A transcription-dependent increase in miniature EPSC frequency accompanies late-phase plasticity in cultured hippocampal neurons

<p>Abstract</p> <p>Background</p> <p>The magnitude and longevity of synaptic activity-induced changes in synaptic efficacy is quantified by measuring evoked responses whose potentiation requires gene transcription to persist for more than 2-3 hours. While miniature EPSCs (mEPSCs) are also increased in amplitude and/or frequency during long-term potentiation (LTP), it is not known how long such changes persist or whether gene transcription is required.</p> <p>Results</p> <p>We use whole-cell patch clamp recordings from dissociated hippocampal cultures to characterise for the first time the persistence and transcription dependency of mEPSC upregulation during synaptic potentiation. The persistence of recurrent action potential bursting in these cultures is transcription-, translation- and NMDA receptor-dependent thus providing an accessible model for long-lasting plasticity. Blockade of GABA_A-receptors with bicuculline for 15 minutes induced action potential bursting in all neurons and was maintained in 50-60% of neurons for more than 6 hours. Throughout this period, the frequency but neither the amplitude of mEPSCs nor whole-cell AMPA currents was markedly increased. The transcription blocker actinomycin D abrogated, within 2 hours of burst induction, both action potential bursting and the increase in mEPSCs. Reversible blockade of action potentials during, but not after this 2 hour transcription period suppressed the increase in mEPSC frequency and the recovery of burst activity at a time point 6 hours after induction.</p> <p>Conclusion</p> <p>These results indicate that increased mEPSC frequency persists well beyond the 2 hour transcription-independent phase of plasticity in this model. This long-lasting mEPSC upregulation is transcription-dependent and requires ongoing action potential activity during the initial 2 hour period but not thereafter. Thus mEPSC upregulation may underlie the long term, transcription-dependent persistence of action potential bursting. This provides mechanistic insight to link gene candidates already identified by gene chip analysis to long lasting plasticity in this in vitro model.</p

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 (iGluf) and ultrafast (iGluu) variants with comparable brightness but increased Kd for glutamate (137 μM and 600 μM, respectively). Compared with iGluSnFR, iGluu 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 iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu 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

Automated analysis of spine dynamics on live CA1 pyramidal cells

Dendritic spines may be tiny in volume, but are of major importance for neuroscience. They are the main receivers for excitatory synaptic connections, and their constant changes in number and in shape reflect the dynamic connectivity of the brain. Two-photon microscopy allows following the fate of individual spines in brain slice preparations and in live animals. The diffraction-limited and non-isotropic resolution of this technique, however, makes detection of such tiny structures rather challenging, especially along the optical axis (z-direction). Here we present a novel spine detection algorithm based on a statistical dendrite intensity model and a corresponding spine probability model. To quantify the fidelity of spine detection, we generated correlative datasets: Following two-photon imaging of live pyramidal cell dendrites, we used Serial Block-Face Scanning Electron Microscopy (SBEM) to reconstruct dendritic ultrastructure in 3D. Statistical models were trained on synthetic fluorescence images generated from SBEM datasets via Point Spread Function (PSF) convolution. After the training period, we tested automatic spine detection on real two-photon datasets and compared the result to ground truth (correlative SBEM data). The performance of our algorithm allowed tracking changes in spine volume automatically over several hours. Using a second fluorescent protein targeted to the endoplasmic reticulum, we could analyze the motion of this organelle inside individual spines. Furthermore, we show that it is possible to distinguish activated spines from non-stimulated neighbors by detection of fluorescently labeled presynaptic vesicle clusters. These examples illustrate how automatic segmentation in 5D (x, y, z, t, λ) allows us to investigate brain dynamics at the level of individual synaptic connections

Calcium imaging and electrophysiology of hippocampal activity under anesthesia and natural sleep in mice

Large-scale, annotated collection of 2-photon calcium imaging data and electrophysiological recordings in CA1 of the murine hippocampus under three distinct anesthetics (Isoflurane, Keta/Xyl and MMF), during natural sleep, and during wakefulness. Direct link to the published dataset: https://doi.gin.g-node.org/10.12751/g-node.lkx6kk

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 that allow investigating 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 of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse