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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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Arcuate Angiotensin II increases arterial pressure via coordinated increases in sympathetic nerve activity and vasopressin secretion

eNeuro

2021 Dec 17

Shi, Z;Stornetta, DS;Stornetta, RL;Brooks, VL;
PMID: 34937769 | DOI: 10.1523/ENEURO.0404-21.2021

The arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII); however, the cellular mechanisms and downstream neurocircuitry are unclear. Here we show that ArcN AngII increases AP in female rats via two phases, both of which are mediated via activation of AngII type 1 receptors (AT1aR): initial vasopressin-induced vasoconstriction, followed by slowly developing increases in sympathetic nerve activity (SNA) and heart rate (HR). In male rats, ArcN AngII evoked a similarly slow increase in SNA, but the initial pressor response was variable. In females, the effects of ArcN AngII varied during the estrus cycle, with significant increases in SNA, HR, and AP occurring during diestrus and estrus, but only increased AP during proestrus. Pregnancy markedly increased the expression of AT1aR in the ArcN with parallel substantial AngII-induced increases in SNA and MAP. In both sexes, the sympathoexcitation relied on suppression of tonic ArcN sympathoinhibitory Neuropeptide Y inputs, and activation of pro-opiomelanocortin (POMC) projections, to the paraventricular nucleus (PVN). Few or no NPY or POMC neurons expressed the AT1aR, suggesting that AngII increases AP and SNA at least in part indirectly via local interneurons, which express tyrosine hydroxylase (TH) and VGat (i.e. GABAergic). ArcN TH neurons release GABA locally, and central AT1aR and TH neurons mediate stress responses; therefore, we propose that TH AT1aR neurons are well situated to locally coordinate the regulation of multiple modalities within the ArcN in response to stress.SIGNIFICANCEThe arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII). Here we show that ArcN AngII activates AT1aR to increase AP in male and female rats by slowly increasing sympathetic nerve activity. In females, ArcN AngII also evoked an initial pressor response mediated by vasopressin-induced vasoconstriction. Pregnant and estrus females responded more than males, in association with higher ArcN AT1aR expression. AT1aR were identified in ArcN interneurons that express tyrosine hydroxylase (TH) and GABA. Since brain AT1aR and TH mediate stress responses, ArcN AT1aR TH neurons are well situated to locally coordinate autonomic, hormonal, and behavioral responses to stress.
VEGFA and VEGFR2 RNAscope determination in gastric cancer.

J Mol Histol.

2018 May 14

Tamma R, Annese T, Ruggieri S, Marzullo A, Nico B, Ribatti D.
PMID: 29761299 | DOI: 10.1007/s10735-018-9777-0

Gastric cancer is the fifth most common cancer and third leading cause of cancer-related death worldwide. Several studies on angiogenic blocking agents in gastric cancer revealing promising results by the use of monoclonal antibodies against VEGFA or its receptor VEGFR2 or against VEGFA activating pathway. The validation of biomarkers useful to better organize the clinical trials involving anti-angiogenic therapies is crucial. Molecular markers such as RNA are increasingly used for cancer diagnosis, prognosis, and therapy guidance as in the case of the targeted therapies concerning the inhibition of angiogenesis. The aim of this study is to set the conditions for evaluating the expression of VEGFA and VEGFR2 in gastric cancer specimens and in healthy gastric mucosa by the use of RNAscope, a novel RNA in situ hybridization (ISH) method that allows the visualization of a specific gene expression in individual cells. We found the increased expression of VEGFA in the tubular glands and VEGFR2 in the endothelium of gastric cancer samples mainly in the T2, T3 and T4 stages of tumor progression as compared to the healthy controls. These results obtained by the application of this highly sensitive method for oligonucleotide detection the role of angiogenesis in gastric cancer progression already highlighted by conventional immunohistochemical methods, and offer significant promise as a new platform for developing and implementing RNA-based molecular diagnostics also in the conditions in which immunohistochemistry is not applicable.

Patients with sporadic FTLD exhibit similar increases in lysosomal proteins and storage material as patients with FTD due to GRN mutations

Acta neuropathologica communications

2023 Apr 28

Davis, SE;Cook, AK;Hall, JA;Voskobiynyk, Y;Carullo, NV;Boyle, NR;Hakim, AR;Anderson, KM;Hobdy, KP;Pugh, DA;Murchison, CF;McMeekin, LJ;Simmons, M;Margolies, KA;Cowell, RM;Nana, AL;Spina, S;Grinberg, LT;Miller, BL;Seeley, WW;Arrant, AE;
PMID: 37118844 | DOI: 10.1186/s40478-023-01571-4

Loss of function progranulin (GRN) mutations are a major autosomal dominant cause of frontotemporal dementia (FTD). Patients with FTD due to GRN mutations (FTD-GRN) develop frontotemporal lobar degeneration with TDP-43 pathology type A (FTLD-TDP type A) and exhibit elevated levels of lysosomal proteins and storage material in frontal cortex, perhaps indicating lysosomal dysfunction as a mechanism of disease. To investigate whether patients with sporadic FTLD exhibit similar signs of lysosomal dysfunction, we compared lysosomal protein levels, transcript levels, and storage material in patients with FTD-GRN or sporadic FTLD-TDP type A. We analyzed samples from frontal cortex, a degenerated brain region, and occipital cortex, a relatively spared brain region. In frontal cortex, patients with sporadic FTLD-TDP type A exhibited similar increases in lysosomal protein levels, transcript levels, and storage material as patients with FTD-GRN. In occipital cortex of both patient groups, most lysosomal measures did not differ from controls. Frontal cortex from a transgenic mouse model of TDP-opathy had similar increases in cathepsin D and lysosomal storage material, showing that TDP-opathy and neurodegeneration can drive these changes independently of progranulin. To investigate these changes in additional FTLD subtypes, we analyzed frontal cortical samples from patients with sporadic FTLD-TDP type C or Pick's disease, an FTLD-tau subtype. All sporadic FTLD groups had similar increases in cathepsin D activity, lysosomal membrane proteins, and storage material as FTD-GRN patients. However, patients with FTLD-TDP type C or Pick's disease did not have similar increases in lysosomal transcripts as patients with FTD-GRN or sporadic FTLD-TDP type A. Based on these data, accumulation of lysosomal proteins and storage material may be a common aspect of end-stage FTLD. However, the unique changes in gene expression in patients with FTD-GRN or sporadic FTLD-TDP type A may indicate distinct underlying lysosomal changes among FTLD subtypes.
Somatostatin Interneurons of the Insula Mediate QR2-Dependent Novel Taste Memory Enhancement

eNeuro

2021 Sep 29

Gould, NL;Kolatt Chandran, S;Kayyal, H;Edry, E;Rosenblum, K;
PMID: 34518366 | DOI: 10.1523/ENEURO.0152-21.2021

Forming long-term memories is crucial for adaptive behavior and survival in changing environments. The molecular consolidation processes which underlie the formation of these long-term memories are dependent on protein synthesis in excitatory and SST-expressing neurons. A centrally important, parallel process to this involves the removal of the memory constraint quinone reductase 2 (QR2), which has been recently shown to enhance memory consolidation for novel experiences in the cortex and hippocampus, via redox modulation. However, it is unknown within which cell type in the cortex removal of QR2 occurs, nor how this affects neuronal function. Here, we use novel taste learning in the mouse anterior insular cortex (aIC) to show that similarly to mRNA translation, QR2 removal occurs in excitatory and SST-expressing neurons. Interestingly, both novel taste and QR2 inhibition reduce excitability specifically within SST, but not excitatory neurons. Furthermore, reducing QR2 expression in SST, but not in PV or excitatory neurons, is sufficient to enhance taste memory. Thus, QR2 mediated intrinsic property changes of SST interneurons in the aIC is a central removable factor to allow novel taste memory formation. This previously unknown involvement of QR2 and SST interneurons in resetting aIC activity hours following learning, describes a molecular mechanism to define cell circuits for novel information. Therefore, the QR2 pathway in SST interneurons provides a fresh new avenue by which to tackle age-related cognitive deficits, while shedding new light onto the functional machinations of long-term memory formation for novel information.
VEGFA amplification/increased gene copy number and VEGFA mRNA expression in renal cell carcinoma with TFEB gene alterations

Mod Pathol.

2018 Sep 11

Caliò A, Brunelli M, Segala D, Pedron S, Doglioni C, Argani P, Martignoni G.
PMID: 30206412 | DOI: 10.1038/s41379-018-0128-1

Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3-4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.

Downregulation of kainate receptors regulating GABAergic transmission in amygdala after early life stress is associated with anxiety-like behavior in rodents

Translational psychiatry

2021 Oct 18

Englund, J;Haikonen, J;Shteinikov, V;Amarilla, SP;Atanasova, T;Shintyapina, A;Ryazantseva, M;Partanen, J;Voikar, V;Lauri, SE;
PMID: 34663781 | DOI: 10.1038/s41398-021-01654-7

Early life stress (ELS) is a well-characterized risk factor for mood and anxiety disorders. GABAergic microcircuits in the amygdala are critically implicated in anxiety; however, whether their function is altered after ELS is not known. Here we identify a novel mechanism by which kainate receptors (KARs) modulate feedforward inhibition in the lateral amygdala (LA) and show that this mechanism is downregulated after ELS induced by maternal separation (MS). Specifically, we show that in control rats but not after MS, endogenous activity of GluK1 subunit containing KARs disinhibit LA principal neurons during activation of cortical afferents. GluK1 antagonism attenuated excitability of parvalbumin (PV)-expressing interneurons, resulting in loss of PV-dependent inhibitory control and an increase in firing of somatostatin-expressing interneurons. Inactivation of Grik1 expression locally in the adult amygdala reduced ongoing GABAergic transmission and was sufficient to produce a mild anxiety-like behavioral phenotype. Interestingly, MS and GluK1-dependent phenotypes showed similar gender specificity, being detectable in male but not female rodents. Our data identify a novel KAR-dependent mechanism for cell-type and projection-specific functional modulation of the LA GABAergic microcircuit and suggest that the loss of GluK1 KAR function contributes to anxiogenesis after ELS.
GABA Neuronal Deletion of Shank3 Exons 14-16 in Mice Suppresses Striatal Excitatory Synaptic Input and Induces Social and Locomotor Abnormalities.

Front Cell Neurosci. 2018 Oct 9;12:341.

2018 Oct 09

Yoo T, Cho H, Lee J, Park H, Yoo YE, Yang E, Kim JY, Kim H, Kim E.
PMID: 30356810 | DOI: 10.3389/fncel.2018.00341

Shank3 is an excitatory postsynaptic scaffolding protein implicated in multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). Although previous neurobiological studies on Shank3 and Shank3-mutant mice have revealed diverse roles of Shank3 in the regulation of synaptic, neuronal and brain functions, whether Shank3 expression in specific cell types distinctly contributes to mouse phenotypes remains largely unclear. In the present study, we generated two Shank3-mutant mouse lines (exons 14-16) carrying global and GABA neuron-specific deletions and characterized their electrophysiological and behavioral phenotypes. These mouse lines show similar decreases in excitatory synaptic input onto dorsolateral striatal neurons. In addition, the abnormal social and locomotor behaviors observed in global Shank3-mutant mice are strongly mimicked by GABA neuron-specific Shank3-mutant mice, whereas the repetitive and anxiety-like behaviors are only partially mimicked. These results suggest that GABAergic Shank3 (exons 14-16) deletion has strong influences on striatal excitatory synaptic transmission and social and locomotor behaviors in mice.
Sox6 expression distinguishes dorsally and ventrally biased dopamine neurons in the substantia nigra with distinctive properties and embryonic origins

Cell reports

2021 Nov 09

Pereira Luppi, M;Azcorra, M;Caronia-Brown, G;Poulin, JF;Gaertner, Z;Gatica, S;Moreno-Ramos, OA;Nouri, N;Dubois, M;Ma, YC;Ramakrishnan, C;Fenno, L;Kim, YS;Deisseroth, K;Cicchetti, F;Dombeck, DA;Awatramani, R;
PMID: 34758317 | DOI: 10.1016/j.celrep.2021.109975

Dopamine (DA) neurons in the ventral tier of the substantia nigra pars compacta (SNc) degenerate prominently in Parkinson's disease, while those in the dorsal tier are relatively spared. Defining the molecular, functional, and developmental characteristics of each SNc tier is crucial to understand their distinct susceptibility. We demonstrate that Sox6 expression distinguishes ventrally and dorsally biased DA neuron populations in the SNc. The Sox6+ population in the ventral SNc includes an Aldh1a1+ subset and is enriched in gene pathways that underpin vulnerability. Sox6+ neurons project to the dorsal striatum and show activity correlated with acceleration. Sox6- neurons project to the medial, ventral, and caudal striatum and respond to rewards. Moreover, we show that this adult division is encoded early in development. Overall, our work demonstrates a dual origin of the SNc that results in DA neuron cohorts with distinct molecular profiles, projections, and functions.
Accelerated tumor metastasis due to IFNγ receptor-mediated dissociation of perivascular cells from blood vessels.

J Pathol.

2017 Apr 18

Ni C, Ma P, Qu L, Wu F, Hao J, Wang R, Lu Y, Yang W, Erben U, Qin Z.
PMID: 28418194 | DOI: 10.1002/path.4907

Angiostasis mediated by IFNγ is a key mechanism of anti-tumor immunity; however, the effect of IFNγ on host VEGFA-expressing cells during tumor progression is still elusive. Here, we developed transgenic mice with IFNγ receptor (IFNγR) expression under control of the Vegfa promoter (V-γR). In these mice, the IFNγ responsiveness of VEGFA -expressing cells led to a dramatic growth suppression of transplanted lung carcinoma cells. Surprisingly, increased mortality and tumor metastasis were observed in the tumor-bearing V-γR mice, in comparison to the control wild type and IFNγR-deficient mice. Further study showed that perivascular cells were VEGFA-expressing cells and potential IFNγ targets. In vivo, tumor vascular perfusion and pericyte association with blood vessels were massively disrupted in V-γR mice. In vitro, IFNγ inhibited TGF-β-signaling through upregulating SMAD7 and therefore, down-regulated N-cadherin expression in pericytes. Importantly, IFNγ neutralization in vivo using a monoclonal antibody reduced tumor metastasis. Together, the results suggest that IFNγR-mediated dissociation of perivascular cells from blood vessels contributes to the acceleration of tumor metastasis. Thus the inhibition of tumor growth via IFNγ-induced angiostasis might also accelerate tumor metastasis.

Cerebellar dopamine D2 receptors regulate social behaviors

Nature neuroscience

2022 Jun 16

Cutando, L;Puighermanal, E;Castell, L;Tarot, P;Belle, M;Bertaso, F;Arango-Lievano, M;Ango, F;Rubinstein, M;Quintana, A;Chédotal, A;Mameli, M;Valjent, E;
PMID: 35710984 | DOI: 10.1038/s41593-022-01092-8

The cerebellum, a primary brain structure involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. Although the regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown. By combining cell-type-specific transcriptomics, histological analyses, three-dimensional imaging and patch-clamp recordings, we demonstrate that cerebellar dopamine D2 receptors (D2Rs) in mice are preferentially expressed in Purkinje cells (PCs) and regulate synaptic efficacy onto PCs. Moreover, we found that changes in D2R levels in PCs of male mice during adulthood alter sociability and preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel roles for D2R in PC function and causally link cerebellar D2R levels of expression to social behaviors.
Single-cell and single-nucleus RNA-seq uncovers shared and distinct axes of variation in dorsal LGN neurons in mice, non-human primates, and humans

eLife

2021 Sep 02

Bakken, TE;van Velthoven, CT;Menon, V;Hodge, RD;Yao, Z;Nguyen, TN;Graybuck, LT;Horwitz, GD;Bertagnolli, D;Goldy, J;Yanny, AM;Garren, E;Parry, S;Casper, T;Shehata, SI;Barkan, ER;Szafer, A;Levi, BP;Dee, N;Smith, KA;Sunkin, SM;Bernard, A;Phillips, J;Hawrylycz, MJ;Koch, C;Murphy, GJ;Lein, E;Zeng, H;Tasic, B;
PMID: 34473054 | DOI: 10.7554/eLife.64875

Abundant evidence supports the presence of at least three distinct types of thalamocortical (TC) neurons in the primate dorsal lateral geniculate nucleus (dLGN) of the thalamus, the brain region that conveys visual information from the retina to the primary visual cortex (V1). Different types of TC neurons in mice, humans, and macaques have distinct morphologies, distinct connectivity patterns, and convey different aspects of visual information to the cortex. To investigate the molecular underpinnings of these cell types, and how these relate to differences in dLGN between human, macaque, and mice, we profiled gene expression in single nuclei and cells using RNA-sequencing. These efforts identified four distinct types of TC neurons in the primate dLGN: magnocellular (M) neurons, parvocellular (P) neurons, and two types of koniocellular (K) neurons. Despite extensively documented morphological and physiological differences between M and P neurons, we identified few genes with significant differential expression between transcriptomic cell types corresponding to these two neuronal populations. Likewise, the dominant feature of TC neurons of the adult mouse dLGN is high transcriptomic similarity, with an axis of heterogeneity that aligns with core vs. shell portions of mouse dLGN. Together, these data show that transcriptomic differences between principal cell types in the mature mammalian dLGN are subtle relative to the observed differences in morphology and cortical projection targets. Finally, alignment of transcriptome profiles across species highlights expanded diversity of GABAergic neurons in primate versus mouse dLGN and homologous types of TC neurons in primates that are distinct from TC neurons in mouse.
GABAergic Neurons in the Rat Medial Septal Complex Express Relaxin-3 Receptor (RXFP3) mRNA

Front. Neuroanat.

2018 Jan 17

Albert-Gascó H, Ma S, Ros-Bernal F, Sánchez-Pérez AM, Gundlach AL, Olucha-Bordonau FE.
PMID: - | DOI: 10.3389/fnana.2017.00133

The medial septum (MS) complex modulates hippocampal function and related behaviors. Septohippocampal projections promote and control different forms of hippocampal synchronization. Specifically, GABAergic and cholinergic projections targeting the hippocampal formation from the MS provide bursting discharges to promote theta rhythm, or tonic activity to promote gamma oscillations. In turn, the MS is targeted by ascending projections from the hypothalamus and brainstem. One of these projections arises from the nucleus incertus in the pontine tegmentum, which contains GABA neurons that co-express the neuropeptide relaxin-3 (Rln3). Both stimulation of the nucleus incertus and septal infusion of Rln3 receptor agonist peptides promotes hippocampal theta rhythm. The Gi/o-protein-coupled receptor, relaxin-family peptide receptor 3 (RXFP3), is the cognate receptor for Rln3 and identification of the transmitter phenotype of neurons expressing RXFP3 in the septohippocampal system can provide further insights into the role of Rln3 transmission in the promotion of septohippocampal theta rhythm. Therefore, we used RNAscope multiplex in situ hybridization to characterize the septal neurons expressing Rxfp3mRNA in the rat. Our results demonstrate that Rxfp3 mRNA is abundantly expressed in vesicular GABA transporter (vGAT) mRNA- and parvalbumin (PV) mRNA-positive GABA neurons in MS, whereas ChATmRNA-positive acetylcholine neurons lack Rxfp3 mRNA. Approximately 75% of Rxfp3 mRNA-positive neurons expressed vGAT mRNA (and 22% were PV mRNA-positive), while the remaining 25% expressed Rxfp3 mRNA only, consistent with a potential glutamatergic phenotype. Similar proportions were observed in the posterior septum. The occurrence of RXFP3 in PV-positive GABAergic neurons gives support to a role for the Rln3-RXFP3 system in septohippocampal theta rhythm.

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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
Pool/Pan
Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)
A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp
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

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