Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network

Mechanisms that entrain and pace rhythmic epileptiform discharges remain debated. Traditionally, the quest to understand them has focused on interneuronal networks driven by synaptic GABAergic connections. However, synchronized interneuronal discharges could also trigger the transient elevations of extracellular GABA across the tissue volume, thus raising tonic conductance (Gtonic) of synaptic and extrasynaptic GABA receptors in multiple cells. Here, we monitor extracellular GABA in hippocampal slices using patch-clamp GABA "sniffer" and a novel optical GABA sensor, showing that periodic epileptiform discharges are preceded by transient, region-wide waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to a cycle of GABA-driven network inhibition and disinhibition underpinning this relationship. We test and validate this hypothesis using simultaneous patch-clamp recordings from multiple neurons and selective optogenetic stimulation of fast-spiking interneurons. Critically, reducing GABA uptake in order to decelerate extracellular GABA fluctuations-without affecting synaptic GABAergic transmission or resting GABA levels-slows down rhythmic activity. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA in pacing regenerative rhythmic activity in brain networks

Within the fold: assessing differential expression measures and reproducibility in microarray assays

BACKGROUND: 'Fold-change' cutoffs have been widely used in microarray assays to identify genes that are differentially expressed between query and reference samples. More accurate measures of differential expression and effective data-normalization strategies are required to identify high-confidence sets of genes with biologically meaningful changes in transcription. Further, the analysis of a large number of expression profiles is facilitated by a common reference sample, the construction of which must be carefully addressed. RESULTS: We carried out a series of 'self-self' hybridizations in which aliquots of the same RNA sample were labeled separately with Cy3 and Cy5 fluorescent dyes and co-hybridized to the same microarray. From this, we can analyze the intensity-dependent behavior of microarray data, define a statistically significant measure of differential expression that exploits the structure of the fluorescent signals, and measure the inherent reproducibility of the technique. We also devised a simple procedure for identifying and eliminating low-quality data for replicates within and between slides. We examine the properties required of a universal reference RNA sample and show how pooling a small number of samples with a diverse representation of expressed genes can outperform more complex mixtures as a reference sample. CONCLUSION: Analysis of cell-line samples can identify systematic structure in measured gene-expression levels. A general procedure for analyzing cDNA microarray data is proposed and validated. We show that pooled reference samples should be based not only on the expression of individual genes in each cell line but also on the expression levels of genes within cell lines

Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network

Funding Information: This work was supported by Wellcome Principal Fellowship (212251/Z/18/Z and 212285/Z/18/Z), Wellcome Collaborative Award (UNS120639 - 223131/Z/21/Z), Epilepsy Research UK (F1901), MRC (MR/W019752/1, MR/V013556/1, and MR/V034758/1), NC3Rs (NC/X001067/1), and ERC Advanced Grant (323113). Optimization and parallelization of ARACHNE algorithms for extended neural network simulations was provided by AMC Bridge (Waltham, MA) and web security by Cyber Curio (Berkhamsted, UK). L.P.S. and D.A.R. narrated the study. I.P. and V.M. designed and carried out electrophysiological and optogenetic studies. N.C. O.K. and T.P.J. designed, carried out, and analyzed iGABASnFR2 imaging experiments. S.S. carried out sniffer-patch experiments. O.T. carried out control-probing K+ imaging tests. L.P.S. designed and carried out network modeling studies and data analyses. J.S.M. L.L.L. J.P.H. and I.K. supplied optical GABA sensors and related protocols. The development of iGABASnFR2 was conducted under the aegis of the HHMI Janelia GENIE Project at Janelia Research Campus. D.M.K. and M.C.W. designed optogenetic experiments. D.A.R. designed selected experiments and simulations, carried out selected analyses, and wrote the manuscript draft, with contributions from V.M. L.P.S. and all other authors. The authors declare no competing interests. We support inclusive, diverse, and equitable conduct of research. Publisher Copyright: © 2023 The Author(s)Peer reviewedPublisher PD

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.ISSN:1548-7105ISSN:1548-709

A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators

<div><p>Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.</p> </div

Fast and sensitive GCaMP calcium indicators for imaging neural populations

Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation

Segmentation of neuronal cell bodies for image analysis.

<p>(<b>A</b>) Bright field and epifluorescence images showing GCaMP3 fluorescence channel, nls-mCherry fluorescence channel, and red and green merged fluorescence channels. Scale bar: 150 µm. (<b>B</b>) nls-mCherry fluorescence channel after low-pass frequency filtering with a circular kernel to identify putative nuclei. (<b>C</b>) Partially segmented image where local intensity maxima were identified using adaptively defined thresholds followed by cutting of image into a Voronoi diagram based on seeds identified by maxima. (<b>D</b>) Images from inset in (<b>C</b>) before and after adaptive thresholding in the GCaMP and mCherry channels within each sub-region to define pixels that belong to cytosol and nuclei. (<b>E</b>) Images before and after final segmentation, where regions of interest were excluded if the average mCherry level did not reach a predefined threshold or if the regions of interest touched the image boundary. (<b>F</b>) GCaMP3 10 AP ∆F/F₀ response before and after segmentation (36 wells).</p

Neuronal culture platform and purified protein measurements for GCaMP variants.

<p>Purified protein measurements do not predict GCaMP variant performance in neurons. Each variant is represented as a circle. The calcium affinity and fluorescence response from purified protein measurements are plotted on the x-axis and y-axis, respectively. Variant performance in neurons (fluorescence change or decay kinetics) is shown by the area of each circle. (<b>A</b>) Neuronal ∆F/F₀ (10 AP; 4 to 249 wells) of variants (circle area) compared with protein ∆F_sat/F_apo and apparent calcium affinity (K_d). (<b>B</b>) Neuronal decay time (τ_1/2, 10 AP) of variants (circle area) compared with protein ∆F_sat/F_apo and apparent calcium affinity (K_d). GCaMP3 (gray circle), GCaMP5G (cyan circle).</p

GCaMP3 fluorescence responses in neuronal culture.

<p>(<b>A</b>) 3 AP fluorescence response for GCaMP3. Scale bar: 150 µm. (<b>B</b>) 10 AP. (<b>C</b>) 160 AP. (<b>D</b>) 3 AP ∆F/F₀ traces for 78 regions of interest (gray). Median trace (red). Stimulus duration (black line). (<b>E</b>) 10 AP. (<b>F</b>) 160 AP. </p

Primary neuron stimulus and imaging screening platform.

<p>(<b>A</b>) Flow chart for GECI optimization on screening platform. (<b>B</b>) Prolentiviral vector with human <i>synapsin-1</i> promoter (syn), GCaMP variant, internal ribosome entry site (IRES), nuclear localization signal fused with mCherry (nls-mCherry), and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). (<b>C</b>) Schematic of screening platform. (<b>D</b>) Schematic of electrodes evoking APs from cultured neurons. Photo of 24-well cap stimulator with pairs of parallel platinum wires.</p