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Probes for INS

ACD can configure probes for the various manual and automated assays for INS for RNAscope Assay, or for Basescope Assay compatible for your species of interest.

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RNA Sequencing of Single Human Islet Cells Reveals Type 2 Diabetes Genes

Cell Metab.

2016 Sep 09

Xin Y, Kim J, Okamoto H, Ni M, Wei Y, Adler C, Murphy AJ, Yancopoulos GD, Lin C, Gromada J.
PMID: 27667665 | DOI: 10.1016/j.cmet.2016.08.018

Pancreatic islet cells are critical for maintaining normal blood glucose levels, and their malfunction underlies diabetes development and progression. We used single-cell RNA sequencing to determine the transcriptomes of 1,492 human pancreatic α, β, δ, and PP cells from non-diabetic and type 2 diabetes organ donors. We identified cell-type-specific genes and pathways as well as 245 genes with disturbed expression in type 2 diabetes. Importantly, 92% of the genes have not previously been associated with islet cell function or growth. Comparison of gene profiles in mouse and human α and β cells revealed species-specific expression. All data are available for online browsing and download and will hopefully serve as a resource for the islet research community.

Deletion of KCNQ2/3 potassium channels from PV+ interneurons leads to homeostatic potentiation of excitatory transmission.

Elife.

2018 Nov 01

Soh H, Park S, Ryan K, Springer K, Maheshwari A, Tzingounis AV.
PMID: 30382937 | DOI: 10.7554/eLife.38617

KCNQ2/3 channels, ubiquitously expressed neuronal potassium channels, have emerged as indispensable regulators of brain network activity. Despite their critical role in brain homeostasis, the mechanisms by which KCNQ2/3 dysfunction lead to hypersychrony are not fully known. Here, we show that deletion of KCNQ2/3 channels changed PV+ interneurons', but not SST+ interneurons', firing properties. We also find that deletion of either KCNQ2/3 or KCNQ2 channels from PV+ interneurons led to elevated homeostatic potentiation of fast excitatory transmission in pyramidal neurons. Pvalb-Kcnq2 null-mice showed increased seizure susceptibility, suggesting that decreases in interneuron KCNQ2/3 activity remodels excitatory networks, providing a new function for these channels.

Oxytocin-receptor-expressing neurons in the parabrachial nucleus regulate fluid intake

Nature Neuroscience

2017 Nov 13

Ryan PJ, Ross SI, Campos CA, Derkach VA, Palmiter RD.
PMID: - | DOI: 10.1038/s41593-017-0014-z

Brain regions that regulate fluid satiation are not well characterized, yet are essential for understanding fluid homeostasis. We found that oxytocin-receptor-expressing neurons in the parabrachial nucleus of mice (OxtrPBN neurons) are key regulators of fluid satiation. Chemogenetic activation of OxtrPBN neurons robustly suppressed noncaloric fluid intake, but did not decrease food intake after fasting or salt intake following salt depletion; inactivation increased saline intake after dehydration and hypertonic saline injection. Under physiological conditions, OxtrPBN neurons were activated by fluid satiation and hypertonic saline injection. OxtrPBN neurons were directly innervated by oxytocin neurons in the paraventricular hypothalamus (OxtPVH neurons), which mildly attenuated fluid intake. Activation of neurons in the nucleus of the solitary tract substantially suppressed fluid intake and activated OxtrPBN neurons. Our results suggest that OxtrPBN neurons act as a key node in the fluid satiation neurocircuitry, which acts to decrease water and/or saline intake to prevent or attenuate hypervolemia and hypernatremia.

Kappa Opioid Receptor Distribution and Function in Primary Afferents.

Neuron.

2018 Sep 19

Snyder LM, Chiang MC, Loeza-Alcocer E, Omori Y, Hachisuka J, Sheahan TD, Gale JR, Adelman PC, Sypek EI, Fulton SA, Friedman RL, Wright MC, Duque MG, Lee YS, Hu Z, Huang H, Cai X, Meerschaert KA, Nagarajan V, Hirai T, Scherrer G, Kaplan DH, Porreca F, Davi
PMID: 30236284 | DOI: 10.1016/j.neuron.2018.08.044

Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and ex vivo recordings, we show that KOR is expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca2+ influx, which we observed in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation and nociceptor sensitization by inflammatory mediators. Finally, peripherally restricted KOR agonists selectively reduce pain and itch behaviors, as well as mechanical hypersensitivity associated with a surgical incision. These experiments provide a rationale for the use of peripherally restricted KOR agonists for therapeutic treatment.

Learning-Related Plasticity in Dendrite-Targeting Layer 1 Interneurons

Neuron

2018 Sep 27

Abs E, Poorthuis RB, Apelblat D, Muhammad K, Pardi MB, Enke L, Kushinsky D, Pu DL, Eizinger MF, Conzelmann KK, Spiegel I, Letzkus JJ.
PMID: - | DOI: 10.1016/j.neuron.2018.09.001

A wealth of data has elucidated the mechanisms by which sensory inputs are encoded in the neocortex, but how these processes are regulated by the behavioral relevance of sensory information is less understood. Here, we focus on neocortical layer 1 (L1), a key location for processing of such top-down information. Using Neuron-Derived Neurotrophic Factor(NDNF) as a selective marker of L1 interneurons (INs) and in vivo 2-photon calcium imaging, electrophysiology, viral tracing, optogenetics, and associative memory, we find that L1 NDNF-INs mediate a prolonged form of inhibition in distal pyramidal neuron dendrites that correlates with the strength of the memory trace. Conversely, inhibition from Martinotti cells remains unchanged after conditioning but in turn tightly controls sensory responses in NDNF-INs. These results define a genetically addressable form of dendritic inhibition that is highly experience dependent and indicate that in addition to disinhibition, salient stimuli are encoded at elevated levels of distal dendritic inhibition.

Brs3 neurons in the mouse dorsomedial hypothalamus regulate body temperature, energy expenditure, and heart rate, but not food intake.

Nat Neurosci. 2018 Nov;21(11):1530-1540.

2018 Oct 22

Piñol RA, Zahler SH, Li C, Saha A, Tan BK, Škop V, Gavrilova O, Xiao C, Krashes MJ, Reitman ML.
PMID: 30349101 | DOI: 10.1038/s41593-018-0249-3

Bombesin-like receptor 3 (BRS3) is an orphan G-protein-coupled receptor that regulates energy homeostasis and heart rate. We report that acute activation of Brs3-expressing neurons in the dorsomedial hypothalamus (DMHBrs3) increased body temperature (Tb), brown adipose tissue temperature, energy expenditure, heart rate, and blood pressure, with no effect on food intake or physical activity. Conversely, activation of Brs3 neurons in the paraventricular nucleus of the hypothalamus had no effect on Tb or energy expenditure, but suppressed food intake. Inhibition of DMHBrs3 neurons decreased Tb and energy expenditure, suggesting a necessary role in Tb regulation. We found that the preoptic area provides major input (excitatory and inhibitory) to DMHBrs3 neurons. Optogenetic stimulation of DMHBrs3 projections to the raphe pallidus increased Tb. Thus, DMHBrs3→raphe pallidus neurons regulate Tb, energy expenditure, and heart rate, and Brs3 neurons in the paraventricular nucleus of the hypothalamus regulate food intake. Brs3 expression is a useful marker for delineating energy metabolism regulatory circuitry.
Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration.

Cell Stem Cell.

2019 May 09

Pepe-Mooney BJ, Dill MT, Alemany A, Ordovas-Montanes J, Matsushita Y, Rao A, Sen A, Miyazaki M, Anakk S, Dawson PA, Ono N, Shalek AK, van Oudenaarden A, Camargo FD.
PMID: 31080134 | DOI: 10.1016/j.stem.2019.04.004

The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasisand after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.

SARS-CoV-2 Disease Severity in the Golden Syrian Hamster Model of Infection is Related to the Volume of Intranasal Inoculum.

preprints

2023 Feb 10

Handley, A;Ryan, K;Davies, E;Bewley, K;Carnell, O;Challis, A;Coombes, N;Fotheringham, S;Gooch, K;Charlton, M;Harris, D;Kennard, C;Ngabo, D;Weldon, T;Salguero, F;Funnell, S;Hall, Y;
| DOI: 10.20944/preprints202302.0171.v1

The Golden Syrian hamster (Mesocricetus auratus) is now commonly used in preclinical research for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the assessment of vaccines, drugs and therapeutics. Here we show that hamsters inoculated via the intranasal route with the same infectious virus dose of prototypical SARS-CoV-2 administered in a different volume present with different clinical signs, weight loss and viral shedding, with a reduced volume resulting in reduced severity of disease similar to that obtained by a 500-fold reduction in challenge dose. The tissue burden of virus and the severity of pulmonary pathology were also significantly affected by different challenge inoculum volumes. These findings suggest that direct comparison between the severity of SARS-CoV-2 variants or studies assessing the efficacy of treatments determined by hamster studies cannot be made unless both the challenge dose and inoculation volume are matched when using the intranasal route. Additionally, analysis of sub-genomic and total genomic RNA PCR data demonstrated no link between sub-genomic and live viral titres and that sub-genomic analyses do not provide any information beyond that provided by more sensitive total genomic PCR.
PIEZO1 transduces mechanical itch in mice

Nature

2022 Jun 22

Hill, RZ;Loud, MC;Dubin, AE;Peet, B;Patapoutian, A;
PMID: 35732741 | DOI: 10.1038/s41586-022-04860-5

Itch triggers scratching, a behavioural defence mechanism that aids in the removal of harmful irritants and parasites1. Chemical itch is triggered by many endogenous and exogenous cues, such as pro-inflammatory histamine, which is released during an allergic reaction1. Mechanical itch can be triggered by light sensations such as wool fibres or a crawling insect2. In contrast to chemical itch pathways, which have been extensively studied, the mechanisms that underlie the transduction of mechanical itch are largely unknown. Here we show that the mechanically activated ion channel PIEZO1 (ref. 3) is selectively expressed by itch-specific sensory neurons and is required for their mechanically activated currents. Loss of PIEZO1 function in peripheral neurons greatly reduces mechanically evoked scratching behaviours and both acute and chronic itch-evoked sensitization. Finally, mice expressing a gain-of-function Piezo1 allele4 exhibit enhanced mechanical itch behaviours. Our studies reveal the polymodal nature of itch sensory neurons and identify a role for PIEZO1 in the sensation of itch.
Dissociable Structural and Functional Hippocampal Outputs via Distinct Subiculum Cell Classes

Cell.

2018 Apr 10

Cembrowski MS, Phillips MG, DiLisio SF, Shields BC, Winnubst J, Chandrashekar J, Bas E, Spruston N.
PMID: 29681453 | DOI: 10.1016/j.cell.2018.03.031

The mammalian hippocampus, comprised of serially connected subfields, participates in diverse behavioral and cognitive functions. It has been postulated that parallel circuitry embedded within hippocampal subfields may underlie such functional diversity. We sought to identify, delineate, and manipulate this putatively parallel architecture in the dorsal subiculum, the primary output subfield of the dorsal hippocampus. Population and single-cell RNA-seq revealed that the subiculum can be divided into two spatially adjacent subregions associated with prominent differences in pyramidal cell gene expression. Pyramidal cells occupying these two regions differed in their long-range inputs, local wiring, projection targets, and electrophysiological properties. Leveraging gene-expression differences across these regions, we use genetically restricted neuronal silencing to show that these regions differentially contribute to spatial working memory. This work provides a coherent molecular-, cellular-, circuit-, and behavioral-level demonstration that the hippocampus embeds structurally and functionally dissociable streams within its serial architecture.

A neural network for intermale aggression to establish social hierarchy

Nature Neuroscience

2018 May 25

Stagkourakis S, Spigolon G, Williams P, Protzmann J, Fisone G, Broberger C.
PMID: - | DOI: 10.1038/s41593-018-0153-x

Intermale aggression is used to establish social rank. Several neuronal populations have been implicated in aggression, but the circuit mechanisms that shape this innate behavior and coordinate its different components (including attack execution and reward) remain elusive. We show that dopamine transporter-expressing neurons in the hypothalamic ventral premammillary nucleus (PMvDAT neurons) organize goal-oriented aggression in male mice. Activation of PMvDATneurons triggers attack behavior; silencing these neurons interrupts attacks. Regenerative PMvDAT membrane conductances interacting with recurrent and reciprocal excitation explain how a brief trigger can elicit a long-lasting response (hysteresis). PMvDAT projections to the ventrolateral part of the ventromedial hypothalamic and the supramammillary nuclei control attack execution and aggression reward, respectively. Brief manipulation of PMvDAT activity switched the dominance relationship between males, an effect persisting for weeks. These results identify a network structure anchored in PMvDAT neurons that organizes aggressive behavior and, as a consequence, determines intermale hierarchy.

A single-cell transcriptomic inventory of murine smooth muscle cells

Developmental cell

2022 Oct 24

Muhl, L;Mocci, G;Pietilä, R;Liu, J;He, L;Genové, G;Leptidis, S;Gustafsson, S;Buyandelger, B;Raschperger, E;Hansson, EM;Björkegren, JLM;Vanlandewijck, M;Lendahl, U;Betsholtz, C;
PMID: 36283392 | DOI: 10.1016/j.devcel.2022.09.015

Smooth muscle cells (SMCs) execute important physiological functions in numerous vital organ systems, including the vascular, gastrointestinal, respiratory, and urogenital tracts. SMC differ morphologically and functionally at these different anatomical locations, but the molecular underpinnings of the differences remain poorly understood. Here, using deep single-cell RNA sequencing combined with in situ gene and protein expression analysis in four murine organs-heart, aorta, lung, and colon-we identify a molecular basis for high-level differences among vascular, visceral, and airway SMC, as well as more subtle differences between, for example, SMC in elastic and muscular arteries and zonation of elastic artery SMC along the direction of blood flow. Arterial SMC exhibit extensive organotypic heterogeneity, whereas venous SMC are similar across organs. We further identify a specific SMC subtype within the pulmonary vasculature. This comparative SMC cross-organ resource offers insight into SMC subtypes and their specific functions.

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Description
sense
Example: Hs-LAG3-sense
Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron#
Example: Mm-Htt-intron2
Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
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Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)
A mixture of multiple probe sets targeting multiple genes or transcripts
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Example: Hs-PDGFB-No-XMm
Does not cross detect with the species (Sp)
XSp
Example: Rn-Pde9a-XMm
designed to cross detect with the species (Sp)
O#
Example: Mm-Islr-O1
Alternative design targeting different regions of the same transcript or isoforms
CDS
Example: Hs-SLC31A-CDS
Probe targets the protein-coding sequence only
EnEmProbe targets exons n and m
En-EmProbe targets region from exon n to exon m
Retired Nomenclature
tvn
Example: Hs-LEPR-tv1
Designed to target transcript variant n
ORF
Example: Hs-ACVRL1-ORF
Probe targets open reading frame
UTR
Example: Hs-HTT-UTR-C3
Probe targets the untranslated region (non-protein-coding region) only
5UTR
Example: Hs-GNRHR-5UTR
Probe targets the 5' untranslated region only
3UTR
Example: Rn-Npy1r-3UTR
Probe targets the 3' untranslated region only
Pan
Example: Pool
A mixture of multiple probe sets targeting multiple genes or transcripts

Enabling research, drug development (CDx) and diagnostics

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