Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs.

Adaptation to different levels of illumination is central to the function of the retina. Here, we demonstrate that levels of the miR-183/96/182 cluster, miR-204, and miR-211 are regulated by different light levels in the mouse retina. Concentrations of these microRNAs were downregulated during dark adaptation and upregulated in light-adapted retinas, with rapid decay and increased transcription being responsible for the respective changes. We identified the voltage-dependent glutamate transporter Slc1a1 as one of the miR-183/96/182 targets in photoreceptor cells. We found that microRNAs in retinal neurons decay much faster than microRNAs in nonneuronal cells. The high turnover is also characteristic of microRNAs in hippocampal and cortical neurons, and neurons differentiated from ES cells in vitro. Blocking activity reduced turnover of microRNAs in neuronal cells while stimulation with glutamate accelerated it. Our results demonstrate that microRNA metabolism in neurons is higher than in most other cells types and linked to neuronal activity.

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.

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.

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.

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.

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.

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.

Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses.

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ∼10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGlu f ) and ultrafast (iGlu u ) variants with comparable brightness but increased Kd for glutamate (137 µM and 600 µM, respectively). Compared with iGluSnFR, iGlu u has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGlu u is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGlu u responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.

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.

Long-term depression triggers the selective elimination of weakly integrated synapses.

Long-term depression (LTD) weakens synaptic transmission in an activity-dependent manner. It is not clear, however, whether individual synapses are able to maintain a depressed state indefinitely, as intracellular recordings rarely exceed 1 h. Here, we combine optogenetic stimulation of identified Schaffer collateral axons with two-photon imaging of postsynaptic calcium signals and follow the fate of individual synapses for 7 d after LTD induction. Optogenetic stimulation of CA3 pyramidal cells at 1 Hz led to strong and reliable depression of postsynaptic calcium transients in CA1. NMDA receptor activation was necessary for successful induction of LTD. We found that, in the days following LTD, many depressed synapses and their "neighbors" were eliminated from the hippocampal circuit. The average lifetime of synapses on nonstimulated dendritic branches of the same neurons remained unaffected. Persistence of individual depressed synapses was highly correlated with reliability of synaptic transmission, but not with spine size or the amplitude of spine calcium transients. Our data suggest that LTD initially leads to homogeneous depression of synaptic function, followed by selective removal of unreliable synapses and recovery of function in the persistent fraction.

High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels.

Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise induction of action potentials with short light pulses. A limiting factor for many optophysiological experiments is the relatively small photocurrent induced by ChR2. We screened a large number of ChR2 point mutants and discovered a dramatic increase in photocurrent amplitude after threonine-to-cysteine substitution at position 159. When we tested the T159C mutant in hippocampal pyramidal neurons, action potentials could be induced at very low light intensities, where currently available channelrhodopsins were unable to drive spiking. Biophysical characterization revealed that the kinetics of most ChR2 variants slows down considerably at depolarized membrane potentials. We show that the recently published E123T substitution abolishes this voltage sensitivity and speeds up channel kinetics. When we combined T159C with E123T, the resulting double mutant delivered fast photocurrents with large amplitudes and increased the precision of single action potential induction over a broad range of frequencies, suggesting it may become the standard for light-controlled activation of neurons.

AMPA receptors gate spine Ca(2+) transients and spike-timing-dependent potentiation.

Spike timing-dependent long-term potentiation (t-LTP) is the embodiment of Donald Hebb's postulated rule for associative memory formation. Pre- and postsynaptic action potentials need to be precisely correlated in time to induce this form of synaptic plasticity. NMDA receptors have been proposed to detect correlated activity and to trigger synaptic plasticity. However, the slow kinetic of NMDA receptor currents is at odds with the millisecond precision of coincidence detection. Here we show that AMPA receptors are responsible for the extremely narrow time window for t-LTP induction. Furthermore, we visualized synergistic interactions between AMPA and NMDA receptors and back-propagating action potentials on the level of individual spines. Supralinear calcium signals were observed for spike timings that induced t-LTP and were most pronounced in spines well isolated from the dendrite. We conclude that AMPA receptors gate the induction of associative synaptic plasticity by regulating the temporal precision of coincidence detection.

NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1.

GABAB receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABAB receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca2+ influx through NMDA receptors when activated by spillover of GABA from neighboring GABAergic terminals. Here, we show that an excitatory signaling cascade enables spines to counteract this GABAB-mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamin-dependent endocytosis of GABAB receptors. NMDA-dependent internalization of GABAB receptors requires activation of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), which associates with GABAB receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABAB1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABAB receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABAB receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABAB1b subunit isoform, which is the isoform that clusters with inhibitory effector K+ channels in the spines. Consistent with this, NMDA receptor activation in neurons impairs the ability of GABAB receptors to activate K+ channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABAB receptors through CaMKII-mediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABAB receptors.

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.

Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa.

The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.

Differential distribution of endoplasmic reticulum controls metabotropic signaling and plasticity at hippocampal synapses.

Synaptic plasticity is considered essential for learning and storage of new memories. Whether all synapses on a given neuron have the same ability to express long-term plasticity is not well understood. Synaptic microanatomy could affect the function of local signaling cascades and thus differentially regulate the potential for plasticity at individual synapses. Here, we investigate how the presence of endoplasmic reticulum (ER) in dendritic spines of CA1 pyramidal neurons affects postsynaptic signaling. We show that the ER is targeted selectively to large spines containing strong synapses. In ER-containing spines, we frequently observed synaptically triggered calcium release events of very large amplitudes. Low-frequency stimulation of these spines induced a permanent depression of synaptic potency that was independent of NMDA receptor activation and specific to the stimulated synapses. In contrast, no functional changes were induced in the majority of spines lacking ER. Both calcium release events and long-term depression depended on the activation of metabotropic glutamate receptors and inositol trisphosphate receptors. In summary, spine microanatomy is a reliable indicator for the presence of specific signaling cascades that govern plasticity on a micrometer scale.

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.

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.