scholarly journals Pain and itch processing by subpopulations of molecularly diverse spinal and trigeminal projection neurons

2020 ◽  
Author(s):  
R. Wercberger ◽  
J.M. Braz ◽  
J.A. Weinrich ◽  
A.I. Basbaum
Keyword(s):  
Affinity Purification ◽  
Relevant Information ◽  
Projection Neuron ◽  
Molecular Signature ◽  
Projection Neurons ◽  
Rna Seq ◽  
Functional Heterogeneity ◽  
Neurokinin 1 ◽  
The Many

ABSTRACTA remarkable molecular and functional heterogeneity of the primary sensory neurons and dorsal horn interneurons transmits pain- and or itch-relevant information, but the molecular signature of the projection neurons that convey the messages to the brain is unclear. Here, using retro-TRAP (translating ribosome affinity purification) and RNA-seq we reveal extensive molecular diversity of spino- and trigeminoparabrachial projection neurons, which to date are almost exclusively defined by their expression of the neurokinin 1 receptor (NK1R). Among the many genes identified, we highlight distinct subsets of Cck+, Nptx2+, Nmb+, and Crh+ expressing projection neurons. By combining in situ hybridization of retrogradely labeled neurons with Fos-based assays we also demonstrate significant functional heterogeneity, including both convergence and segregation of pain- and itch-provoking inputs onto molecularly diverse subsets of NK1R- and non-NK1R-expressing projection neurons. The current study provides the first comprehensive investigation into the molecular profiles and functional properties of projection neuron subtypes.

scholarly journals Pain and itch processing by subpopulations of molecularly diverse spinal and trigeminal projection neurons

2021 ◽  
Vol 118 (28) ◽  
pp. e2105732118
Author(s):  
Racheli Wercberger ◽  
Joao M. Braz ◽  
Jarret A. Weinrich ◽  
Allan I. Basbaum
Keyword(s):  
Sensory Neurons ◽  
Molecular Diversity ◽  
Affinity Purification ◽  
Relevant Information ◽  
Molecular Signature ◽  
Projection Neurons ◽  
Functional Heterogeneity ◽  
The Many ◽  
The Brain

A remarkable molecular and functional heterogeneity of the primary sensory neurons and dorsal horn interneurons transmits pain- and or itch-relevant information, but the molecular signature of the projection neurons that convey the messages to the brain is unclear. Here, using retro-TRAP (translating ribosome affinity purification) and RNA sequencing, we reveal extensive molecular diversity of spino- and trigeminoparabrachial projection neurons. Among the many genes identified, we highlight distinct subsets of Cck+-, Nptx2+-, Nmb+-, and Crh+-expressing projection neurons. By combining in situ hybridization of retrogradely labeled neurons with Fos-based assays, we also demonstrate significant functional heterogeneity, including both convergence and segregation of pain- and itch-provoking inputs into molecularly diverse subsets of NK1R- and non–NK1R-expressing projection neurons.

scholarly journals Molecular characterization of projection neuron subtypes in the mouse olfactory bulb

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sara Zeppilli ◽  
Tobias Ackels ◽  
Robin Attey ◽  
Nell Klimpert ◽  
Dr. Kimberly Ritola ◽  
...  
Keyword(s):  
Olfactory Bulb ◽  
Projection Neuron ◽  
Mitral Cell ◽  
Cell Populations ◽  
Projection Neurons ◽  
Functional Roles ◽  
Entry Points ◽  
Single Nucleus ◽  
Tufted Cell

Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA in situ hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data, and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

scholarly journals The neurokinin-1 receptor is expressed with gastrin-releasing peptide receptor in spinal interneurons and modulates itch

2020 ◽  
Author(s):  
Tayler D. Sheahan ◽  
Charles A. Warwick ◽  
Louis G. Fanien ◽  
Sarah E. Ross
Keyword(s):  
Dorsal Horn ◽  
Projection Neurons ◽  
Spinal Neurons ◽  
Neurokinin 1 Receptor ◽  
Peptide Receptor ◽  
Gastrin Releasing Peptide ◽  
Dorsal Horn Neurons ◽  
Endogenous Expression ◽  
Neurokinin 1

AbstractThe neurokinin-1 receptor (NK1R, encoded by Tacr1) is expressed in spinal dorsal horn neurons and has been suggested to mediate itch. However, previous studies relied heavily on neurotoxic ablation of NK1R spinal neurons, which limited further dissection of their function in spinal itch circuitry. Thus, we leveraged a newly developed Tacr1CreER mouse line to characterize the role of NK1R spinal neurons in itch. We show that pharmacological activation of spinal NK1R and chemogenetic activation of Tacr1CreER spinal neurons increases itch behavior, whereas pharmacological inhibition of spinal NK1R suppresses itch behavior. We use fluorescence in situ hybridization to characterize the endogenous expression of Tacr1 throughout the superficial and deeper dorsal horn, as well as the lateral spinal nucleus.Retrograde labeling studies from the parabrachial nucleus show that less than 20% of superficial Tacr1CreER dorsal horn neurons are spinal projection neurons, and thus the majority of Tacr1CreER are local interneurons. We then use a combination of in situ hybridization and ex vivo two-photon Ca2+ imaging of the spinal cord to establish that NK1R and the gastrin-releasing peptide receptor (GRPR) are coexpressed within a subpopulation of excitatory superficial dorsal horn neurons. These findings are the first to describe a role for NK1R interneurons in itch and extend our understanding of the complexities of spinal itch circuitry.

scholarly journals Role of the lncRNA BACE1-AS in shared disease mechanisms between heart failure and Alzheimer's disease

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Greco ◽  
A Made' ◽  
A.S Tascini ◽  
J Garcia Manteiga ◽  
S Castelvecchio ◽  
...  
Keyword(s):  
Heart Failure ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
In Situ Hybridization ◽  
Cell Apoptosis ◽  
Common Disease ◽  
Funding Source ◽  
Mrna Levels ◽  
Rna Seq

Abstract Background BACE1 encodes for β-secretase, the key enzyme involved in β-amyloid (βA) generation, a peptide well known for its involvement in Alzheimer's disease (AD). Of note, heart failure (HF) and AD share several risk factors and effectors. We recently showed that, in the heart of ischemic HF patients, the levels of both BACE1, its antisense RNA BACE1-AS and βA are all increased. BACE1-AS positively regulates the expression of BACE1, triggering βA intracellular accumulation, and its overexpression or βA administration induce cardiovascular-cell apoptosis. Aim To characterize the transcripts of the BACE1 locus and to investigate the molecular mechanisms underpinning BACE1-AS regulation of cell vitality. Methods By PCR and sequencing, we studied in the heart the expression of a variety of antisense BACE1 transcripts predicted by FANTOM CAT Epigenome. We studied BACE1 RNA stability by BrdU pulse chase experiments (BRIC assay). The cellular localization of BACE1-AS RNA was investigated by in situ hybridization assay. BACE1-AS binding RNAs were evaluated by BACE1-AS-MS2-Tag pull-down in AC16 cardiomyocytes followed by RNA-seq. Enriched RNAs were validated by qPCR and analysed by bioinformatics comparison with publicly available gene expression datasets of AD brains. Results We readily detected several antisense BACE1 transcripts expressed in AC16 cardiomyocytes; however, only BACE1-AS RNAs overlapping exon 6 of BACE1 positively regulated BACE1 mRNA levels, acting by increasing its stability. BACE1 silencing reverted cell apoptosis induced by BACE1-AS expression, indicating that BACE1 is a functional target of BACE1-AS. However, in situ hybridization experiments indicated a mainly nuclear localization for BACE1-AS, which displayed a punctuated distribution, compatible with chromatin association and indicative of potential additional targets. To identify other BACE1-AS binding RNAs, a BACE1-AS-MS2-tag pull-down was performed and RNA-seq of the enriched RNAs identified 698 BACE1-AS interacting RNAs in cardiomyocytes. Gene ontology of the BACE1-AS binding RNAs identified categories of relevance for cardiovascular or neurological diseases, such as dopaminergic synapse, glutamatergic synapse, calcium signalling pathway and voltage-gated channel activity. In spite of the differences between brain and heart transcriptomes, BACE1-AS-interacting RNAs identified in cardiomyocytes were significantly enriched in transcripts differentially expressed in AD brains as well as in RNAs expressed by enhancer genomic regions that are significantly hypomethylated in AD brains. Conclusions These data shed a new light on the complexity of BACE1-AS locus and on the existence of RNAs interacting with BACE1-AS with a potential as enhancer-RNAs. Moreover, the dysregulation of the BACE1-AS/BACE1/βA pathway may be a common disease mechanism shared by cardiovascular and neurological degenerative diseases. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Italian Health Ministery_Ricerca Corrente 2020

scholarly journals Loss of Wnt16 Leads to Skeletal Deformities and Downregulation of Bone Developmental Pathway in Zebrafish

2021 ◽  
Vol 22 (13) ◽  
pp. 6673
Author(s):  
Xiaochao Qu ◽  
Mei Liao ◽  
Weiwei Liu ◽  
Yisheng Cai ◽  
Qiaorong Yi ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Integration Site ◽  
Skeletal Development ◽  
Rna Seq ◽  
Developmental Pathway ◽  
Mineral Density ◽  
Zebrafish Model ◽  
Protein Protein Interaction ◽  
Ct Analysis

Wingless-type MMTV integration site family, member 16 (wnt16), is a wnt ligand that participates in the regulation of vertebrate skeletal development. Studies have shown that wnt16 can regulate bone metabolism, but its molecular mechanism remains largely undefined. We obtained the wnt16-/- zebrafish model using the CRISPR-Cas9-mediated gene knockout screen with 11 bp deletion in wnt16, which led to the premature termination of amino acid translation and significantly reduced wnt16 expression, thus obtaining the wnt16-/- zebrafish model. The expression of wnt16 in bone-related parts was detected via in situ hybridization. The head, spine, and tail exhibited significant deformities, and the bone mineral density and trabecular bone decreased in wnt16-/- using light microscopy and micro-CT analysis. RNA sequencing was performed to explore the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the down-regulated DEGs are mainly concentrated in mTOR, FoxO, and VEGF pathways. Protein–protein interaction (PPI) network analysis was performed with the detected DEGs. Eight down-regulated DEGs including akt1, bnip4, ptena, vegfaa, twsg1b, prkab1a, prkab1b, and pla2g4f.2 were validated by qRT-PCR and the results were consistent with the RNA-seq data. Overall, our work provides key insights into the influence of wnt16 gene on skeletal development.

scholarly journals Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 964
Author(s):  
Sarka Benesova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
High Sensitivity ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Liquid Biopsies ◽  
Comprehensive Overview ◽  
Rna Molecules ◽  
Novel Mirna ◽  
The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

scholarly journals In situ RNA-seq

2013 ◽  
Vol 14 (9) ◽  
pp. 596-596 ◽  
Author(s):  
Hannah Stower
Keyword(s):  
Rna Seq

Differential Involvement of Projection Neurons During Emergence of Spontaneous Activity in the Developing Avian Hindbrain

2009 ◽  
Vol 101 (2) ◽  
pp. 591-602 ◽  
Author(s):  
Hiraku Mochida ◽  
Gilles Fortin ◽  
Jean Champagnat ◽  
Joel C. Glover
Keyword(s):  
Spontaneous Activity ◽  
Projection Neuron ◽  
Projection Neurons ◽  
Charge Coupled Device ◽  
Neuron Populations ◽  
Motor Nerves ◽  
Differential Involvement ◽  
A Charge ◽  
The Brain ◽  
Calcium Green

To better characterize the emergence of spontaneous neuronal activity in the developing hindbrain, spontaneous activity was recorded optically from defined projection neuron populations in isolated preparations of the brain stem of the chicken embryo. Ipsilaterally projecting reticulospinal (RS) neurons and several groups of vestibuloocular (VO) neurons were labeled retrogradely with Calcium Green-1 dextran amine and spontaneous calcium transients were recorded using a charge-coupled-device camera mounted on a fluorescence microscope. Simultaneous extracellular recordings were made from one of the trigeminal motor nerves (nV) to register the occurrence of spontaneous synchronous bursts of activity. Two types of spontaneous activity were observed: synchronous events (SEs), which occurred in register with spontaneous bursts in nV once every few minutes and were tetrodotoxin (TTX) dependent, and asynchronous events (AEs), which occurred in the intervals between SEs and were TTX resistant. AEs occurred developmentally before SEs and were in general smaller and more variable in amplitude than SEs. SEs appeared at the same stage as nV bursts early on embryonic day 4, first in RS neurons and then in VO neurons. All RS neurons participated equally in SEs from the outset, whereas different subpopulations of VO neurons participated differentially, both in terms of the proportion of neurons that exhibited SEs, the fidelity with which the SEs in individual neurons followed the nV bursts, and the developmental stage at which SEs appeared and matured. The results show that spontaneous activity is expressed heterogeneously among hindbrain projection neuron populations, suggesting its differential involvement in the formation of different functional neuronal circuits.

Action of substance P (neurokinin-1) receptor activation on rat neostriatal projection neurons

Synapse ◽  
1999 ◽  
Vol 33 (1) ◽  
pp. 26-35 ◽  
Author(s):  
Elvira Galarraga ◽  
Salvador Hern�ndez-L�pez ◽  
Dagoberto Tapia ◽  
Arturo Reyes ◽  
Jos� Bargas
Keyword(s):  
Substance P ◽  
Receptor Activation ◽  
Projection Neurons ◽  
Neurokinin 1 Receptor ◽  
Neurokinin 1

scholarly journals OsMYB80 Regulates Anther Development and Pollen Fertility by Targeting Multiple Biological Pathways

2020 ◽  
Vol 61 (5) ◽  
pp. 988-1004 ◽  
Author(s):  
Xiaoying Pan ◽  
Wei Yan ◽  
Zhenyi Chang ◽  
Yingchao Xu ◽  
Ming Luo ◽  
...  
Keyword(s):  
Pollen Development ◽  
Male Fertility ◽  
Gene Networks ◽  
Affinity Purification ◽  
Anther Development ◽  
Rna Seq ◽  
Male Sterile ◽  
New Information ◽  
Gel Shift Assay

Abstract Pollen development is critical to the reproductive success of flowering plants, but how it is regulated is not well understood. Here, we isolated two allelic male-sterile mutants of OsMYB80 and investigated how OsMYB80 regulates male fertility in rice. OsMYB80 was barely expressed in tissues other than anthers, where it initiated the expression during meiosis, reached the peak at the tetrad-releasing stage and then quickly declined afterward. The osmyb80 mutants exhibited premature tapetum cell death, lack of Ubisch bodies, no exine and microspore degeneration. To understand how OsMYB80 regulates anther development, RNA-seq analysis was conducted to identify genes differentially regulated by OsMYB80 in rice anthers. In addition, DNA affinity purification sequencing (DAP-seq) analysis was performed to identify DNA fragments interacting with OsMYB80 in vitro. Overlap of the genes identified by RNA-seq and DAP-seq revealed 188 genes that were differentially regulated by OsMYB80 and also carried an OsMYB80-interacting DNA element in the promoter. Ten of these promoter elements were randomly selected for gel shift assay and yeast one-hybrid assay, and all showed OsMYB80 binding. The 10 promoters also showed OsMYB80-dependent induction when co-expressed in rice protoplast. Functional annotation of the 188 genes suggested that OsMYB80 regulates male fertility by directly targeting multiple biological processes. The identification of these genes significantly enriched the gene networks governing anther development and provided much new information for the understanding of pollen development and male fertility.

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