scholarly journals TRPM3 Is Expressed in Afferent Bladder Neurons and Is Upregulated during Bladder Inflammation

2021 ◽  
Vol 23 (1) ◽  
pp. 107
Author(s):  
Matthias Vanneste ◽  
Marie Mulier ◽  
Ana Cristina Nogueira Freitas ◽  
Nele Van Ranst ◽  
Axelle Kerstens ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Single Molecule ◽  
Drg Neurons ◽  
Functional Responses ◽  
Internal Organs ◽  
Rna In Situ Hybridization ◽  
Deficient Mice ◽  
Heat Hyperalgesia

The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established.

scholarly journals A122 THE INFLUENCE OF SEX AND THE GUT MICROBIOTA ON VISCERAL PAIN SENSITIVITY IN MICE

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 142-143
Author(s):  
J Pujo ◽  
G De Palma ◽  
J Lu ◽  
S M Collins ◽  
P Bercik
Keyword(s):  
Abdominal Pain ◽  
Gut Microbiota ◽  
Sensory Neurons ◽  
Neuronal Response ◽  
Calcium Mobilization ◽  
Visceral Sensitivity ◽  
Drg Neurons ◽  
E Coli ◽  
Germ Free

Abstract Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Its treatment is of limited efficacy as the pathophysiology is largely unknown. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to have altered pain signaling, which normalizes after colonization. Sex also appears to play a key role in visceral sensitivity, as abdominal pain is diagnosed predominantly in female patients. Thus, both gut bacteria and sex are important in the regulation of gut nociception, but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota and sex in abdominal pain. Methods We used primary cultures of sensory neurons from dorsal root ganglia (DRG) of female and male conventional mice (SPF) or germ-free (GF) mice, age 7–18 weeks. To study the visceral afferent activity in vitro, calcium mobilization in DRG sensory neurons was measured by inverted fluorescence microscope using a fluorescent calcium probe Fluo-4 (1mM). Two parameters were considered: i) percentage of responding neurons and ii) intensity of neuronal response. First, DRG sensory neurons were stimulated by a TRPV1 agonist capsaicin (12.5nM, 125nM and 1.25µM) or by an agonist mix of G-protein coupled receptors (GPCR: bradykinin, histamine and serotonin; 1µM, 10µM and 100µM). We next cultured Escherichia coli JM83 (E. coli) and Enterobacter aerogenes (E. aer) overnight in LB and LDMIIG medium, respectively. Bacterial supernatant of 1010 CFU/mL was diluted to 1% in Krebs Ringer solution to treat DRG neurons from GF/SPF mice and calcium mobilization was measured. Results The percentage of neurons responding to capsaicin and GPCR agonist was similar in SPF male and SPF female. In contrast, the intensity of the neuronal response was higher in SPF male compared to SPF female mice in response to capsaicin (125nM p<0.035 and 1.25µM p<0.038) but not in response to GPCR. The same trend was observed in GF mice. Neuronal activation induced by capsaicin or GPCR agonist was similar in SPF and GF mice. While bacterial supernatant from E. coli did not affect the activity of sensory neurons, the bacterial supernatant from E. aer induced changes in calcium mobilization in DRG neurons. Conclusions Our data suggest that at the level of DRG neurons from healthy mice, female sex and the absence of gut microbiota do not predispose to visceral hypersensitivity. In fact, the intensity of neuronal responses to capsaicin appear to be higher in DRGs from male mice. Furthermore, we show that metabolites from certain bacteria can activate sensory neurons. Thus, further studies are needed to investigate the role of gut microbiota and sex in visceral sensitivity Funding Agencies CIHR

scholarly journals Atg5-Deficient Mice Infected with Francisella tularensis LVS Demonstrate Increased Survival and Less Severe Pathology in Internal Organs

2020 ◽  
Vol 8 (10) ◽  
pp. 1531
Author(s):  
Ina Kelava ◽  
Mirna Mihelčić ◽  
Mateja Ožanič ◽  
Valentina Marečić ◽  
Maša Knežević ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Cell Types ◽  
Internal Organs ◽  
Tularensis Subsp ◽  
Cell Cytosol ◽  
Severe Pathology ◽  
Deficient Mice

Francisella tularensis is a highly virulent intracellular pathogen that proliferates within various cell types and can infect a multitude of animal species. Francisella escapes the phagosome rapidly after infection and reaches the host cell cytosol where bacteria undergo extensive replication. Once cytosolic, Francisella becomes a target of an autophagy-mediated process. The mechanisms by which autophagy plays a role in replication of this cytosolic pathogen have not been fully elucidated. In vitro, F. tularensis avoids degradation via autophagy and the autophagy process provides nutrients that support its intracellular replication, but the role of autophagy in vivo is unknown. Here, we investigated the role of autophagy in the pathogenesis of tularemia by using transgenic mice deficient in Atg5 in the myeloid lineage. The infection of Atg5-deficient mice with Francisella tularensis subsp. holarctica live vaccine strain (LVS) resulted in increased survival, significantly reduced bacterial burden in the mouse organs, and less severe histopathological changes in the spleen, liver and lung tissues. The data highlight the contribution of Atg5 in the pathogenesis of tularemia in vivo.

scholarly journals Expression of the Fgf6 gene is restricted to developing skeletal muscle in the mouse embryo

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 601-611 ◽  
Author(s):  
O. deLapeyriere ◽  
V. Ollendorff ◽  
J. Planche ◽  
M.O. Ott ◽  
S. Pizette ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Skeletal Muscles ◽  
Terminal Differentiation ◽  
Developmentally Regulated ◽  
Putative Receptor ◽  
Rna In Situ Hybridization ◽  
Additional Support ◽  
Insight Into

Fgf6, a member of the Fibroblast Growth Factor (FGF) family, is developmentally regulated and its expression is highly restricted in the adult. To gain further insight into the role of Fgf6, we studied its expression during embryogenesis using RNA in situ hybridization. Fgf6 expression is restricted to developing skeletal muscle. Fgf6 transcripts are first detected in the somites at 9.5 days post-conceptus, and expression continues in developing skeletal muscles up to at least 16.5 days post-conceptus. Fgfr4 is a putative receptor for FGF6. Its pattern of expression during myogenesis overlaps that of Fgf6, but both genes are not expressed in exactly the same population of cells. In addition, recombinant FGF6 protein is able to repress the terminal differentiation of myoblasts in culture, providing additional support to the concept that FGF6 plays an important role in myogenesis.

scholarly journals The mechanosensitive ion channel Piezo2 mediates sensitivity to mechanical pain in mice

2018 ◽  
Vol 10 (462) ◽  
pp. eaat9897 ◽  
Author(s):  
Swetha E. Murthy ◽  
Meaghan C. Loud ◽  
Ihab Daou ◽  
Kara L. Marshall ◽  
Frederick Schwaller ◽  
...  
Keyword(s):  
Nerve Injury ◽  
Sensory Neurons ◽  
Mechanical Allodynia ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Mechanical Stimuli ◽  
C Fiber ◽  
Mechanosensitive Ion Channel ◽  
Deficient Mice

The brush of a feather and a pinprick are perceived as distinct sensations because they are detected by discrete cutaneous sensory neurons. Inflammation or nerve injury can disrupt this sensory coding and result in maladaptive pain states, including mechanical allodynia, the development of pain in response to innocuous touch. However, the molecular mechanisms underlying the alteration of mechanical sensitization are poorly understood. In mice and humans, loss of mechanically activated PIEZO2 channels results in the inability to sense discriminative touch. However, the role of Piezo2 in acute and sensitized mechanical pain is not well defined. Here, we showed that optogenetic activation ofPiezo2-expressing sensory neurons induced nociception in mice. Mice lackingPiezo2in caudal sensory neurons had impaired nocifensive responses to mechanical stimuli. Consistently, ex vivo recordings in skin-nerve preparations from these mice showed diminished Aδ-nociceptor and C-fiber firing in response to mechanical stimulation. Punctate and dynamic allodynia in response to capsaicin-induced inflammation and spared nerve injury was absent in Piezo2-deficient mice. These results indicate that Piezo2 mediates inflammation- and nerve injury–induced sensitized mechanical pain, and suggest that targeting PIEZO2 might be an effective strategy for treating mechanical allodynia.

scholarly journals Expression of brain-derived neurotrophic factor in basolateral amygdala inputs to lateral septum is necessary for mice to identify socially novel individuals

2021 ◽  
Author(s):  
Lionel A Rodriguez ◽  
Sun-Hong Kim ◽  
Stephanie Cerceo Page ◽  
Claudia V Nguyen ◽  
Elizabeth A Pattie ◽  
...  
Keyword(s):  
Single Molecule ◽  
Neurotrophic Factor ◽  
Basolateral Amygdala ◽  
Brain Derived Neurotrophic Factor ◽  
Social Recognition ◽  
Lateral Septum ◽  
Projection Neurons ◽  
Bdnf Expression

Background: The lateral septum (LS) is a critical social behavior node. The LS is activated by social novelty, and perturbing LS activity impairs social recognition. However, the neural circuits and cell signaling pathways that converge on the LS to mediate this behavior aren't known. We identify brain-derived neurotrophic factor (BDNF) in projection neurons from the basolateral amgydala (BLA) to the LS, and tropomyosin kinase B receptor (TrkB) signaling in LS neurons, as required for social novelty recognition. Methods: We used single-molecule fluorescent in-situ hybridization to quantify Ntrk2 expression in LS. Viral transgenesis was used to induce cre-recombinase mediated TrkB knockdown in LS to evaluate impact on social recognition behavior, and social novelty-induced c-Fos expression in the LS. We used viral approaches for projection-specific ablation and BDNF-depletion in BLA-LS projection neurons to assess the role of this circuit in social recognition behavior. Results: The majority of GABAergic neurons in LS express TrkB. TrkB knockdown in LS abolishes social novelty recognition, and decreases LS activation in response to social novelty. Selectively ablating BLA-LS projection neurons abolishes social novelty recognition behavior, an effect that is phenocopied by depleting BDNF in this circuit. Conclusions: BLA-LS projection neurons depend on BDNF expression to regulate social recognition in the mouse. Within the LS, neurons require intact TrkB receptor signaling to mediate this behavior.

scholarly journals Single-molecule imaging suggests compact and spliceosome dependent organization of long introns

2021 ◽  
Author(s):  
Srivathsan Adivarahan ◽  
A.M.S.Kalhara Abeykoon ◽  
Daniel Zenklusen
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Splice Site ◽  
Spatial Organization ◽  
Spliceosome Assembly ◽  
U2 Snrnp ◽  
Rna In Situ Hybridization ◽  
Intron Removal ◽  
In Cells

Intron removal from pre-mRNAs is a critical step in the processing of RNA polymerase II transcripts, required to create translation competent mRNAs. In humans, introns account for large portions of the pre-mRNA, with intronic sequences representing about 95% of most pre-mRNA. Intron length varies considerably; introns can be as short as a few to hundreds of thousands of nucleotides in length. How nascent long intronic RNA is organized during transcription to facilitate the communication between 5′ and 3′ splice-sites required for spliceosome assembly however is still poorly understood. Here, we use single-molecule fluorescent RNA in situ hybridization (smFISH) to investigate the spatial organization of co- and post-transcriptional long introns in cells. Using two long introns within the POLA1 pre-mRNA as a model, we show that introns are packaged into compact assemblies, and when fully transcribed, are organized in a looped conformation with their ends in proximity. This organization is observed for nascent and nucleoplasmic pre-mRNAs and requires spliceosome assembly, as disruption of U2 snRNP binding results in introns with separated 5′ and 3′ ends. Moreover, interrogating the spatial organization of partially transcribed co-transcriptional POLA1 intron 35 indicates that the 5′ splice site is maintained proximal to the 3′ splice site during transcription, supporting a model that 5′ splice site tethering to the elongating polymerase might contribute to spliceosome assembly at long introns. Together, our study reveals details of intron and pre-mRNA organization in cells and provides a tool to investigate mechanisms of splicing for long introns.

Morphologic Features and Development of Granulomatous Vasculitis in Feline Infectious Peritonitis

2005 ◽  
Vol 42 (3) ◽  
pp. 321-330 ◽  
Author(s):  
A. Kipar ◽  
H. May ◽  
S. Menger ◽  
M. Weber ◽  
W. Leukert ◽  
...  
Keyword(s):  
Systemic Disease ◽  
Feline Infectious Peritonitis ◽  
Granulomatous Vasculitis ◽  
Perivascular Macrophages ◽  
Rna In Situ Hybridization ◽  
General Activation ◽  
Activated Monocytes ◽  
Factor Α

Feline infectious peritonitis (FIP) is a fatal, Coronavirus (CoV)-induced systemic disease in cats, characterized by granulomas in organs and granulomatous vasculitis. This study describes the morphologic features of granulomatous vasculitis in FIP as well as its development in the course of monocyte-associated feline CoV (FCoV) viremia in five naturally infected Domestic Shorthair cats with FIP. Monocyte-associated FCoV viremia was demonstrated by immunohistology, RNA in situ hybridization, and electron micropscopy. Granulomatous phlebitis at different stages of development was observed. Vasculitic processes ranged from attachment and emigration of FCoV-infected monocytes to vascular/perivascular granulomatous infiltrates with destruction of the vascular basal lamina. Monocytes as well as perivascular macrophages were activated because they were strongly positive for CD18 and expressed cytokines (tumor necrosis factor-α and interleukin-1β) and matrix metalloproteinase-9. In addition, general activation of endothelial cells, represented by major histocompatibility complex II upregulation, was observed in all cases. These results confirm FIP as a monocyte-triggered systemic disease and demonstrate the central role of activated monocytes in FIP vasculitis.

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