Summary Understanding how neural information is processed in physiological and pathological states would benefit from precise detection, localization and quantification of the activity of all neurons across the entire brain, which has not to date been achieved in the mammalian brain. We introduce a pipeline for high speed acquisition of brain activity at cellular resolution through profiling immediate early gene expression using immunostaining and light-sheet fluorescence imaging, followed by automated mapping and analysis of activity by an open-source software program we term ClearMap. We validate the pipeline first by analysis of brain regions activated in response to Haloperidol. Next, we report new cortical regions downstream of whisker-evoked sensory processing during active exploration. Lastly, we combine activity mapping with axon tracing to uncover new brain regions differentially activated during parenting behavior. This pipeline is widely applicable to different experimental paradigms, including animal species for which transgenic activity reporters are not readily available.

Here we describe an automated method, which we call serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in 3D, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders.

The partial purification of mouse mammary gland stem cells (MaSCs) using combinatorial cell surface markers (Lin − CD24 + CD29 h CD49f h ) has improved our understanding of their role in normal development and breast tumorigenesis. Despite the significant improvement in MaSC enrichment, there is presently no methodology that adequately isolates pure MaSCs. Seeking new markers of MaSCs, we characterized the stem-like properties and expression signature of label-retaining cells from the mammary gland of mice expressing a controllable H2b-GFP transgene. In this system, the transgene expression can be repressed in a doxycycline-dependent fashion, allowing isolation of slowly dividing cells with retained nuclear GFP signal. Here, we show that H2b-GFP h cells reside within the predicted MaSC compartment and display greater mammary reconstitution unit frequency compared with H2b-GFP neg MaSCs. According to their transcriptome profile, H2b-GFP h MaSCs are enriched for pathways thought to play important roles in adult stem cells. We found Cd1d, a glycoprotein expressed on the surface of antigen-presenting cells, to be highly expressed by H2b-GFP h MaSCs, and isolation of Cd1d + MaSCs further improved the mammary reconstitution unit enrichment frequency to nearly a single-cell level. Additionally, we functionally characterized a set of MaSC-enriched genes, discovering factors controlling MaSC survival. Collectively, our data provide tools for isolating a more precisely defined population of MaSCs and point to potentially critical factors for MaSC maintenance.

Kadiri LR, Kwan AC, Webb WW, Harris-Warrick RM. Dopamineinduced oscillations of the pyloric pacemaker neuron rely on release of calcium from intracellular stores. J Neurophysiol 106: 1288-1298, 2011. First published June 15, 2011 doi:10.1152/jn.00456.2011.-Endogenously bursting neurons play central roles in many aspects of nervous system function, ranging from motor control to perception. The properties and bursting patterns generated by these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents. In the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, the anterior burster (AB) neuron is a conditional oscillator in the presence of dopamine (DA) and other neuromodulators and serves as the pacemaker to drive rhythmic output from the pyloric network. We analyzed the mechanisms by which DA evokes bursting in the AB neuron. Previous work showed that DA-evoked bursting is critically dependent on external calcium (Harris-Warrick RM, Flamm RE. J Neurosci 7: [2113][2114][2115][2116][2117][2118][2119][2120][2121][2122][2123][2124][2125][2126][2127][2128] 1987). Using two-photon microscopy and calcium imaging, we show that DA evokes the release of calcium from intracellular stores well before the emergence of voltage oscillations. When this release from intracellular stores is blocked by antagonists of ryanodine or inositol trisphosphate (IP 3 ) receptor channels, DA fails to evoke AB bursting. We further demonstrate that DA enhances the calcium-activated inward current, I CAN , despite the fact that it significantly reduces voltage-activated calcium currents. This suggests that DA-induced release of calcium from intracellular stores activates I CAN , which provides a depolarizing ramp current that underlies endogenous bursting in the AB neuron.calcium-activated inward current; ryanodine; voltage clamp; central pattern generator RHYTHMIC ACTIVITY plays central roles in many aspects of nervous system function, ranging from motor control to perception. The neural mechanisms underlying rhythmogenesis are not well understood but include both network-driven oscillators and neuronal oscillators (Harris-Warrick 2010). Neuronal oscillators are single neurons that generate an endogenous oscillatory voltage pattern based on the ionic currents they express. The bursting properties of these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents (Harris-Warrick and Johnson 2010;Lotshaw et al. 1986;Marder and Bucher 2001;Marder et al. 2005;Partridge et al. 1990).The pyloric network in the crustacean stomatogastric ganglion (STG) is an important system for studying the mechanisms of rhythmogenesis (Harris-Warrick 1993; Marder and Bucher 2001; Selverston and Ayers 2006). The pyloric circuit consists of 1 interneuron (the anterior burster, or AB) and 13 motoneurons. The AB neuron is a conditional oscillator: under the appropriate modul...

Background Quickly detecting and isolating individuals positive for SARS-CoV-2 is essential for limiting virus spread. Policy makers rely on the number of active cases to make decisions, and individuals use this information to evaluate risk should they return to public spaces. Robust testing strategies have been plagued with limited authorized diagnostic assays and high test prices, with large-scale implementation hampered by worldwide supply chain issues. Methods Having identified its potential early in the pandemic, we simplified saliva-based COVID-19 diagnostic testing by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens for SARS-CoV-2 in dualplex RT-qPCR. Moreover, we validated this approach (“SalivaDirect”) with reagents and instruments from multiple vendors to circumvent supply chain disruptions. Results SalivaDirect’s simplified protocol does not compromise on sensitivity. In our hospital cohort, we found a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial RT-qPCR kit. With the National Basketball Association we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (< 0.05%) results. Using comparative assays and sample types, we also demonstrated SalivaDirect to efficiently detect SARS-CoV-2 in asymptomatic individuals. SalivaDirect is a simplified method for SARS-CoV-2 detection (A) Schematic overview of SalivaDirect workflow depicting the main steps of mixing saliva with proteinase K, heat inactivation, and dualplex qRT-PCR testing. Figure created with Biorender.com. (B) SARS-CoV-2 is stable in saliva for at least 7 days at 4C, room temperature (RT; 19C), and 30C without addition of stabilizing buffers. Spiked-in saliva samples of low virus concentrations (12, 25, and 50 SARS-CoV-2 copies/mL) were kept at the indicated temperature for 7 days and then tested with SalivaDirect. N1 cycle threshold (Ct) values were lower when kept for 7 days at 30C as compared to fresh specimens (Kruskal-Wallis; p = 0.03). Horizontal bars indicate the median. (C) Comparing Ct values for saliva treated with proteinase K and heat as compared to nucleic extraction yields higher N1 Ct values without extraction (Wilcoxon; p < 0.01). (D) Testing extracted nucleic acid from saliva with the N1 primer-probe set (singleplex) as compared to a multiplex assay showed stronger N1 detection in multiplex (Wilcoxon; p < 0.01). The dotted line in (B)–(D) indicates the limit of detection. Conclusion Saliva is a valid alternative to swabs for SARS-CoV-2 screening. Importantly, SalivaDirect enables labs to utilize existing infrastructure, improving test implementation time and requiring limited investment to scale-up to meet mass testing needs. With the safe and reliable self-collection of saliva, our vision is to help provide accessible and equitable testing solutions, especially in low-resource and remote settings. Disclosures Anne Wyllie, PhD, Global Diagnostic Systems (Consultant)Pfizer (Advisor or Review Panel member, Research Grant or Support)PPS Health (Consultant)Tempus Labs, Inc (Research Grant or Support) Nathan D. Grubaugh, PhD, Tempus Labs (Consultant)