Ablation of Growth Hormone Receptor in GABAergic Neurons Leads to Increased Pulsatile Growth Hormone Secretion

Endocrinology

2022 Aug 01

Dos Santos, WO;Wasinski, F;Tavares, MR;Campos, AMP;Elias, CF;List, EO;Kopchick, JJ;Szawka, RE;Donato, J;
PMID: 35803590 | DOI: 10.1210/endocr/bqac103

Growth hormone (GH) acts in several hypothalamic neuronal populations to modulate metabolism and the autoregulation of GH secretion via negative-feedback loops. However, few studies have investigated whether GH receptor (GHR) expression in specific neuronal populations is required for the homeostatic control of GH secretion and energy homeostasis. In the present study, we investigated the consequences of the specific GHR ablation in GABAergic (VGAT-expressing) or glutamatergic (VGLUT2-expressing) cells. GHR ablation in GABAergic neurons led to increased GH secretion, lean mass, and body growth in male and female mice. VGAT-specific GHR knockout (KO) male mice also showed increased serum insulin-like growth factor-1, hypothalamic Ghrh, and hepatic Igf1 messenger RNA levels. In contrast, normal GH secretion, but reduced lean body mass, was observed in mice carrying GHR ablation in glutamatergic neurons. GHR ablation in GABAergic cells increased weight loss and led to decreased blood glucose levels during food restriction, whereas VGLUT2-specific GHR KO mice showed blunted feeding response to 2-deoxy-D-glucose both in males and females, and increased relative food intake, oxygen consumption, and serum leptin levels in male mice. Of note, VGLUT2-cre female mice, independently of GHR ablation, exhibited a previously unreported phenotype of mild reduction in body weight without further metabolic alterations. The autoregulation of GH secretion via negative-feedback loops requires GHR expression in GABAergic cells. Furthermore, GHR ablation in GABAergic and glutamatergic neuronal populations leads to distinct metabolic alterations. These findings contribute to the understanding of the neuronal populations responsible for mediating the neuroendocrine and metabolic effects of GH.

Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior

eLife

2022 Apr 07

Weera, MM;Agoglia, AE;Douglass, E;Jiang, Z;Rajamanickam, S;Shackett, RS;Herman, MA;Justice, NJ;Gilpin, NW;
PMID: 35389341 | DOI: 10.7554/eLife.67822

Corticotropin-releasing factor type-1 (CRF1) receptors are critical to stress responses because they allow neurons to respond to CRF released in response to stress. Our understanding of the role of CRF1-expressing neurons in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF1+ cells in rats. Here, we describe the generation and validation of a transgenic CRF1-Cre-tdTomato rat. We report that Crhr1 and Cre mRNA expression are highly colocalized in both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in tdTomato+ neurons are similar to those previously reported in GFP+ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be targeted to CeA CRF1+ cells via virally delivered Cre-dependent transgenes, that transfected Cre/tdTomato+ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like and nocifensive behaviors. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the brain, and that the expression pattern of Cre-tdTomato cells is similar to the known expression pattern of CRF1 cells. Given the accuracy of expression in the CRF1-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF1+ neurons can now be performed in assays that require the use of rats as the model organism.

Type I IFN-Driven Immune Cell Dysregulation in Rat Autoimmune Diabetes

ImmunoHorizons

2021 Oct 26

Qaisar, N;Arowosegbe, A;Derr, AG;Kucukural, A;Satish, B;Racicot, R;Guo, Z;Trombly, MI;Wang, JP;
PMID: 34702762 | DOI: 10.4049/immunohorizons.2100088

Type 1 diabetes is a chronic autoimmune disease, characterized by the immune-mediated destruction of insulin-producing β cells of pancreatic islets. Essential components of the innate immune antiviral response, including type I IFN and IFN receptor (IFNAR)-mediated signaling pathways, likely contribute to human type 1 diabetes susceptibility. We previously showed that LEW.1WR1 Ifnar1 -/- rats have a significant reduction in diabetes frequency following Kilham rat virus (KRV) infection. To delineate the impact of IFNAR loss on immune cell populations in KRV-induced diabetes, we performed flow cytometric analysis in spleens from LEW.1WR1 wild-type (WT) and Ifnar1 -/- rats after viral infection but before the onset of insulitis and diabetes. We found a relative decrease in CD8+ T cells and NK cells in KRV-infected LEW.1WR1 Ifnar1 -/- rats compared with KRV-infected WT rats; splenic regulatory T cells were diminished in WT but not Ifnar1 -/- rats. In contrast, splenic neutrophils were increased in KRV-infected Ifnar1 -/- rats compared with KRV-infected WT rats. Transcriptional analysis of splenic cells from KRV-infected rats confirmed a reduction in IFN-stimulated genes in Ifnar1 -/- compared with WT rats and revealed an increase in transcripts related to neutrophil chemotaxis and MHC class II. Single-cell RNA sequencing confirmed that MHC class II transcripts are increased in monocytes and macrophages and that numerous types of splenic cells harbor KRV. Collectively, these findings identify dynamic shifts in innate and adaptive immune cells following IFNAR disruption in a rat model of autoimmune diabetes, providing insights toward the role of type I IFNs in autoimmunity.

Crosstalk between transforming growth factor β-2 and Autotaxin in trabecular meshwork and different subtypes of glaucoma

Journal of biomedical science

2021 Jun 17

Igarashi, N;Honjo, M;Yamagishi, R;Kurano, M;Yatomi, Y;Igarashi, K;Kaburaki, T;Aihara, M;
PMID: 34140021 | DOI: 10.1186/s12929-021-00745-3

Elevated transforming growth factor (TGF)-β2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-β2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators.The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-β levels were measured using the Bio-Plex Pro TGF-β Assay. RNA scope was used to assess the expression of ATX and TGF-β2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-β2 regulates ATX expressions.TGF-β2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-β2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-β2 with concentration-dependent manner. In counter, ATX also induced TGF-β1, TGF-β2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-β and ATX inhibition.Trans-signaling of TGF-β2 regulates ATX expressions and thereby induced upregulations of TGF-βs or fibrosis of hTM. TGF-β2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.

Analgesic effect of central relaxin receptor activation on persistent inflammatory pain in mice: behavioral and neurochemical data

Pain reports

2021 Jun 16

Abboud, C;Brochoire, L;Drouet, A;Hossain, MA;Hleihel, W;Gundlach, AL;Landry, M;
PMID: 34159282 | DOI: 10.1097/PR9.0000000000000937

The relaxin peptide signaling system is involved in diverse physiological processes, but its possible roles in the brain, including nociception, are largely unexplored.In light of abundant expression of relaxin receptor (RXFP1) mRNA/protein in brain regions involved in pain processing, we investigated the effects of central RXFP1 activation on nociceptive behavior in a mouse model of inflammatory pain and examined the neurochemical phenotype and connectivity of relaxin and RXFP1 mRNA-positive neurons.Mice were injected with Complete Freund Adjuvant (CFA) into a hind paw. After 4 days, the RXFP1 agonist peptides, H2-relaxin or B7-33, ± the RXFP1 antagonist, B-R13/17K-H2, were injected into the lateral cerebral ventricle, and mechanical and thermal sensitivity were assessed at 30 to 120 minutes. Relaxin and RXFP1 mRNA in excitatory and inhibitory neurons were examined using multiplex, fluorescent in situ hybridization. Relaxin-containing neurons were detected using immunohistochemistry and their projections assessed using fluorogold retrograde tract-tracing.Both H2-relaxin and B7-33 produced a strong, but transient, reduction in mechanical and thermal sensitivity of the CFA-injected hind paw alone, at 30 minutes postinjection. Notably, coinjection of B-R13/17K-H2 blocked mechanical, but not thermal, analgesia. In the claustrum, cingulate cortex, and subiculum, RXFP1 mRNA was expressed in excitatory neurons. Relaxin immunoreactivity was detected in neurons in forebrain and midbrain areas involved in pain processing and sending projections to the RXFP1-rich, claustrum and cingulate cortex. No changes were detected in CFA mice.Our study identified a previously unexplored peptidergic system that can control pain processing in the brain and produce analgesia.

Resolving the cellular specificity of TSPO imaging in a rat model of peripherally-induced neuroinflammation

Brain, behavior, and immunity

2021 May 27

Vicente-Rodríguez, M;Singh, N;Turkheimer, F;Peris-Yague, A;Randall, K;Veronese, M;Simmons, C;Karim Haji-Dheere, A;Bordoloi, J;Sander, K;Awais, RO;Årstad, E;Consortium, N;Cash, D;Parker, CA;
PMID: 34052363 | DOI: 10.1016/j.bbi.2021.05.025

the increased expression of 18kDa Translocator protein (TSPO) is one of the few available biomarkers of neuroinflammation that can be assessed in humans in vivo by positron emission tomography (PET). TSPO PET imaging of the central nervous system (CNS) has been widely undertaken, but to date no clear consensus has been reached about its utility in brain disorders. One reason for this could be because the interpretation of TSPO PET signal remains challenging, given the cellular heterogeneity and ubiquity of TSPO in the brain. the aim of the current study was to ascertain if TSPO PET imaging can be used to detect neuroinflammation induced by a peripheral treatment with endotoxin lipopolysaccharide (LPS) in a rat model (ip LPS), and investigate the origin of TSPO signal changes in terms of their cellular sources and regional distribution. An initial pilot study utilising both [18F]DPA-714 and [11C]PK11195 demonstrated [18F]DPA-714 to exhibit a significantly higher lesion-related signal in the intracerebral LPS rat model (ic LPS) than [11C]PK11195. Subsequently, [18F]DPA-714 was selected for use in the ip LPS study. twenty-four hours after ip LPS, there was an increased uptake of [18F]DPA-714 across the whole brain. Further analyses of regions of interest, using immunohistochemistry and RNAscope Multiplex fluorescence V2 in situ hybridization technology, showed TSPO expression in microglia, monocyte derived-macrophages, astrocytes, neurons and endothelial cells. The expression of TSPO was significantly increased after ip LPS in a region-dependent manner; with microglia, monocyte-derived macrophages and astrocytes in the substantia nigra, in contrast to the hippocampus where TSPO was mostly confined to microglia and astrocytes. in summary, our data demonstrate the robust detection of peripherally-induced neuroinflammation in the CNS utilizing the TSPO radioligand [18F]DPA-714, and importantly, confirm that the TSPO signal increase arises mostly from a combination of microglia, astrocytes and monocyte-derived macrophages.

Single-nucleus RNA sequencing of human pancreatic islets identifies novel gene sets and distinguishes β-cell subpopulations with dynamic transcriptome profiles

Genome medicine

2023 May 01

Kang, RB;Li, Y;Rosselot, C;Zhang, T;Siddiq, M;Rajbhandari, P;Stewart, AF;Scott, DK;Garcia-Ocana, A;Lu, G;
PMID: 37127706 | DOI: 10.1186/s13073-023-01179-2

Single-cell RNA sequencing (scRNA-seq) provides valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo. On the other hand, single-nucleus RNA sequencing (snRNA-seq) has compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and affords enhanced information from intronic sequences that can be leveraged to identify pre-mRNA transcripts.We obtained nuclear preparations from fresh human islet cells and generated snRNA-seq datasets. We compared these datasets to scRNA-seq output obtained from human islet cells from the same donor. We employed snRNA-seq to obtain the transcriptomic profile of human islets engrafted in immunodeficient mice. In both analyses, we included the intronic reads in the snRNA-seq data with the GRCh38-2020-A library.First, snRNA-seq analysis shows that the top four differentially and selectively expressed genes in human islet endocrine cells in vitro and in vivo are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT (β-cells); PTPRT, FAP, PDK4, LOXL4 (α-cells); LRFN5, ADARB2, ERBB4, KCNT2 (δ-cells); and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 (γ-cells). Second, by integrating information from scRNA-seq and snRNA-seq of human islet cells, we distinguish three β-cell sub-clusters: an INS pre-mRNA cluster (β3), an intermediate INS mRNA cluster (β2), and an INS mRNA-rich cluster (β1). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA-rich cluster (β1) becomes the predominant sub-cluster in vivo.In summary, snRNA-seq and pre-mRNA analysis of human islet cells can accurately identify human islet cell populations, subpopulations, and their dynamic transcriptome profile in vivo.

The CysLT2R receptor mediates leukotriene C4-driven acute and chronic itch

Proceedings of the National Academy of Sciences of the United States of America

2021 Mar 30

Voisin, T;Perner, C;Messou, MA;Shiers, S;Ualiyeva, S;Kanaoka, Y;Price, TJ;Sokol, CL;Bankova, LG;Austen, KF;Chiu, IM;
PMID: 33753496 | DOI: 10.1073/pnas.2022087118

Acute and chronic itch are burdensome manifestations of skin pathologies including allergic skin diseases and atopic dermatitis, but the underlying molecular mechanisms are not well understood. Cysteinyl leukotrienes (CysLTs), comprising LTC4, LTD4, and LTE4, are produced by immune cells during type 2 inflammation. Here, we uncover a role for LTC4 and its signaling through the CysLT receptor 2 (CysLT2R) in itch. Cysltr2 transcript is highly expressed in dorsal root ganglia (DRG) neurons linked to itch in mice. We also detected CYSLTR2 in a broad population of human DRG neurons. Injection of leukotriene C4 (LTC4) or its nonhydrolyzable form NMLTC4, but neither LTD4 nor LTE4, induced dose-dependent itch but not pain behaviors in mice. LTC4-mediated itch differed in bout duration and kinetics from pruritogens histamine, compound 48/80, and chloroquine. NMLTC4-induced itch was abrogated in mice deficient for Cysltr2 or when deficiency was restricted to radioresistant cells. Itch was unaffected in mice deficient for Cysltr1, Trpv1, or mast cells (WSh mice). CysLT2R played a role in itch in the MC903 mouse model of chronic itch and dermatitis, but not in models of dry skin or compound 48/80- or Alternaria-induced itch. In MC903-treated mice, CysLT levels increased in skin over time, and Cysltr2-/- mice showed decreased itch in the chronic phase of inflammation. Collectively, our study reveals that LTC4 acts through CysLT2R as its physiological receptor to induce itch, and CysLT2R contributes to itch in a model of dermatitis. Therefore, targeting CysLT signaling may be a promising approach to treat inflammatory itch.

Extensive GJD2 Expression in the Song Motor Pathway Reveals the Extent of Electrical Synapses in the Songbird Brain

Biology

2021 Oct 25

Alcami, P;Totagera, S;Sohnius-Wilhelmi, N;Leitner, S;Grothe, B;Frankl-Vilches, C;Gahr, M;
| DOI: 10.3390/biology10111099

Birdsong is a precisely timed animal behavior. The connectivity of song premotor neural networks has been proposed to underlie the temporal patterns of neuronal activity that control vo-cal muscle movements during singing. Although the connectivity of premotor nuclei via chemical synapses has been characterized, electrical synapses and their molecular identity remain unex-plored. We show with in situ hybridizations that GJD2 mRNA, coding for the major channel-form-ing electrical synapse protein in mammals, connexin 36, is expressed in the two nuclei that control song production, HVC and RA from canaries and zebra finches. In canaries’ HVC, GJD2 mRNA is extensively expressed in GABAergic and only a fraction of glutamatergic cells. By contrast, in RA, GJD2 mRNA expression is widespread in glutamatergic and GABAergic neurons. Remarkably, GJD2 expression is similar in song nuclei and their respective embedding brain regions, revealing the widespread expression of GJD2 in the avian brain. Inspection of a single-cell sequencing data-base from zebra and Bengalese finches generalizes the distributions of electrical synapses across cell types and song nuclei that we found in HVC and RA from canaries, reveals a differential GJD2 mRNA expression in HVC glutamatergic subtypes and its transient increase along the neurogenic lineage. We propose that songbirds are a suitable model to investigate the contribution of electrical synapses to motor skill learning and production.

Involvement of Scratch2 in GalR1-mediated depression-like behaviors in the rat ventral periaqueductal gray

Proceedings of the National Academy of Sciences of the United States of America

2021 Jun 15

Yang, Y;Li, Y;Liu, B;Li, C;Liu, Z;Deng, J;Luo, H;Li, X;Wu, J;Li, H;Wang, CY;Zhao, M;Wu, H;Lallemend, F;Svenningsson, P;Hökfelt, TGM;Xu, ZD;
PMID: 34108238 | DOI: 10.1073/pnas.1922586118

Galanin receptor1 (GalR1) transcript levels are elevated in the rat ventral periaqueductal gray (vPAG) after chronic mild stress (CMS) and are related to depression-like behavior. To explore the mechanisms underlying the elevated GalR1 expression, we carried out molecular biological experiments in vitro and in animal behavioral experiments in vivo. It was found that a restricted upstream region of the GalR1 gene, from -250 to -220, harbors an E-box and plays a negative role in the GalR1 promoter activity. The transcription factor Scratch2 bound to the E-box to down-regulate GalR1 promoter activity and lower expression levels of the GalR1 gene. The expression of Scratch2 was significantly decreased in the vPAG of CMS rats. Importantly, local knockdown of Scratch2 in the vPAG caused elevated expression of GalR1 in the same region, as well as depression-like behaviors. RNAscope analysis revealed that GalR1 mRNA is expressed together with Scratch2 in both GABA and glutamate neurons. Taking these data together, our study further supports the involvement of GalR1 in mood control and suggests a role for Scratch2 as a regulator of depression-like behavior by repressing the GalR1 gene in the vPAG.

Corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis exhibit sex-specific pain encoding in mice

Scientific reports

2021 Jun 14

Yu, W;Caira, CM;Del R Rivera Sanchez, N;Moseley, GA;Kash, TL;
PMID: 34127705 | DOI: 10.1038/s41598-021-91672-8

The bed nucleus of the stria terminalis (BNST) plays an emerging role in pain regulation. Pharmacological studies have found that inhibiting corticotropin-releasing factor (CRF) signaling in the BNST can selectively mitigate the sensory and affective-motivational components of pain. However, mechanistic insight on the source of CRF that drives BNST responses to these harmful experiences remains unknown. In the present study, we used a series of genetic approaches to show that CRF in the BNST is engaged in the processing and modulation of pain. We conducted cell-type specific in vivo calcium imaging in CRF-Cre mice and found robust and synchronized recruitment of BNSTCRF neurons during acute exposures to noxious heat. Distinct patterns of recruitment were observed by sex, as the magnitude and timing of heat responsive activity in BNSTCRF neurons differed for male and female mice. We then used a viral approach in Floxed-CRF mice to selectively reduce CRF expression in the BNST and found it decreased nociceptive sensitivity for both sexes and increased paw attending for females. Together, these findings reveal that CRF in the BNST influences multiple facets of the pain experience to impact the sex-specific expression of pain-related behaviors.

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

Sci Transl Med. 2018 Oct 10;10(462).

2018 Oct 10

Murthy SE, Loud MC, Daou I, Marshall KL, Schwaller F, Kühnemund J, Francisco AG, Keenan WT, Dubin AE, Lewin GR, Patapoutian A.
PMID: 30305457 | DOI: 10.1126/scitranslmed.aat9897

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 of Piezo2-expressing sensory neurons induced nociception in mice. Mice lacking Piezo2 in 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.

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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
EnEm	Probe targets exons n and m
En-Em	Probe 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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