Modeling sources of interlaboratory variability in electrophysiological properties of mammalian neurons.

Patch-clamp electrophysiology is widely used to characterize neuronal electrical phenotypes. However, there are no standard experimental conditions for in vitro whole cell patch-clamp electrophysiology, complicating direct comparisons between data sets. In this study, we sought to understand how basic experimental conditions differ among laboratories and how these differences might impact measurements of electrophysiological parameters. We curated the compositions of external bath solutions (artificial cerebrospinal fluid), internal pipette solutions, and other methodological details such as animal strain and age from 509 published neurophysiology articles studying rodent neurons. We found that very few articles used the exact same experimental solutions as any other, and some solution differences stem from recipe inheritance from advisor to advisee as well as changing trends over the years. Next, we used statistical models to understand how the use of different experimental conditions impacts downstream electrophysiological measurements such as resting potential and action potential width. Although these experimental condition features could explain up to 43% of the study-to-study variance in electrophysiological parameters, the majority of the variability was left unexplained. Our results suggest that there are likely additional experimental factors that contribute to cross-laboratory electrophysiological variability, and identifying and addressing these will be important to future efforts to assemble consensus descriptions of neurophysiological phenotypes for mammalian cell types. NEW & NOTEWORTHY This article describes how using different experimental methods during patch-clamp electrophysiology impacts downstream physiological measurements. We characterized how methodologies and experimental solutions differ across articles. We found that differences in methods can explain some, but not all, of the study-to-study variance in electrophysiological measurements. Explicitly accounting for methodological differences using statistical models can help correct downstream electrophysiological measurements for cross-laboratory methodology differences.

Transcriptomic correlates of neuron electrophysiological diversity.

How neuronal diversity emerges from complex patterns of gene expression remains poorly understood. Here we present an approach to understand electrophysiological diversity through gene expression by integrating pooled- and single-cell transcriptomics with intracellular electrophysiology. Using neuroinformatics methods, we compiled a brain-wide dataset of 34 neuron types with paired gene expression and intrinsic electrophysiological features from publically accessible sources, the largest such collection to date. We identified 420 genes whose expression levels significantly correlated with variability in one or more of 11 physiological parameters. We next trained statistical models to infer cellular features from multivariate gene expression patterns. Such models were predictive of gene-electrophysiological relationships in an independent collection of 12 visual cortex cell types from the Allen Institute, suggesting that these correlations might reflect general principles relating expression patterns to phenotypic diversity across very different cell types. Many associations reported here have the potential to provide new insights into how neurons generate functional diversity, and correlations of ion channel genes like Gabrd and Scn1a (Nav1.1) with resting potential and spiking frequency are consistent with known causal mechanisms. Our work highlights the promise and inherent challenges in using cell type-specific transcriptomics to understand the mechanistic origins of neuronal diversity.

Unravelling human hematopoietic progenitor cell diversity through association with intrinsic regulatory factors.

Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34- cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and in vitro clonal differentiation assays. Overall, we demonstrate the utility of using molecular regulators as surrogates for cellular identity and functional potential, providing a framework for description, prospective isolation, and cross-tissue comparison of HSPCs in humans.

Spatial proteomics reveals human microglial states shaped by anatomy and neuropathology.

Microglia are implicated in aging, neurodegeneration, and Alzheimer's disease (AD). Traditional, low-plex, imaging methods fall short of capturing in situ cellular states and interactions in the human brain. We utilized Multiplexed Ion Beam Imaging (MIBI) and data-driven analysis to spatially map proteomic cellular states and niches in healthy human brain, identifying a spectrum of microglial profiles, called the microglial state continuum (MSC). The MSC ranged from senescent-like to active proteomic states that were skewed across large brain regions and compartmentalized locally according to their immediate microenvironment. While more active microglial states were proximal to amyloid plaques, globally, microglia significantly shifted towards a, presumably, dysfunctional low MSC in the AD hippocampus, as confirmed in an independent cohort (n=26). This provides an in situ single cell framework for mapping human microglial states along a continuous, shifting existence that is differentially enriched between healthy brain regions and disease, reinforcing differential microglial functions overall.

An optimized protocol for phenotyping human granulocytes by mass cytometry.

Granulocytes encompass diverse roles, from fighting off pathogens to regulating inflammatory processes in allergies. These roles are represented by distinct cellular phenotypes that we captured with mass cytometry (CyTOF). Our protocol enables simultaneous evaluation of human basophils, eosinophils, and neutrophils under homeostasis and upon immune activation by anti-Immunoglobulin E (anti-IgE) or interleukin-3 (IL-3). Granulocyte integrity and detection of protein markers were optimized so that rare granulocyte populations could be deeply characterized by single cell mass cytometry. For complete details on the use and execution of this protocol, please refer to Vivanco Gonzalez et al. (2020).

Multiplexed Ion Beam Imaging: Insights into Pathobiology.

Next-generation tools for multiplexed imaging have driven a new wave of innovation in understanding how single-cell function and tissue structure are interrelated. In previous work, we developed multiplexed ion beam imaging by time of flight, a highly multiplexed platform that uses secondary ion mass spectrometry to image dozens of antibodies tagged with metal reporters. As instrument throughput has increased, the breadth and depth of imaging data have increased as well. To extract meaningful information from these data, we have developed tools for cell identification, cell classification, and spatial analysis. In this review, we discuss these tools and provide examples of their application in various contexts, including ductal carcinoma in situ, tuberculosis, and Alzheimer's disease. We hope the synergy between multiplexed imaging and automated image analysis will drive a new era in anatomic pathology and personalized medicine wherein quantitative spatial signatures are used routinely for more accurate diagnosis, prognosis, and therapeutic selection.

Single-cell spatial proteomic imaging for human neuropathology.

Neurodegenerative disorders are characterized by phenotypic changes and hallmark proteopathies. Quantifying these in archival human brain tissues remains indispensable for validating animal models and understanding disease mechanisms. We present a framework for nanometer-scale, spatial proteomics with multiplex ion beam imaging (MIBI) for capturing neuropathological features. MIBI facilitated simultaneous, quantitative imaging of 36 proteins on archival human hippocampus from individuals spanning cognitively normal to dementia. Customized analysis strategies identified cell types and proteopathies in the hippocampus across stages of Alzheimer's disease (AD) neuropathologic change. We show microglia-pathologic tau interactions in hippocampal CA1 subfield in AD dementia. Data driven, sample independent creation of spatial proteomic regions identified persistent neurons in pathologic tau neighborhoods expressing mitochondrial protein MFN2, regardless of cognitive status, suggesting a survival advantage. Our study revealed unique insights from multiplexed imaging and data-driven approaches for neuropathologic analysis and serves broadly as a methodology for spatial proteomic analysis of archival human neuropathology. TEASER: Multiplex Ion beam Imaging enables deep spatial phenotyping of human neuropathology-associated cellular and disease features.

Single-synapse analyses of Alzheimer's disease implicate pathologic tau, DJ1, CD47, and ApoE.

Synaptic molecular characterization is limited for Alzheimer's disease (AD). Our newly invented mass cytometry­based method, synaptometry by time of flight (SynTOF), was used to measure 38 antibody probes in approximately 17 million single-synapse events from human brains without pathologic change or with pure AD or Lewy body disease (LBD), nonhuman primates (NHPs), and PS/APP mice. Synaptic molecular integrity in humans and NHP was similar. Although not detected in human synapses, Aß was in PS/APP mice single-synapse events. Clustering and pattern identification of human synapses showed expected disease-specific differences, like increased hippocampal pathologic tau in AD and reduced caudate dopamine transporter in LBD, and revealed previously unidentified findings including increased hippocampal CD47 and lowered DJ1 in AD and higher ApoE in AD with dementia. Our results were independently supported by multiplex ion beam imaging of intact tissue. This highlights the higher depth and breadth of insight on neurodegenerative diseases obtainable through SynTOF.

Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity.

Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45+HLA-DR-CD123+). Through CyTOF and fluorescence-activated cell sorting (FACS), we classified these four PBG subpopulations as (I) CD16lowFcεRIhighCD244high (88.5 ± 1.2%), (II) CD16highFcεRIhighCD244high (9.1 ± 0.4%), (III) CD16lowFcεRIlowCD244low (2.3 ± 1.3), and (IV) CD16highFcεRIlowCD244low (0.4 ± 0.1%). Prospective isolation confirmed basophilic-morphology of PBG I-III, but neutrophilic-morphology of PBG IV. Functional interrogation via IgE-crosslinking or IL-3 stimulation demonstrated that PBG I-II had significant increases in CD203c expression, whereas PBG III-IV remained unchanged compared with media-alone conditions. Thus, PBG III-IV could serve roles in non-IgE-mediated immunity. Our findings offer new perspectives in human basophil heterogeneity and the varying functional potential of these new subsets in health and disease.

Mass synaptometry: High-dimensional multi parametric assay for single synapses.

BACKGROUND: Synaptic alterations, especially presynaptic changes, are cardinal features of neurodegenerative diseases and strongly correlate with cognitive decline. NEW METHOD: We report "Mass Synaptometry" for the high-dimensional analysis of individual human synaptosomes, enriched nerve terminals from brain. This method was adapted from cytometry by time-of-flight mass spectrometry (CyTOF), which is commonly used for single-cell analysis of immune and blood cells. RESULT: Here we overcome challenges for single synapse analysis by optimizing synaptosome preparations, generating a 'SynTOF panel,' recalibrating acquisition settings, and applying computational analyses. Through the analysis of 390,000 individual synaptosomes, we also provide proof-of principle validation by characterizing changes in synaptic diversity in Lewy Body Disease (LBD), Alzheimer's disease and normal brain. COMPARISON WITH EXISTING METHOD(S): Current imaging methods to study synapses in humans are capable of analyzing a limited number of synapses, and conventional flow cytometric techniques are typically restricted to fewer than 6 parameters. Our method allows for the simultaneous detection of 34 parameters from tens of thousands of individual synapses. CONCLUSION: We applied Mass Synaptometry to analyze 34 parameters simultaneously on more than 390,000 synaptosomes from 13 human brain samples. This new approach revealed regional and disease-specific changes in synaptic phenotypes, including validation of this method with the expected changes in the molecular composition of striatal dopaminergic synapses in Lewy body disease and Alzheimer's disease. Mass synaptometry enables highly parallel molecular profiling of individual synaptic terminals.