Changes in Neuroimmunological Synapses During Cerebral Ischemia.

The direct interplay between the immune and nervous systems is now well established. Within the brain, these interactions take place between neurons and resident glial cells, i.e., microglia and astrocytes, or infiltrating immune cells, influenced by systemic factors. A special form of physical cell-cell interactions is the so-called "neuroimmunological (NI) synapse." There is compelling evidence that the same signaling pathways that regulate inflammatory responses to injury or ischemia also play potent roles in brain development, plasticity, and function. Proper synaptic wiring is as important during development as it is during disease states, as it is necessary for activity-dependent refinement of neuronal circuits. Since the process of forming synaptic connections in the brain is highly dynamic, with constant changes in strength and connectivity, the immune component is perfectly suited for the regulatory task as it is in constant turnover. Many cellular and molecular players in this interaction remain to be uncovered, especially in pathological states. In this review, we discuss and propose possible communication hubs between components of the adaptive and innate immune systems and the synaptic element in ischemic stroke pathology.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and a key component of the cytoskeleton. A range of small molecules have emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Amongst these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390 - 490 nm pulsed light and rapidly relaxes to the inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated by live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

ΔFosB accumulation in hippocampal granule cells drives cFos pattern separation during spatial learning.

Mice display signs of fear when neurons that express cFos during fear conditioning are artificially reactivated. This finding gave rise to the notion that cFos marks neurons that encode specific memories. Here we show that cFos expression patterns in the mouse dentate gyrus (DG) change dramatically from day to day in a water maze spatial learning paradigm, regardless of training level. Optogenetic inhibition of neurons that expressed cFos on the first training day affected performance days later, suggesting that these neurons continue to be important for spatial memory recall. The mechanism preventing repeated cFos expression in DG granule cells involves accumulation of ΔFosB, a long-lived splice variant of FosB. CA1 neurons, in contrast, repeatedly expressed cFos. Thus, cFos-expressing granule cells may encode new features being added to the internal representation during the last training session. This form of timestamping is thought to be required for the formation of episodic memories.

Vesicular release probability sets the strength of individual Schaffer collateral synapses.

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. The potency of individual boutons is highly correlated with their vesicular release probability while the number of releasable vesicles affects synaptic output only under high pves conditions.

Presynaptic cGMP sets synaptic strength in the striatum and is important for motor learning.

The striatum is a subcortical brain region responsible for the initiation and termination of voluntary movements. Striatal spiny projection neurons receive major excitatory synaptic input from neocortex and thalamus, and cyclic nucleotides have long been known to play important roles in striatal function. Yet, the precise mechanism of action is unclear. Here, we combine optogenetic stimulation, 2-photon imaging, and genetically encoded scavengers to dissect the regulation of striatal synapses in mice. Our data show that excitatory striatal inputs are tonically depressed by phosphodiesterases (PDEs), in particular PDE1. Blocking PDE activity boosts presynaptic calcium entry and glutamate release, leading to strongly increased synaptic transmission. Although PDE1 degrades both cAMP and cGMP, we uncover that the concentration of cGMP, not cAMP, controls the gain of striatal inputs. Disturbing this gain control mechanism in vivo impairs motor skill learning in mice. The tight dependence of striatal excitatory synapses on PDE1 and cGMP offers a new perspective on the molecular mechanisms regulating striatal activity.

A glibenclamide-sensitive TRPM4-mediated component of CA1 excitatory postsynaptic potentials appears in experimental autoimmune encephalomyelitis.

The transient receptor potential melastatin 4 (TRPM4) channel contributes to disease severity in the murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and to neuronal cell death in models of excitotoxicity and traumatic brain injury. As TRPM4 is activated by intracellular calcium and conducts monovalent cations, we hypothesized that TRPM4 may contribute to and boost excitatory synaptic transmission in CA1 pyramidal neurons of the hippocampus. Using single-spine calcium imaging and electrophysiology, we found no effect of the TRPM4 antagonists 9-phenanthrol and glibenclamide on synaptic transmission in hippocampal slices from healthy mice. In contrast, glibenclamide but not 9-phenanthrol reduced excitatory synaptic potentials in slices from EAE mice, an effect that was absent in slices from EAE mice lacking TRPM4. We conclude that TRPM4 plays little role in basal hippocampal synaptic transmission, but a glibenclamide-sensitive TRPM4-mediated contribution to excitatory postsynaptic responses is upregulated at the acute phase of EAE.

Orthogonally-polarized excitation for improved two-photon and second-harmonic-generation microscopy, applied to neurotransmitter imaging with GPCR-based sensors.

Fluorescent proteins are excited by light that is polarized parallel to the dipole axis of the chromophore. In two-photon microscopy, polarized light is used for excitation. Here we reveal surprisingly strong polarization sensitivity in a class of genetically encoded, GPCR-based neurotransmitter sensors. In tubular structures such as dendrites, this effect led to a complete loss of membrane signal in dendrites running parallel to the polarization direction of the excitation beam. To reduce the sensitivity to dendritic orientation, we designed an optical device that generates interleaved pulse trains of orthogonal polarization. The passive device, which we inserted in the beam path of an existing two-photon microscope, removed the strong direction bias from fluorescence and second-harmonic (SHG) images. We conclude that for optical measurements of transmitter concentration with GPCR-based sensors, orthogonally polarized excitation is essential.

Spike-timing-dependent plasticity rewards synchrony rather than causality.

Spike-timing-dependent plasticity (STDP) is a candidate mechanism for information storage in the brain, but the whole-cell recordings required for the experimental induction of STDP are typically limited to 1 h. This mismatch of time scales is a long-standing weakness in synaptic theories of memory. Here we use spectrally separated optogenetic stimulation to fire precisely timed action potentials (spikes) in CA3 and CA1 pyramidal cells. Twenty minutes after optogenetic induction of STDP (oSTDP), we observed timing-dependent depression (tLTD) and timing-dependent potentiation (tLTP), depending on the sequence of spiking. As oSTDP does not require electrodes, we could also assess the strength of these paired connections three days later. At this late time point, late tLTP was observed for both causal (CA3 before CA1) and anticausal (CA1 before CA3) timing, but not for asynchronous activity patterns (Δt = 50 ms). Blocking activity after induction of oSTDP prevented stable potentiation. Our results confirm that neurons wire together if they fire together, but suggest that synaptic depression after anticausal activation (tLTD) is a transient phenomenon.

Imaging Synaptic Glutamate Release with Two-Photon Microscopy in Organotypic Slice Cultures.

The strength of an excitatory synapse relies on the amount of glutamate it releases and on the amount of postsynaptic receptors responding to the released glutamate. Here we describe a strategy to investigate presynaptic release independently of postsynaptic receptors, using a genetically encoded glutamate indicator (GEGI) such as iGluSnFR to measure synaptic transmission in rodent organotypic slice cultures. We express the iGluSnFR in CA3 pyramidal cells and perform two-photon glutamate imaging on individual Schaffer collateral boutons in CA1. Sparse labeling is achieved via transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser scanning microscopy. A spiral scan path over an individual presynaptic bouton allows to sample at high temporal resolution the local release site in order to capture the peak of iGluSnFR transients.

PACmn for improved optogenetic control of intracellular cAMP.

BACKGROUND: Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. But low activity in the dark might raise resting cAMP in cells expressing bPAC, and most eukaryotic cyclases are membrane-targeted rather than soluble. Our aim was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated. RESULTS: Using a streamlined method based on expression in Xenopus oocytes, we compared natural PACs and confirmed bPAC as the best starting point for protein engineering efforts. We identified several modifications that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceed the responses to forskolin-induced activation of endogenous cyclases and return to baseline within seconds (cAMP) or ~10 min (PKA). CONCLUSIONS: PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types.

Freeze-Frame Imaging of Dendritic Calcium Signals With TubuTag.

The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.

Photoactivatable CaMKII: Rewiring the Brain, One Synapse at a Time.

A recent article by Shibata et al. introduces the engineered photoactivatable enzyme paCaMKII. Activation of this new tool is sufficient to induce long-term potentiation (LTP) of hippocampal synapses in slice culture and in intact animals, thereby expanding the existing toolkit for light-induced modification of brain connectivity at the synaptic level.

The role of microglia membrane potential in chemotaxis.

Microglia react to danger signals by rapid and targeted extension of cellular processes towards the source of the signal. This positive chemotactic response is accompanied by a hyperpolarization of the microglia membrane. Here, we show that optogenetic depolarization of microglia has little effect on baseline motility, but significantly slows down the chemotactic response. Reducing the extracellular Ca2+ concentration mimics the effect of optogenetic depolarization. As the membrane potential sets the driving force for Ca2+ entry, hyperpolarization is an integral part of rapid stimulus-response coupling in microglia. Compared to typical excitable cells such as neurons, the sign of the activating response is inverted in microglia, leading to inhibition by depolarizing channelrhodopsins.

Endoplasmic reticulum visits highly active spines and prevents runaway potentiation of synapses.

In hippocampal pyramidal cells, a small subset of dendritic spines contain endoplasmic reticulum (ER). In large spines, ER frequently forms a spine apparatus, while smaller spines contain just a single tubule of smooth ER. Here we show that the ER visits dendritic spines in a non-random manner, targeting spines during periods of high synaptic activity. When we blocked ER motility using a dominant negative approach against myosin V, spine synapses became stronger compared to controls. We were not able to further potentiate these maxed-out synapses, but long-term depression (LTD) was readily induced by low-frequency stimulation. We conclude that the brief ER visits to active spines have the important function of preventing runaway potentiation of individual spine synapses, keeping most of them at an intermediate strength level from which both long-term potentiation (LTP) and LTD are possible.

Freeze-frame imaging of synaptic activity using SynTagMA.

Information within the brain travels from neuron to neuron across billions of synapses. At any given moment, only a small subset of neurons and synapses are active, but finding the active synapses in brain tissue has been a technical challenge. Here we introduce SynTagMA to tag active synapses in a user-defined time window. Upon 395-405 nm illumination, this genetically encoded marker of activity converts from green to red fluorescence if, and only if, it is bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we show how to identify and track the fluorescence of thousands of individual synapses in an automated fashion. Together, these tools provide an efficient method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior.

Myosin V regulates synaptopodin clustering and localization in the dendrites of hippocampal neurons.

The spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca2+ homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaft-associated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site.

High-speed imaging of glutamate release with genetically encoded sensors.

The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.

The kinetic mechanisms of fast-decay red-fluorescent genetically encoded calcium indicators.

Genetically encoded calcium indicators (GECIs) are useful reporters of cell-signaling, neuronal, and network activities. We have generated novel fast variants and investigated the kinetic mechanisms of two recently developed red-fluorescent GECIs (RGECIs), mApple-based jRGECO1a and mRuby-based jRCaMP1a. In the formation of fluorescent jRGECO1a and jRCaMP1a complexes, calcium binding is followed by rate-limiting isomerization. However, fluorescence decay of calcium-bound jRGECO1a follows a different pathway from its formation: dissociation of calcium occurs first, followed by the peptide, similarly to GCaMP-s. In contrast, fluorescence decay of calcium-bound jRCaMP1a occurs by the reversal of the on-pathway: peptide dissociation is followed by calcium. The mechanistic differences explain the generally slower off-kinetics of jRCaMP1a-type indicators compared with GCaMP-s and jRGECO1a-type GECI: the fluorescence decay rate of f-RCaMP1 was 21 s-1, compared with 109 s-1 for f-RGECO1 and f-RGECO2 (37 °C). Thus, the CaM-peptide interface is an important determinant of the kinetic responses of GECIs; however, the topology of the structural link to the fluorescent protein demonstrably affects the internal dynamics of the CaM-peptide complex. In the dendrites of hippocampal CA3 neurons, f-RGECO1 indicates calcium elevation in response to a 100 action potential train in a linear fashion, making the probe particularly useful for monitoring large-amplitude, fast signals, e.g. those in dendrites, muscle cells, and immune cells.