Postsynaptic Ca2 Research Articles

Absence of plateau potentials in dLGN cells leads to a breakdown in retinogeniculate refinement.

The link between neural activity and the refinement of projections from retina to the dorsal lateral geniculate nucleus (dLGN) of thalamus is based largely on studies that disrupt presynaptic retinogeniculate activity. Postsynaptic mechanisms responsible for implementing the activity-dependent remodeling in dLGN remain unknown. We tested whether L-type Ca(2+) channel activity in the form of synaptically evoked plateau potentials in dLGN cells is needed for remodeling by using a mutant mouse that lacks the ancillary β3 subunit and, as a consequence, has highly reduced L-type channel expression and attenuated L-type Ca(2+) currents. In the dLGNs of β3-null mice, glutamatergic postsynaptic activity evoked by optic tract stimulation was normal, but plateau potentials were rarely observed. The few plateaus that were evoked required high rates of retinal stimulation, but were still greatly attenuated compared with those recorded in age-matched wild-type mice. While β3-null mice exhibit normal stage II and III retinal waves, their retinogeniculate projections fail to segregate properly and dLGN cells show a high degree of retinal convergence even at late postnatal ages. These structural and functional defects were also accompanied by a reduction in CREB phosphorylation, a signaling event that has been shown to be essential for retinogeniculate axon segregation. Thus, postsynaptic L-type Ca(2+) activity plays an important role in mediating the refinement of the retinogeniculate pathway.

Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X1, P2X2, P2X3 channels, P2Y and A3 metabotropic receptors in neurotransmission

RationaleThe role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. Experimental approachLSCM-Fluo-4/(Ca2+)-imaging of postsynaptic Ca2+ transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). ResultsSynaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca2+ transients or Ca2+ oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1–7, P2Y1,2,12–14R. ConclusionsPurines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.

The CA3 region of the hippocampus: how is it? What is it for? How does it do it?

The CA3 region of the hippocampus: how is it? What is it for? How does it do it?

Long-term potentiation at spinal C-fiber synapses: a target for pathological pain.

Long-term potentiation (LTP), referring to a lasting increase in efficacy of synaptic transmission, is a common mechanism of memory storage in central nervous system (CNS). LTP at C-fiber synapses in spinal dorsal horn is considered as a synaptic model of pathological pain, as the spinal LTP is only induced by noxious electrical and natural stimuli but not by innoxious ones and LTPinducible stimulation is capable of leading to lasting behavioral signs of pathological pain in human and in animals. The molecular mechanisms of spinal LTP at C-fiber synapses are similar to hippocampal LTP in following aspects. Induction of LTP depends on postsynaptic Ca(2+) rise resulting from opening of N-methyl-D-aspartate channels (NMDA) and voltage-gated calcium channels (VGCCs), and Ca(2+) release from intracellular store; Early-phase LTP (<3h) needs activation of intracellular protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII), phospholipase C (PLC) and release of nitric oxide (NO); Late-phase LTP (>3h) is dependent on de novo protein synthesis; Activation of either dopamine D1 receptors or PKA, and extrogenous brain-derived neurotrophic factor (BDNF) or ATP directly induces late-phase LTP. Therefore, the drugs targeting at the above molecules may impair memory function of hippocampus. The striking difference between hippocampal LTP and spinal LTP at C-fiber synapses is that activation of glial cells and the over-expression of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin- beta (IL-1β), inhibit LTP in hippocampus, but promote LTP in spinal dorsal horn. The drugs targeting at the neuroinflammatory process may not only attenuate pathological pain but also improve memory in hippocampus.

Compartment-specific modulation of GABAergic synaptic transmission by TRPV1 channels in the dentate gyrus.

The transient receptor potential TRPV1 or vanilloid receptor is a nonselective ligand-gated channel highly expressed in primary sensory neurons where it mediates nociception. TRPV1 is also expressed in the brain where its activation depresses excitatory synaptic transmission. Whether TRPV1 also regulates inhibitory synapses in the brain is unclear. Here, using a combination of pharmacology, electrophysiology, and an in vivo knockdown strategy, we report that TRPV1 activation by capsaicin or by the endocannabinoid anandamide depresses somatic, but not dendritic inhibitory transmission in both rat and mouse dentate gyrus. The effect on somatic inhibition was absent in TRPV1 knock-out mice and was also eliminated by two different TRPV1 shRNAs expressed in dentate granule cells, strongly supporting a functional role for TRPV1 in modulating GABAergic synaptic function. Moreover, TRPV1-mediated depression occurs independently of GABA release, requires postsynaptic Ca(2+) rise and activation of calcineurin, and is likely due to clathrin-dependent internalization of GABA receptors. Altogether, these findings reveal a novel form of compartment-specific regulation whereby TRPV1 channels can modify synaptic function in the brain.

The initiation of synaptic 2-AG mobilization requires both an increased supply of diacylglycerol precursor and increased postsynaptic calcium

On-demand postsynaptic synthesis and release of endocannabinoid lipids and subsequent binding to presynaptic CB1 receptors (CB1Rs) mediates short and long-term depression (LTD) of excitatory transmission in many brain regions. However, mechanisms involved in the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) by diacylglycerol lipase α (DGLα) are poorly understood. Since Gq-coupled receptor activation can stimulate production of a major DGL substrate 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) by PLCβ, we sought to determine if 2-AG biosynthesis was limited only by a lack of substrate availability, or if other pathways, such as Ca2+ signaling, also need to be simultaneously engaged. To address this question, we loaded medium spiny neurons of the dorsolateral striatum with SAG while monitoring excitatory synaptic inputs. SAG-loading had no significant effect on evoked excitatory synaptic currents when cells were voltage-clamped at −80 mV. However, depolarization of MSNs to −50 mV revealed a SAG-loading dependent decrease in the amplitude of excitatory currents that was accompanied by an increase in paired pulse ratio, consistent with decreased glutamate release. Both effects of loading SAG at −50 mV were blocked by chelation of postsynaptic Ca2+ using BAPTA or by bath application of tetrahydrolipstatin (THL), a DGL inhibitor. Loading of SAG into glutamatergic pyramidal neurons of the amygdala similarly inhibited excitatory synaptic inputs and increased the PPR. SAG-induced depression was absent in both regions from mice lacking CB1Rs. These data show that increasing substrate availability alone is insufficient to drive 2-AG mobilization and that DGL-dependent synaptic depression via CB1R activation requires postsynaptic Ca2+ signals.

Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles.

Presynaptic terminals release neurotransmitters spontaneously in a manner that can be regulated by Ca(2+). However, the mechanisms underlying this regulation are poorly understood because the inherent stochasticity and low probability of spontaneous fusion events has curtailed their visualization at individual release sites. Here, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single spontaneous fusion events and found that they are retrieved extremely rapidly with faster re-acidification kinetics than their action potential-evoked counterparts. These fusion events were coupled to postsynaptic NMDA receptor-driven Ca(2+) signals, and at elevated Ca(2+) concentrations there was an increase in the number of vesicles that would undergo fusion. Furthermore, spontaneous vesicle fusion propensity in a synapse was Ca(2+)-dependent but regulated autonomously: independent of evoked fusion probability at the same synapse. Taken together, these results expand classical quantal analysis to incorporate endocytic and exocytic phases of single fusion events and uncover autonomous regulation of spontaneous fusion.

Presynaptic Ca2+-permeable AMPA-receptors modulate paired-pulse depression in nociceptive sensory synapses

The role of Ca2+-permeable AMPA-receptors (CP-AMPARs) in the induction of paired-pulse depression (PPD) at glutamatergic nociceptive sensory synapses was examined in co-culture of rat’s dorsal root ganglion and dorsal horn neurons. CP-AMPARs make a considerable contribution to excitatory postsynaptic currents recorded in DH neurons following action potential generation in nociceptive DRG neurons. Activation of CP-AMPARs during fast synaptic transmission induces strong PPD (with a 200ms inter-pulse interval). Blockage of CP-AMPARs with Naspm (100μM) results in most cases in a significant reduction of the PPD magnitude, also reversing to paired-pulse facilitation in some cases. However, loading of postsynaptic DH neurons with BAPTA (10mM) did not alter the effect of Naspm (100μM) on the paired-pulse ratio. Our data provide evidence that presynaptic CP-AMPARs in nociceptive sensory synapses regulate the magnitude of PPD and that postsynaptic Ca2+-permeable glutamatergic receptors are not required for PPD induction.

Pannexin 1 regulates bidirectional hippocampal synaptic plasticity in adult mice

The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca2+ concentration and NMDA receptor (NMDAR) composition of GluN2 subunits. Pannexin 1 (Panx1), a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LTP), it remains unknown whether these channels also modulate long-term depression (LTD) or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LTP, whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory.

Joint CP-AMPA and group I mGlu receptor activation is required for synaptic plasticity in dentate gyrus fast-spiking interneurons.

Hippocampal principal cell (PC) assemblies provide the brain with a mnemonic representation of space. It is assumed that the formation of cell assemblies is supported by long-lasting modification of glutamatergic synapses onto perisomatic inhibitory interneurons (PIIs), which provide powerful feedback inhibition to neuronal networks. Repetitive activation of dentate gyrus PIIs by excitatory mossy fiber (MF) inputs induces Hebbian long-term potentiation (LTP). In contrast, long-term depression (LTD) emerges in the absence of PII activity. However, little is known about the molecular mechanisms underlying synaptic plasticity in PIIs. Here, we examined the role of group I metabotropic glutamate receptors 1 and 5 (mGluRs1/5) in inducing plastic changes at MF-PII synapses. We found that mGluRs1/5 are located perisynaptically and that pharmacological block of mGluR1 or mGluR5 abolished MF-LTP. In contrast, their exogenous activation was insufficient to induce MF-LTP but cleared MF-LTD. No LTP could be elicited in PIIs loaded with blockers of G protein signaling and Ca(2+)-dependent PKC. Two-photon imaging revealed that the intracellular Ca(2+) rise necessary for MF-LTP was largely mediated by Ca(2+)-permeable AMPA receptors (CP-AMPARs), but less by NMDA receptors or mGluRs1/5. Thus, our data indicate that fast Ca(2+) signaling via CP-AMPARs and slow G protein-mediated signaling via mGluRs1/5 converge to a PKC-dependent molecular pathway to induce Hebbian MF-LTP. We further propose that Hebbian activation of mGluRs1/5 gates PIIs into a "readiness mode" to promote MF-LTP, which, in turn, will support timed PII recruitment, thereby assisting in PC assembly formation.

Theta-burst stimulation induces LTP at excitatory and inhibitory synapses in the spinal trigeminal subnucleus interpolaris

Long-lasting synaptic modifications of excitatory and inhibitory synaptic transmissions induced by theta-burst stimulation (TBS) were examined in the spinal trigeminal subnucleus interpolaris (Vi). We found that conditioning afferents of another subnucleus caudalis (Vc) to the Vi with TBS produced long-term depression (LTD). However, when GABAA and glycine receptors were blocked, the same stimulation paradigm produced long-term potentiation (LTP). The induction of LTP involved neither NMDA receptors nor a presynaptic change. The expression of LTP was obviously suppressed by the activation of group I mGluRs because its magnitude increased in the presence of antagonists for group I mGluRs. Besides the LTP at excitatory synapses, TBS also induced LTP at inhibitory GABAergic synapses, which required the activation of NMDA receptors and NO-cGMP signaling but was not involved in the increase of postsynaptic Ca2+ concentration. Therefore, this study shows, for the first time, an activity-dependent plasticity at excitatory and inhibitory synapses in the Vi by the same conditioning stimulation.

Ionotropic NMDA Receptor Signaling Is Required for the Induction of Long-Term Depression in the Mouse Hippocampal CA1 Region

Previous studies have provided strong support for the notion that NMDAR-mediated increases in postsynaptic Ca(2+) have a crucial role in the induction of long-term depression (LTD). This view has recently been challenged, however, by findings suggesting that LTD induction is instead attributable to an ion channel-independent, metabotropic form of NMDAR signaling. Thus, to explore the role of ionotropic versus metabotropic NMDAR signaling in LTD, we examined the effects of varying extracellular Ca(2+) levels or blocking NMDAR channel ion fluxes with MK-801 on LTD and NMDAR signaling in the mouse hippocampal CA1 region. We find that the induction of LTD in the adult hippocampus is highly sensitive to extracellular Ca(2+) levels and that MK-801 blocks NMDAR-dependent LTD in the hippocampus of both adult and immature mice. Moreover, MK-801 inhibits NMDAR-mediated activation of p38-MAPK and dephosphorylation of AMPAR GluA1 subunits at sites implicated in LTD. Thus, our results indicate that the induction of LTD in the hippocampal CA1 region is dependent on ionotropic, rather than metabotropic, NMDAR signaling.

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.

Synaptic excitation is regulated by the postsynaptic dSK channel at theDrosophilalarval NMJ

In the mammalian central nervous system, the postsynaptic small-conductance Ca(2+)-dependent K(+) (SK) channel has been shown to reduce postsynaptic depolarization and limit Ca(2+) influx through N-methyl-d-aspartate receptors. To examine further the role of the postsynaptic SK channel in synaptic transmission, we studied its action at the Drosophila larval neuromuscular junction (NMJ). Repetitive synaptic stimulation produced an increase in postsynaptic membrane conductance leading to depression of excitatory postsynaptic potential amplitude and hyperpolarization of the resting membrane potential (RMP). This reduction in synaptic excitation was due to the postsynaptic Drosophila SK (dSK) channel; synaptic depression, increased membrane conductance and RMP hyperpolarization were reduced in dSK mutants or after expressing a Ca(2+) buffer in the muscle. Ca(2+) entering at the postsynaptic membrane was sufficient to activate dSK channels based upon studies in which the muscle membrane was voltage clamped to prevent opening voltage-dependent Ca(2+) channels. Increasing external Ca(2+) produced an increase in resting membrane conductance and RMP that was not seen in dSK mutants or after adding the glutamate-receptor blocker philanthotoxin. Thus it appeared that dSK channels were also activated by spontaneous transmitter release and played a role in setting membrane conductance and RMP. In mammals, dephosphorylation by protein phosphatase 2A (PP2A) increased the Ca(2+) sensitivity of the SK channel; PP2A appeared to increase the sensitivity of the dSK channel since PP2A inhibitors reduced activation of the dSK channel by evoked synaptic activity or increased external Ca(2+). It is proposed that spontaneous and evoked transmitter release activate the postsynaptic dSK channel to limit synaptic excitation and stabilize synapses.

Dendritic Calcium Nonlinearities Switch the Direction of Synaptic Plasticity in Fast-Spiking Interneurons

Postsynaptic calcium (Ca2+) nonlinearities allow neuronal coincidence detection and site-specific plasticity. Whether such events exist in dendrites of interneurons and play a role in regulation of synaptic efficacy remains unknown. Here, we used a combination of whole-cell patch-clamp recordings and two-photon Ca2+ imaging to reveal Ca2+ nonlinearities associated with synaptic integration in dendrites of mouse hippocampal CA1 fast-spiking interneurons. Local stimulation of distal dendritic branches within stratum oriens/alveus elicited fast Ca2+ transients, which showed a steep sigmoidal relationship to stimulus intensity. Supralinear Ca2+ events required Ca2+ entry through AMPA receptors with a subsequent Ca2+ release from internal stores. To investigate the functional significance of supralinear Ca2+ signals, we examined activity-dependent fluctuations in transmission efficacy triggered by Ca2+ signals of different amplitudes at excitatory synapses of interneurons. Subthreshold theta-burst stimulation (TBS) produced small amplitude postsynaptic Ca2+ transients and triggered long-term potentiation. In contrast, the suprathreshold TBS, which was associated with the generation of supralinear Ca2+ events, triggered long-term depression. Blocking group I/II metabotropic glutamate receptors (mGluRs) during suprathreshold TBS resulted in a slight reduction of supralinear Ca2+ events and induction of short-term depression. In contrast, blocking internal stores and supralinear Ca2+ signals during suprathreshold TBS switched the direction of plasticity from depression back to potentiation. These data reveal a novel type of supralinear Ca2+ events at synapses lacking the GluA2 AMPA subtype of glutamate receptors and demonstrate a general mechanism by which Ca2+ -permeable AMPA receptors, together with internal stores and mGluRs, control the direction of plasticity at interneuron excitatory synapses.

Long-term potentiation at excitatory synaptic inputs to the intercalated cell masses of the amygdala

The intercalated cell masses (ITCs) of the amygdala are clusters of GABAergic interneurons that surround the basolateral complex of the amygdala. ITCs have been increasingly implicated in the acquisition and extinction of conditioned fear responses, but the underlying cellular mechanisms remain unexplored. Here, we report that repetitive stimulation of lateral amygdala (LA) afferents with a modified theta burst stimulation (TBS) protocol and induces long-term potentiation (LTP) of excitatory synapses onto medial paracapsular ITC (Imp) neurons. This TBS-induced LTP is; (1) induced and expressed post-synaptically, (2) involves a rise in post-synaptic Ca2+ and the activation of NR2B-containing N-methyl-D-aspartate receptors (NMDARs), (3) dependent on calcium/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase activation, and (4) associated with increased exocytotic delivery of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) to the post-synaptic membrane. Remarkably, auditory fear conditioning led to a persistent increase in AMPAR/NMDAR ratio of glutamatergic synaptic currents and occluded TBS-induced LTP at LA-Imp synapses. Furthermore, extinction training rescued the effect of fear conditioning on AMPAR/NMDAR ratio and LTP induction. These results show that a prominent form of LTP can be elicited at LA-Imp synapses and suggest that this synaptic plasticity may contribute to the expression of fear conditioning.

Long-Term Potentiation at Cerebellar Parallel Fiber–Purkinje Cell Synapses Requires Presynaptic and Postsynaptic Signaling Cascades

Long-term depression (LTD) and long-term potentiation (LTP) at cerebellar parallel fiber-Purkinje cell (PF-PC) synapses play critical roles in motor learning. The 1 Hz stimulation at PF-PC synapses induces a postsynaptically expressed LTP that requires a postsynaptic Ca(2+) transient, phosphatases, and nitric oxide (NO). However, the mechanism underlying 1 Hz PF-LTP remains unclear because none of the known events is related to each other. Here, we demonstrated that 1 Hz PF-LTP requires postsynaptic cytosolic phospholipase A2 α (cPLA2α)/arachidonic acid (AA) signaling and presynaptic endocannabinoid receptors. Using patch-clamp recording in cerebellar slices, we found that 1 Hz PF-LTP was abolished in cPLA2α-knock-out mice. This deficit was effectively rescued by the conjunction of 1 Hz PF stimulation and the local application of AA. 2-Arachidonoylglycerol and the retrograde activation of cannabinoid receptor 1 (CB1R) were also involved in 1 Hz LTP because it was blocked by the hydrolysis of 2-AG or by inhibiting CB1Rs. The amount of NO released was detected using an NO electrode in cultured granule cells and PF terminals. Our results showed that the activation of CB1Rs at PF terminals activated NO synthetase and promoted NO production. The 1 Hz PF-stimuli evoked limited NO, but 100 Hz PF stimulation generated a large amount. Therefore, 1 Hz PF-LTP, distinct from classical postsynaptically expressed plasticity, requires concurrent presynaptic and postsynaptic activity. In addition, NO of sufficient amplitude decides between the weakening and strengthening of PF-PC synapses.

PSD-95 promotes the stabilization of young synaptic contacts

Maintaining a population of stable synaptic connections is probably of critical importance for the preservation of memories and functional circuitry, but the molecular dynamics that underlie synapse stabilization is poorly understood. Here, we use simultaneous time-lapse imaging of post synaptic density-95 (PSD-95) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to investigate the dynamics of protein composition at axodendritic (AD) contacts. Our data reveal that this composition is highly dynamic, with both proteins moving into and out of the same synapse independently, so that synapses cycle rapidly between states in which they are enriched for none, one or both proteins. We assessed how PSD-95 and CaMKII interact at stable and transient AD sites and found that both phospho-CaMKII and PSD-95 are present more often at stable than labile contacts. Finally, we found that synaptic contacts are more stable in older neurons, and this process can be mimicked in younger neurons by overexpression of PSD-95. Taken together, these data show that synaptic protein composition is highly variable over a time-scale of hours, and that PSD-95 is probably a key synaptic protein that promotes synapse stability.

Control of synaptic function by endocannabinoid-mediated retrograde signaling.

Since the first reports in 2001, great advances have been made towards the understanding of endocannabinoid-mediated synaptic modulation. Electrophysiological studies have revealed that one of the two major endocannabinoids, 2-arachidonoylglycerol (2-AG), is produced from membrane lipids upon postsynaptic Ca2+ elevation and/or activation of Gq/11-coupled receptors, and released from postsynaptic neurons. The released 2-AG then acts retrogradely onto presynaptic cannabinoid CB1 receptors and induces suppression of neurotransmitter release either transiently or persistently. These forms of 2-AG-mediated retrograde synaptic modulation are functional throughout the brain. The other major endocannabinoid, anandamide, mediates a certain form of endocannabinoid-mediated long-term depression (LTD). Anandamide also functions as an agonist for transient receptor potential vanilloid receptor type 1 (TRPV1) and mediates endocannabinoid-independent and TRPV1-dependent forms of LTD. It has also been demonstrated that the endocannabinoid system itself is plastic, which can be either up- or down-regulated by experimental or environmental conditions. In this review, I will make an overview of the mechanisms underlying endocannabinoid-mediated synaptic modulation.

Activation of STIM1 by L-Glutamate Rapidly Inhibits L-Type Calcium Channel Current in Cultured Hippocampal Neurons

At glutamatergic synapses, synaptic activity elicits postsynaptic Ca2+ signals that are generated in part by voltage-gated, L-type calcium channels. L-channel Ca2+ signals are known to control transcription of genes involved in synaptic plasticity, such as during late-phase long-term potentiation. Here we report that, in short-term cultured rat hippocampal neurons, 15-sec application of L-glutamate inhibited pharmacologically-isolated L-type Ca2+ current: inhibition ensued with a t1/2 of 49 ± 2 sec, and current was reduced by ∼35%. Measurements made with a genetically-encoded Ca2+ indicator (D1ER) targeted to intracellular stores showed that application of L-glutamate triggered release of Ca2+ from stores. FRET-imaging demonstrated that L-glutamate application also induced interaction of the Ca2+ store-depletion sensor STIM1 (CFP-tagged) with the L-channel scaffolding protein, AKAP79/150 (YFP-tagged), but not another AKAP scaffold protein, AKAP15/18. The time course of L-channel inhibition paralleled that for the increase in STIM1-AKAP79/150 FRET, suggesting a functional relationship between glutamate-triggered STIM1 translocation and inhibition of L-channel current. Supporting this idea, RNAi knockdown of STIM1 abolished glutamate-induced inhibition of L-channel activity. The ability of glutamate to inhibit L-channels in STIM1-knockdown neurons was rescued by co-transfection of rat neurons with the human STIM1 isotype, which was insensitive to the rat RNAi. Our results suggest a model wherein (i) glutamate receptor activation triggers Ca2+ release from endoplasmic reticulum (ER) stores within seconds of glutamate application, (ii) lowered ER Ca2+ activates STIM1 translocation to ER/plasma membrane junctions, and (iii) STIM1 interaction with the L-channel-AKAP79/150 complex down-regulates L-type Ca2+ current. This STIM1-mediated link between glutamate receptors and L-type Ca2+ channels may tune cytoplasmic Ca2+ signals involved in synaptic plasticity of hippocampal neurons.