Spatiotemporal limits of optogenetic manipulations in cortical circuits, bioRxiv, 2019-05-20
AbstractNeuronal inactivation is commonly used to assess the involvement of groups of neurons in specific brain functions. Optogenetic tools allow manipulations of genetically and spatially defined neuronal populations with excellent temporal resolution. However, the targeted neurons are coupled with other neural populations over multiple length scales. As a result, the effects of localized optogenetic manipulations are not limited to the targeted neurons, but produces spatially extended excitation and inhibition with rich dynamics. Here we benchmarked several optogenetic silencers in transgenic mice and with viral gene transduction, with the goal to inactivate excitatory neurons in small regions of neocortex. We analyzed the effects of the perturbations in vivo using electrophysiology. Channelrhodopsin activation of GABAergic neurons produced more effective photoinhibition of pyramidal neurons than direct photoinhibition using light-gated ion pumps. We made transgenic mice expressing the light-dependent chloride channel GtACR under the control of Cre-recombinase. Activation of GtACR produced the most potent photoinhibition. For all methods, localized photostimuli produced photoinhibition that extended substantially beyond the spread of light in tissue, although different methods had slightly different resolution limits (radius of inactivation, 0.5 mm to 1 mm). The spatial profile of photoinhibition was likely shaped by strong coupling between cortical neurons. Over some range of photostimulation, circuits produced the “paradoxical effect”, where excitation of inhibitory neurons reduced activity in these neurons, together with pyramidal neurons, a signature of inhibition-stabilized neural networks. The offset of optogenetic inactivation was followed by rebound excitation in a light dose-dependent manner, which can be mitigated by slowly varying photostimuli, but at the expense of time resolution. Our data offer guidance for the design of in vivo optogenetics experiments and suggest how these experiments can reveal operating principles of neural circuits.
biorxiv neuroscience 100-200-users 2019A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins, bioRxiv, 2019-05-18
AbstractSpecialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a novel, rationally designed epitope tag that serves an exceptionally broad spectrum of applications in life sciences while outperforming established tags like the HA, FLAG or myc tags. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We developed a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in-vivo detection of proteins. By solving the crystal structure of the complex we were able to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.
biorxiv biochemistry 200-500-users 2019High-throughput microcircuit analysis of individual human brains through next-generation multineuron patch-clamp, bioRxiv, 2019-05-18
AbstractComparing neuronal microcircuits across different brain regions, species and individuals can reveal common and divergent principles of network computation. Simultaneous patch-clamp recordings from multiple neurons offer the highest temporal and subthreshold resolution to analyse local synaptic connectivity. However, its establishment is technically complex and the experimental performance is limited by high failure rates, long experimental times and small sample sizes. We introduce an in-vitro multipatch setup with an automated pipette pressure and cleaning system facilitating recordings of up to 10 neurons simultaneously and sequential patching of additional neurons. We present hardware and software solutions that increase the usability, speed and data throughput of multipatch experiments which allowed probing of 150 synaptic connections between 17 neurons in one human cortical slice and screening of over 600 connections in tissue from a single patient. This method will facilitate the systematic analysis of microcircuits and allow unprecedented comparisons at the level of individuals.
biorxiv neuroscience 0-100-users 2019Live-cell STED microscopy of mitochondrial cristae, bioRxiv, 2019-05-18
Mitochondria are highly dynamic organelles that exhibit a complex inner architecture. They exhibit a smooth outer membrane and a highly convoluted inner membrane that forms invaginations called cristae. Imaging cristae in living cells poses a formidable challenge for light microscopy. Relying on a cell line stably expressing the mitochondrial protein Cox8A fused to the SNAP-tag and using STED (stimulated emission depletion) super-resolution microscopy, we demonstrate the visualization of cristae dynamics in cultivated human cells.
biorxiv cell-biology 100-200-users 2019Non-replication of functional connectivity differences in autism spectrum disorder across multiple sites and denoising strategies, bioRxiv, 2019-05-18
AbstractA rapidly growing number of studies on autism spectrum disorder (ASD) have used resting-state fMRI to identify alterations of functional connectivity, with the hope of identifying clinical biomarkers or underlying neural mechanisms. However, results have been largely inconsistent across studies, and there is therefore a pressing need to determine the primary factors influencing replicability. Here, we used resting-state fMRI data from the Autism Brain Imaging Data Exchange to investigate two potential factors denoising strategy and data site (which differ in terms of sample, data acquisition, etc.). We examined the similarity of both group-average functional connectomes and group-level differences (ASD vs. control) across 33 denoising pipelines and four independently-acquired datasets. The group-average connectomes were highly consistent across pipelines (r = 0.92±0.06) and sites (r = 0.88±0.02). However, the group differences, while still consistent within site across pipelines (r = 0.76±0.12), were highly inconsistent across sites regardless of choice of denoising strategies (r = 0.07±0.04), suggesting lack of replication may be strongly influenced by site andor cohort differences. Across-site similarity remained low even when considering the data at a large-scale network level or when considering only the most significant edges. We further show through an extensive literature survey that the parameters chosen in the current study (i.e., sample size, age range, preprocessing methods) are quite representative of the published literature. These results highlight the importance of examining replicability in future studies of ASD, and, more generally, call for extra caution when interpreting alterations in functional connectivity across groups of individuals.
biorxiv neuroscience 100-200-users 2019Assortative mating in hybrid zones is remarkably ineffective in promoting speciation, bioRxiv, 2019-05-17
AbstractAssortative mating and other forms of partial prezygotic isolation are often viewed as being more important than partial postzygotic isolation (low fitness of hybrids) early in the process of speciation. Here we simulate secondary contact between two populations (‘species’) to examine effects of pre- and postzygotic isolation in preventing blending. A small reduction in hybrid fitness (e.g., 10%) produces a narrower hybrid zone than a strong but imperfect mating preference (e.g., 10x stronger preference for conspecific over heterospecific mates). This is because, in the latter case, rare F1 hybrids find each other attractive (due to assortative mating), leading to the gradual buildup of a full continuum of intermediates between the two species. The cline is narrower than would result from purely neutral diffusion over the same number of generations, but this effect is due to the frequency-dependent mating disadvantage of individuals of rare mating types. Hybrids tend to pay this cost of rarity more than pure individuals, meaning there is an induced postzygotic isolation effect of assortative mating. When this induced mating disadvantage is removed, partial assortative mating does not prevent eventual blending of the species. These results prompt a questioning of the concept of partial prezygotic isolation, since it is not very isolating unless there is also postzygotic isolation.
biorxiv evolutionary-biology 0-100-users 2019