Two-photon Glutamate Uncaging Research Articles

VAP spatially stabilizes dendritic mitochondria to locally support synaptic plasticity

Synapses are pivotal sites of plasticity and memory formation. Consequently, synapses are energy consumption hotspots susceptible to dysfunction when their energy supplies are perturbed. Mitochondria are stabilized near synapses via the cytoskeleton and provide the local energy required for synaptic plasticity. However, the mechanisms that tether and stabilize mitochondria to support synaptic plasticity are unknown. We identified proteins exclusively tethering mitochondria to actin near postsynaptic spines. We find that VAP, the vesicle-associated membrane protein-associated protein implicated in amyotrophic lateral sclerosis, stabilizes mitochondria via actin near the spines. To test if the VAP-dependent stable mitochondrial compartments can locally support synaptic plasticity, we used two-photon glutamate uncaging for spine plasticity induction and investigated the induced and adjacent uninduced spines. We find VAP functions as a spatial stabilizer of mitochondrial compartments for up to ~60 min and as a spatial ruler determining the ~30 μm dendritic segment supported during synaptic plasticity.

Mechanisms That Underlie Expression of Estradiol-Induced Excitatory Synaptic Potentiation in the Hippocampus Differ between Males and Females.

17β-estradiol (E2) is synthesized in the hippocampus of both sexes and acutely potentiates excitatory synapses in each sex. Previously, we found that the mechanisms for initiation of E2-induced synaptic potentiation differ between males and females, including in the molecular signaling involved. Here, we used electrical stimulation and two-photon glutamate uncaging in hippocampal slices from adult male and female rats to investigate whether the downstream consequences of distinct molecular signaling remain different between the sexes or converge to the same mechanism(s) of expression of potentiation. This showed that synaptic activity is necessary for expression of E2-induced potentiation in females but not males, which paralleled a sex-specific requirement in females for calcium-permeable AMPARs (cpAMPARs) to stabilize potentiation. Nonstationary fluctuation analysis of two-photon evoked unitary synaptic currents showed that the postsynaptic component of E2-induced potentiation occurs either through an increase in AMPAR conductance or in nonconductive properties of AMPARs (number of channels × open probability) and never both at the same synapse. In females, most synapses (76%) were potentiated via increased AMPAR conductance, whereas in males, more synapses (60%) were potentiated via an increase in nonconductive AMPAR properties. Inhibition of cpAMPARs eliminated E2-induced synaptic potentiation in females, whereas some synapses in males were unaffected by cpAMPAR inhibition; these synapses in males potentiated exclusively via increased AMPAR nonconductive properties. This sex bias in expression mechanisms of E2-induced synaptic potentiation underscores the concept of latent sex differences in mechanisms of synaptic plasticity in which the same outcome in each sex is achieved through distinct underlying mechanisms.SIGNIFICANCE STATEMENT Estrogens are synthesized in the brains of both sexes and potentiate excitatory synapses to the same degree in each sex. Despite this apparent similarity, the molecular signaling that initiates estrogen-induced synaptic potentiation differs between the sexes. Here we show that these differences extend to the mechanisms of expression of synaptic potentiation and result in distinct patterns of postsynaptic neurotransmitter receptor modulation in each sex. Such latent sex differences, in which the same outcome is achieved through distinct underlying mechanisms in males versus females, indicate that molecular mechanisms targeted for drug development may differ between the sexes even in the absence of an overt sex difference in behavior or disease.

Altered integration of excitatory inputs onto the basal dendrites of layer 5 pyramidal neurons in a mouse model of Fragile X syndrome

Layer 5 (L5) pyramidal neurons receive predictive and sensory inputs in a compartmentalized manner at their apical and basal dendrites, respectively. To uncover how integration of sensory inputs is affected in autism spectrum disorders (ASD), we used two-photon glutamate uncaging to activate spines in the basal dendrites of L5 pyramidal neurons from a mouse model of Fragile X syndrome (FXS), the most common genetic cause of ASD. While subthreshold excitatory inputs integrate linearly in wild-type animals, surprisingly those with FXS summate sublinearly, contradicting what would be expected of sensory hypersensitivity classically associated with ASD. We next investigated the mechanism underlying this sublinearity by performing knockdown of the regulatory β4 subunit of BK channels, which rescued the synaptic integration, a result that was corroborated with numerical simulations. Taken together, these findings suggest that there is a differential impairment in the integration of feedforward sensory and feedback predictive inputs in L5 pyramidal neurons in FXS and potentially other forms of ASD, as a result of specifically localized subcellular channelopathies. These results challenge the traditional view that FXS and other ASD are characterized by sensory hypersensitivity, proposing instead a hyposensitivity of sensory inputs and hypersensitivity of predictive inputs onto cortical neurons.

Fast Synaptically Activated Calcium and Sodium Kinetics in Hippocampal Pyramidal Neuron Dendritic Spines.

An accurate assessment of the time course, components, and magnitude of postsynaptic currents is important for a quantitative understanding of synaptic integration and signaling in dendritic spines. These parameters have been studied in some detail in previous experiments, primarily using two-photon imaging of [Ca2+]i changes and two-photon uncaging of glutamate. However, even with these revolutionary techniques, there are some missing pieces in our current understanding, particularly related to the time courses of synaptically evoked [Ca2+]i and [Na+]i changes. In new experiments, we used low-affinity, linear Na+ and Ca2+ indicators, laser fluorescence stimulation, and a sensitive camera-based detection system, combined with electrical stimulation and two-photon glutamate uncaging, to extend measurements of these spine parameters. We found that (1) almost all synaptically activated Na+ currents in CA1 hippocampal pyramidal neuron spines in slices from mice of either sex are through AMPA receptors with little Na+ entry through voltage-gated sodium channels (VGSCs) or NMDA receptor channels; (2) a spectrum of sodium transient decay times was observed, suggesting a spectrum of spine neck resistances, even on the same dendrite; (3) synaptically activated [Ca2+]i changes are very fast and are almost entirely because of Ca2+ entry through NMDA receptors at the time when the Mg2+ block is relieved by the fast AMPA-mediated EPSP; (4) the [Ca2+]i changes evoked by uncaging glutamate are slower than the changes evoked by synaptic release, suggesting that the relative contribution of Ca2+ entering through NMDA receptors at rest following uncaging is higher than following electrical stimulation.

Input rate encoding and gain control in dendrites of neocortical pyramidal neurons.

SUMMARYElucidating how neurons encode network activity is essential to understanding how the brain processes information. Neocortical pyramidal cells receive excitatory input onto spines distributed along dendritic branches. Local dendritic branch nonlinearities can boost the response to spatially clustered and synchronous input, but how this translates into the integration of patterns of ongoing activity remains unclear. To examine dendritic integration under naturalistic stimulus regimes, we use two-photon glutamate uncaging to repeatedly activate multiple dendritic spines at random intervals. In the proximal dendrites of two populations of layer 5 pyramidal neurons in the mouse motor cortex, spatially restricted synchrony is not a prerequisite for dendritic boosting. Branches encode afferent inputs with distinct rate sensitivities depending upon cell and branch type. Thus, inputs distributed along a dendritic branch can recruit supralinear boosting and the window of this nonlinearity may provide a mechanism by which dendrites can preferentially amplify slow-frequency network oscillations.

Mechanical actions of dendritic-spine enlargement on presynaptic exocytosis.

Synaptic transmission involves cell-to-cell communication at the synaptic junction between two neurons, and chemical and electrical forms of this process have been extensively studied. In the brain, excitatory glutamatergic synapses are often made on dendritic spines that enlarge during learning1-5. As dendritic spines and the presynaptic terminals are tightly connected with the synaptic cleft6, the enlargement may have mechanical effects on presynaptic functions7. Here we show that fine and transient pushing of the presynaptic boutons with a glass pipette markedly promotes both the evoked release of glutamate and the assembly of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins8-12-as measured by Förster resonance transfer (FRET) and fluorescence lifetime imaging-in rat slice culture preparations13. Both of these effects persisted for more than 20 minutes. The increased presynaptic FRET was independent of cytosolic calcium (Ca2+), but dependent on the assembly of SNARE proteins and actin polymerization in the boutons. Notably, a low hypertonic solution of sucrose (20 mM) had facilitatory effects on both the FRET and the evoked release without inducing spontaneous release, in striking contrast with a high hypertonic sucrose solution (300 mM), which induced exocytosis by itself14. Finally, spine enlargement induced by two-photon glutamate uncaging enhanced the evoked release and the FRET only when the spines pushed the boutons by their elongation. Thus, we have identified a mechanosensory and transduction mechanism15 in the presynaptic boutons, in which the evoked release of glutamate is enhanced for more than 20 min.

Rapid Ultrastructural Changes in the PSD and Surrounding Membrane after Induction of Structural LTP in Single Dendritic Spines.

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon-spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.

Presynaptic NMDARs cooperate with local spikes toward GABA release from the reciprocal olfactory bulb granule cell spine.

In the rodent olfactory bulb the smooth dendrites of the principal glutamatergic mitral cells (MCs) form reciprocal dendrodendritic synapses with large spines on GABAergic granule cells (GC), where unitary release of glutamate can trigger postsynaptic local activation of voltage-gated Na+-channels (Navs), that is a spine spike. Can such single MC input evoke reciprocal release? We find that unitary-like activation via two-photon uncaging of glutamate causes GC spines to release GABA both synchronously and asynchronously onto MC dendrites. This release indeed requires activation of Navs and high-voltage-activated Ca2+-channels (HVACCs), but also of NMDA receptors (NMDAR). Simulations show temporally overlapping HVACC- and NMDAR-mediated Ca2+-currents during the spine spike, and ultrastructural data prove NMDAR presence within the GABAergic presynapse. This cooperative action of presynaptic NMDARs allows to implement synapse-specific, activity-dependent lateral inhibition, and thus could provide an efficient solution to combinatorial percept synthesis in a sensory system with many receptor channels.

Pathway-specific dysregulation of striatal excitatory synapses by LRRK2 mutations.

LRRK2 is a kinase expressed in striatal spiny projection neurons (SPNs), cells which lose dopaminergic input in Parkinson's disease (PD). R1441C and G2019S are the most common pathogenic mutations of LRRK2. How these mutations alter the structure and function of individual synapses on direct and indirect pathway SPNs is unknown and may reveal pre-clinical changes in dopamine-recipient neurons that predispose toward disease. Here, R1441C and G2019S knock-in mice enabled thorough evaluation of dendritic spines and synapses on pathway-identified SPNs. Biochemical synaptic preparations and super-resolution imaging revealed increased levels and altered organization of glutamatergic AMPA receptors in LRRK2 mutants. Relatedly, decreased frequency of miniature excitatory post-synaptic currents accompanied changes in dendritic spine nano-architecture, and single-synapse currents, evaluated using two-photon glutamate uncaging. Overall, LRRK2 mutations reshaped synaptic structure and function, an effect exaggerated in R1441C dSPNs. These data open the possibility of new neuroprotective therapies aimed at SPN synapse function, prior to disease onset.

A spike-timing-dependent plasticity rule for dendritic spines

The structural organization of excitatory inputs supporting spike-timing-dependent plasticity (STDP) remains unknown. We performed a spine STDP protocol using two-photon (2P) glutamate uncaging (pre) paired with postsynaptic spikes (post) in layer 5 pyramidal neurons from juvenile mice. Here we report that pre-post pairings that trigger timing-dependent LTP (t-LTP) produce shrinkage of the activated spine neck and increase in synaptic strength; and post-pre pairings that trigger timing-dependent LTD (t-LTD) decrease synaptic strength without affecting spine shape. Furthermore, the induction of t-LTP with 2P glutamate uncaging in clustered spines (<5 μm apart) enhances LTP through a NMDA receptor-mediated spine calcium accumulation and actin polymerization-dependent neck shrinkage, whereas t-LTD was dependent on NMDA receptors and disrupted by the activation of clustered spines but recovered when separated by >40 μm. These results indicate that synaptic cooperativity disrupts t-LTD and extends the temporal window for the induction of t-LTP, leading to STDP only encompassing LTP.

The NMDA receptor subunit GluN3A regulates synaptic activity-induced and myocyte enhancer factor 2C (MEF2C)-dependent transcription

N-Methyl-d-aspartate type glutamate receptors (NMDARs) are key mediators of synaptic activity-regulated gene transcription in neurons, both during development and in the adult brain. Developmental differences in the glutamate receptor ionotropic NMDA 2 (GluN2) subunit composition of NMDARs determines whether they activate the transcription factor cAMP-responsive element-binding protein 1 (CREB). However, whether the developmentally regulated GluN3A subunit also modulates NMDAR-induced transcription is unknown. Here, using an array of techniques, including quantitative real-time PCR, immunostaining, reporter gene assays, RNA-Seq, and two-photon glutamate uncaging with calcium imaging, we show that knocking down GluN3A in rat hippocampal neurons promotes the inducible transcription of a subset of NMDAR-sensitive genes. We found that this enhancement is mediated by the accumulation of phosphorylated p38 mitogen-activated protein kinase in the nucleus, which drives the activation of the transcription factor myocyte enhancer factor 2C (MEF2C) and promotes the transcription of a subset of synaptic activity-induced genes, including brain-derived neurotrophic factor (Bdnf) and activity-regulated cytoskeleton-associated protein (Arc). Our evidence that GluN3A regulates MEF2C-dependent transcription reveals a novel mechanism by which NMDAR subunit composition confers specificity to the program of synaptic activity-regulated gene transcription in developing neurons.

Molecular Mechanisms of Non-ionotropic NMDA Receptor Signaling in Dendritic Spine Shrinkage.

Structural plasticity of dendritic spines is a key component of the refinement of synaptic connections during learning. Recent studies highlight a novel role for the NMDA receptor (NMDAR), independent of ion flow, in driving spine shrinkage and LTD. Yet little is known about the molecular mechanisms that link conformational changes in the NMDAR to changes in spine size and synaptic strength. Here, using two-photon glutamate uncaging to induce plasticity at individual dendritic spines on hippocampal CA1 neurons from mice and rats of both sexes, we demonstrate that p38 MAPK is generally required downstream of non-ionotropic NMDAR signaling to drive both spine shrinkage and LTD. In a series of pharmacological and molecular genetic experiments, we identify key components of the non-ionotropic NMDAR signaling pathway driving dendritic spine shrinkage, including the interaction between NOS1AP (nitric oxide synthase 1 adaptor protein) and neuronal nitric oxide synthase (nNOS), nNOS enzymatic activity, activation of MK2 (MAPK-activated protein kinase 2) and cofilin, and signaling through CaMKII. Our results represent a large step forward in delineating the molecular mechanisms of non-ionotropic NMDAR signaling that can drive shrinkage and elimination of dendritic spines during synaptic plasticity.SIGNIFICANCE STATEMENT Signaling through the NMDA receptor (NMDAR) is vitally important for the synaptic plasticity that underlies learning. Recent studies highlight a novel role for the NMDAR, independent of ion flow, in driving synaptic weakening and dendritic spine shrinkage during synaptic plasticity. Here, we delineate several key components of the molecular pathway that links conformational signaling through the NMDAR to dendritic spine shrinkage during synaptic plasticity.

Single Synapse LTP: A Matter of Context?

The most commonly studied form of synaptic plasticity is long-term potentiation (LTP). Over the last 15 years, it has been possible to induce structural and functional LTP in dendritic spines using two-photon glutamate uncaging, allowing for studying the signaling mechanisms of LTP with single synapse resolution. In this review, we compare different stimulation methods to induce single synapse LTP and discuss how LTP is expressed. We summarize the underlying signaling mechanisms that have been studied with high spatiotemporal resolution. Finally, we discuss how LTP in a single synapse can be affected by excitatory and inhibitory synapses nearby. We argue that single synapse LTP is highly dependent on context: the choice of induction method, the history of the dendritic spine and the dendritic vicinity crucially affect signaling pathways and expression of single synapse LTP.

Bidirectional in vivo structural dendritic spine plasticity revealed by two-photon glutamate uncaging in the mouse neocortex

Most excitatory synapses in the brain form on dendritic spines. Two-photon uncaging of glutamate is widely utilized to characterize the structural plasticity of dendritic spines in brain slice preparations in vitro. In the present study, glutamate uncaging was used to investigate spine plasticity, for the first time, in vivo. A caged glutamate compound was applied to the surface of the mouse visual cortex in vivo, revealing the successful induction of spine enlargement by repetitive two-photon uncaging in a magnesium free solution. Notably, this induction occurred in a smaller fraction of spines in the neocortex in vivo (22%) than in hippocampal slices (95%). Once induced, the time course and mean long-term enlargement amplitudes were similar to those found in hippocampal slices. However, low-frequency (1–2 Hz) glutamate uncaging in the presence of magnesium caused spine shrinkage in a similar fraction (35%) of spines as in hippocampal slices, though spread to neighboring spines occurred less frequently than it did in hippocampal slices. Thus, the structural plasticity may occur similarly in the neocortex in vivo as in hippocampal slices, although it happened less frequently in our experimental conditions.

5-HT2A receptor activation enhances NMDA receptor-mediated glutamate responses through Src kinase in the dendrites of rat jaw-closing motoneurons.

5-HT increases the excitability of brainstem and spinal motoneurons, including the jaw-closing motoneurons, by depolarizing the membrane potential and decreasing the medium-duration afterhyperpolarization. In this study, we focused on how 5-HT enhances postsynaptic glutamatergic responses in the dendrites of the jaw-closing motoneurons. We demonstrate that 5-HT augments glutamatergic signalling by enhancing the function of the GluN2A-containing NMDA receptor (NMDAR) through the activation of 5-HT2A receptors (5-HT2A Rs) and Src kinase. To enhance glutamatergic responses, activation of the 5-HT2A Rs must occur within ∼60μm of the location of the glutamate responses. 5-HT inputs to the jaw-closing motoneurons can significantly vary their input-output relationship, which may contribute to wide-range regulation of contractile forces of the jaw-closing muscles. Various motor behaviours are modulated by 5-HT. Although the masseter (jaw-closing) motoneurons receive both glutamatergic and serotonergic inputs, it remains unclear how 5-HT affects the glutamatergic inputs to the motoneuronal dendrites. We examined the effects of 5-HT on postsynaptic responses evoked by single- or two-photon uncaging of caged glutamate (glutamate responses) to the dendrites of masseter motoneurons in postnatal day 2-5 rats of either sex. Application of 5-HT induced membrane depolarization and enhanced the glutamate-response amplitude. This enhancement was mimicked by the 5-HT2A receptor (5-HT2A R) agonist and was blocked by the 5-HT2A/2C R antagonist. However, neither the 5-HT2B R nor the 5-HT2C R agonists altered glutamate responses. Blockade of the NMDA receptors (NMDARs), but not AMPA receptors, abolished the 5-HT-induced enhancement. Furthermore, the selective antagonist for the GluN2A subunit abolished the 5-HT-induced enhancement. 5-HT increased GluN2A phosphorylation, while the Src kinase inhibitor reduced the 5-HT-induced enhancement and GluN2A phosphorylation. When exposure to the 5-HT2A R agonist was targeted to the dendrites, the enhancement of glutamate responses was restricted to the loci of the dendrites near the puff loci. Electron microscopic immunohistochemistry revealed that both the NMDARs and the 5-HT2A Rs were close to each other in the same dendrite. These results suggest that activation of dendritic 5-HT2A Rs enhances the function of local GluN2A-containing NMDARs through Src kinase. Such enhancement of the glutamate responses by 5-HT may contribute to wide-range regulation of contractile forces of the jaw-closing muscles.

Endocannabinoid Signaling Mediates Local Dendritic Coordination between Excitatory and Inhibitory Synapses

Dendritic inhibitory synapses are most efficient in modulating excitatory inputs localized on the same dendrite, but it is unknown whether their location is random or regulated. Here, we show that the formation of inhibitory synapses can be directed by excitatory synaptic activity on the same dendrite. We stimulated dendritic spines close to a GABAergic axon crossing by pairing two-photon glutamate uncaging with postsynaptic depolarization in CA1 pyramidal cells. We found that repeated spine stimulation promoted growth of a GABAergic bouton onto the same dendrite. The dendritic feedback signal required postsynaptic activation of DAGL, which produces the endocannabinoid 2-AG, and was mediated by CB1 receptors. We could also induce inhibitory bouton growth by local, brief applications of 2-AG. Our findings reveal a dendritic signaling mechanism to trigger growth of an inhibitory bouton at dendritic locations with strong excitatory synaptic activity, and this mechanism may serve to ensure inhibitory control over clustered excitatory inputs.

Coincidence Detection within the Excitable Rat Olfactory Bulb Granule Cell Spines.

In the mammalian olfactory bulb, the inhibitory axonless granule cells (GCs) feature reciprocal synapses that interconnect them with the principal neurons of the bulb, mitral, and tufted cells. These synapses are located within large excitable spines that can generate local action potentials (APs) upon synaptic input ("spine spike"). Moreover, GCs can fire global APs that propagate throughout the dendrite. Strikingly, local postsynaptic Ca2+ entry summates mostly linearly with Ca2+ entry due to coincident global APs generated by glomerular stimulation, although some underlying conductances should be inactivated. We investigated this phenomenon by constructing a compartmental GC model to simulate the pairing of local and global signals as a function of their temporal separation Δt. These simulations yield strongly sublinear summation of spine Ca2+ entry for the case of perfect coincidence Δt = 0 ms. Summation efficiency (SE) sharply rises for both positive and negative Δt. The SE reduction for coincident signals depends on the presence of voltage-gated Na+ channels in the spine head, while NMDARs are not essential. We experimentally validated the simulated SE in slices of juvenile rat brain (both sexes) by pairing two-photon uncaging of glutamate at spines and APs evoked by somatic current injection at various intervals Δt while imaging spine Ca2+ signals. Finally, the latencies of synaptically evoked global APs and EPSPs were found to correspond to Δt ≈ 10 ms, explaining the observed approximately linear summation of synaptic local and global signals. Our results provide additional evidence for the existence of the GC spine spike.SIGNIFICANCE STATEMENT Here we investigate the interaction of local synaptic inputs and global activation of a neuron by a backpropagating action potential within a dendritic spine with respect to local Ca2+ signaling. Our system of interest, the reciprocal spine of the olfactory bulb granule cell, is known to feature a special processing mode, namely, a synaptically triggered action potential that is restricted to the spine head. Therefore, coincidence detection of local and global signals follows different rules than in more conventional synapses. We unravel these rules using both simulations and experiments and find that signals coincident within ≈±7 ms around 0 ms result in sublinear summation of Ca2+ entry because of synaptic activation of voltage-gated Na+ channels within the spine.

Two-Photon Uncaging of Glutamate.

Two-photon microscopy produces the excited singlet state of a chromophore with wavelengths approximately double that used for normal excitation. Two photons are absorbed almost simultaneously, via a virtual state, and this makes the excitation technique inherently non-linear. It requires ultra-fast lasers to deliver the high flux density needed to access intrinsically very short lived intermediates, and in combination with lenses of high numerical aperture, this confines axial excitation highly. Since the two-photon excitation volume is similar to a large spine head, the technique has been widely used to study glutamatergic transmission in brain slices. Here I describe the principles of two-photon uncaging of glutamate and provide a practical guide to its application.

Voltage Gated Calcium Channel Activation by Backpropagating Action Potentials Downregulates NMDAR Function.

The majority of excitatory synapses are located on dendritic spines of cortical glutamatergic neurons. In spines, compartmentalized Ca2+ signals transduce electrical activity into specific long-term biochemical and structural changes. Action potentials (APs) propagate back into the dendritic tree and activate voltage gated Ca2+ channels (VGCCs). For spines, this global mode of spine Ca2+ signaling is a direct biochemical feedback of suprathreshold neuronal activity. We previously demonstrated that backpropagating action potentials (bAPs) result in long-term enhancement of spine VGCCs. This activity-dependent VGCC plasticity results in a large interspine variability of VGCC Ca2+ influx. Here, we investigate how spine VGCCs affect glutamatergic synaptic transmission. We combined electrophysiology, two-photon Ca2+ imaging and two-photon glutamate uncaging in acute brain slices from rats. T- and R-type VGCCs were the dominant depolarization-associated Ca2+conductances in dendritic spines of excitatory layer 2 neurons and do not affect synaptic excitatory postsynaptic potentials (EPSPs) measured at the soma. Using two-photon glutamate uncaging, we compared the properties of glutamatergic synapses of single spines that express different levels of VGCCs. While VGCCs contributed to EPSP mediated Ca2+ influx, the amount of EPSP mediated Ca2+ influx is not determined by spine VGCC expression. On a longer timescale, the activation of VGCCs by bAP bursts results in downregulation of spine NMDAR function.

Metaplasticity at CA1 Synapses by Homeostatic Control of Presynaptic Release Dynamics

Hebbian and homeostatic forms of plasticity operate on different timescales to regulate synaptic strength. The degree of mechanistic overlap between these processes and their mutual influence are still incompletely understood. Here, we report that homeostaticsynaptic strengthening induced by prolonged network inactivity compromised the ability of CA1 synapses to exhibit LTP. This effect could not be accounted for by an obvious deficit in the postsynaptic capacity for LTP expression, since neither the fraction of silent synapses nor the ability to induce LTP by two-photon glutamate uncaging were reduced by the homeostatic process. Rather, optical quantal analysis reveals that homeostatically strengthened synapses display a reduced capacity to maintain glutamate release fidelity during repetitive stimulation, ultimately impeding the induction, and thus expression, of LTP. By regulating the short-term dynamics of glutamate release, the homeostatic process thus influences key aspects of dynamic network function and exhibits features of metaplasticity.