcholine in the absence of extracellular Ca(2+) was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca(2+) entry through store-operated Ca(2+) channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd(3+) (2 microM), but not by inhibition of noncapacitative Ca(2+) entry by SB203580 (10 microM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca(2+) influx.