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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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Sexually differentiated microglia and CA1 hippocampal synaptic connectivity

Journal of Neuroendocrinology

2023 Apr 17

Prengel, T;Brunne, B;Habiballa, M;Rune, G;
| DOI: 10.1111/jne.13276

Microglia have been shown to sculpt postnatal circuitry from birth, up to adulthood due to their role in both synapse formation and synaptic pruning, the elimination of weak, redundant synapses. Microglia are differentiated in a sex-dependent manner. In this study, we tested whether sexual differentiation of microglia results in sex-dependent postnatal reorganization of CA1 synaptic connectivity in the hippocampus. The stereological counting of synapses in mice in the electron microscope showed a continuous rise in synapse density until the fourth week, followed by a plateau phase and loss of synapses from the eighth week onwards, with no difference between sexes. This course of alteration in synapse numbers did not differ between sexes. But selectively, on postnatal day (P) 14 the density of synapses was significantly higher in the female than in the male hippocampus. Higher synapse density in females was paralleled by higher activity of microglia, as indicated by morphological changes, CD68 expression, and proximity of microglia to synaptic sites. In Thy1-GFP mice, consistent with increased synapse numbers, bouton density was also clearly increased in females at P14. At this time point, CD47 expression, the “don't eat me” signal of neurons, was similar in males and females. The decrease in bouton density thereafter in conjuction with increased synapse numbers argues for a role of microglia in the formation of multispine boutons (MSB). Our data in females at P14 support the regulatory role of microglia in synapse density. Sexual differentiation of microglia, however, does not substantially affect long-term synaptic reorganization in the hippocampus.
Sexually dimorphic effects of estrogen receptor 2 deletion in the dorsal raphe nucleus on emotional behaviors

Journal of Neuroendocrinology

2022 Aug 19

He, J;Yan, J;Zha, X;Ding, X;Zhang, Y;Lu, Z;Xu, X;
| DOI: 10.1111/jne.13195

Sex differences in emotional behaviors and affective disorders have been widely noted, of which sexually dimorphic secretion of gonadal steroid hormones such as estrogen is suspected to play a role. However, the underlying neural mechanisms remain poorly understood. We noted that the expression of estrogen receptor 2 (Esr2, or ERβ), a key mediator of estrogen signaling in the brain, was enriched in the dorsal raphe nucleus (DRN), a region involved in emotion regulation. To test whether DRN Esr2 expression confers sex-specific susceptibility or vulnerability in emotional behaviors, we generated a conditional allele of Esr2 that allowed for site-specific deletion of Esr2 in DRN via local injection of Cre-expressing viruses. DRN-specific Esr2 deletion mildly increased anxiety behaviors in females, as shown by decreased time spent in the center zone of an open field in knockout females. By contrast, DRN Esr2 deletion had no effects on anxiety levels in males, as demonstrated by knockout males spending comparable time in the center zone of an open field and open arms of an elevated-plus maze. Furthermore, in the tail suspension test, DRN Esr2 deletion reduced immobility, a depression-like behavior, in a male-biased manner. Together these results reveal sex-specific functions of DRN Esr2 in regulating emotional behaviors and suggest targeted manipulation of DRN Esr2 signaling as a potential therapeutic strategy to treat sex-biased affective disorders.
Activation of oxytocin receptors in mouse GABAergic amacrine cells modulates retinal dopaminergic signaling

BMC biology

2022 Sep 21

Hu, S;Wang, Y;Han, X;Dai, M;Zhang, Y;Ma, Y;Weng, S;Xiao, L;
PMID: 36127701 | DOI: 10.1186/s12915-022-01405-0

Oxytocin, secreted by oxytocin neurons in the hypothalamus, is an endogenous neuropeptide involved in modulating multiple sensory information processing pathways, and its roles in the brain have been associated with prosocial, maternal, and feeding-related behaviors. Visual information is necessary for initiating these behaviors, with the retina consisting of the first stage in the visual system mediating external stimulus perception. Oxytocin has been detected in the mammalian retina; however, the expression and possible function of oxytocin receptors (OxtR) in the retina remain unknown. Here, we explore the role of oxytocin in regulating visual information processing in the retina.We observed that OxtR mRNA and protein are expressed in the mouse retina. With Oxtr-Cre transgenic mice, immunostaining, and fluorescence in situ hybridization, we found that OxtRs are mainly expressed in GABAergic amacrine cells (ACs) in both the inner nuclear layer (INL) and ganglion cell layer (GCL). Further immunoreactivity studies showed that GABAergic OxtR+ neurons are mainly cholinergic and dopaminergic neurons in the INL and are cholinergic and corticotrophin-releasing hormone neurons in the GCL. Surprisingly, a high level of Oxtr mRNAs was detected in retinal dopaminergic neurons, and exogenous oxytocin application activated dopaminergic neurons to elevate the retinal dopamine level. Relying on in vivo electroretinographic recording, we found that activating retinal OxtRs reduced the activity of bipolar cells via OxtRs and dopamine receptors.These data indicate the functional expression of OxtRs in retinal GABAergic ACs, especially dopaminergic ACs, and expand the interactions between oxytocinergic and dopaminergic systems. This study suggests that visual perception, from the first stage of information processing in the retina, is modulated by hypothalamic oxytocin signaling.
Effects of the isolated and combined ablation of growth hormone and IGF-1 receptors in somatostatin neurons

Endocrinology

2022 Apr 08

Chaves, FM;Wasinski, F;Tavares, MR;Mansano, NS;Frazão, R;Gusmao, DO;Quaresma, PGF;Pedroso, JAB;Elias, CF;List, EO;Kopchick, JJ;Szawka, RE;Donato, J;
PMID: 35395079 | DOI: 10.1210/endocr/bqac045

Hypophysiotropic somatostatin (SST) neurons in the periventricular hypothalamic area express growth hormone (GH) receptor (GHR) and are frequently considered as the key neuronal population that mediates the negative feedback loop controlling the hypothalamic-GH axis. Additionally, insulin-like growth factor-1 (IGF-1) may also act at the hypothalamic level to control pituitary GH secretion via long-loop negative feedback. However, to the best of our knowledge, no study so far has tested whether GHR or IGF-1 receptor (IGF1R) signaling specifically in SST neurons is required for the homeostatic control of GH secretion. Here we show that GHR ablation in SST neurons did not impact the negative-feedback mechanisms that control pulsatile GH secretion or body growth in male and female mice. The sex difference in hepatic gene expression profile was only mildly affected by GHR ablation in SST neurons. Similarly, IGF1R ablation in SST neurons did not affect pulsatile GH secretion, body growth or hepatic gene expression. In contrast, simultaneous ablation of both GHR and IGF1R in SST-expressing cells increased mean GH levels and pulse amplitude in male and female mice, and partially disrupted the sex differences in hepatic gene expression. Despite the increased GH secretion in double-knockout mice, no alterations in body growth and serum or liver IGF-1 levels were observed. In summary, GHR and IGF1R signaling in SST neurons play a redundant role in the control of GH secretion. Furthermore, our results reveal the importance of GH/IGF-1 negative-feedback mechanisms on SST neurons for the establishment of sex differences in hepatic gene expression profile.
Role and neural regulation of clock genes in the rat pineal gland: Clock modulates amplitude of rhythmic expression of Aanat encoding the melatonin-producing enzyme

Journal of pineal research

2023 Jun 22

Blancas-Velazquez, AS;Bering, T;Bille, S;Rath, MF;
PMID: 37349875 | DOI: 10.1111/jpi.12893

Circadian clock gene expression in the suprachiasmatic nucleus (SCN) controls 24h rhythms in body functions, but clock genes are also expressed in extra-hypothalamic tissues, including the melatonin-producing pineal gland. The nocturnal increase in pineal melatonin synthesis is a hallmark in circadian biology, but the role of local clock gene oscillations in the mammalian pineal gland is unknown. The aim of this work is to determine the role of clock genes in endocrine function of the pineal gland with focus on the Aanat transcript encoding the rhythm-generating enzyme of melatonin synthesis. Using the rat as a model, we here established 24h expression patterns of clock genes in the pineal gland in vivo. Lesion studies showed that rhythmic clock gene expression in the pineal gland to a large extent depends on the SCN; further, clock gene rhythms could be reestablished in cultured pineal cells synchronized by rhythmic stimulation with norepinephrine in 12 h pulses, suggesting that pineal cells house a slave oscillator controlled by adrenergic signaling in the gland. Histological analyses showed that clock genes are expressed in pinealocytes and co-localize with Aanat transcripts, thus potentially enabling clock gene products to control cellular melatonin production. To test this, cultured pineal cells were transfected using small interfering RNA to knock down clock gene expression. While successful knockdown of Per1 had a minor effect on Aanat, Clock knockdown produced a marked overexpression of Aanat in the pinealocytes. Our study suggests that SCN-dependent rhythmic Clock gene expression in the pinealocytes regulates the daily profile of Aanat expression. This article is protected by
Constitutive and conditional PACAP deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of PACAP

Journal of Neuroendocrinology

2023 May 02

Bakalar, D;Gavrilova, O;Jiang, S;Zhang, H;Roy, S;Williams, S;Liu, N;Wisser, S;Usdin, T;Eiden, L;
| DOI: 10.1111/jne.13286

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive PACAP knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in wild-type mice that do not occur in knock-out mice (aPRGs). Comparing constitutive PACAP knock-out mice to a variety of temporally and regionally specific PACAP knock-outs, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knock-out mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.
Sexual differentiation of estrogen receptor alpha subpopulations in the ventromedial nucleus of the hypothalamus

Hormones and behavior

2023 Mar 20

Cortes, LR;Sturgeon, H;Forger, NG;
PMID: 36948113 | DOI: 10.1016/j.yhbeh.2023.105348

Estrogen receptor (ER) α-expressing neurons in the ventrolateral area of the ventromedial hypothalamus (VMHvl) are implicated in the control of many behaviors and physiological processes, some of which are sex-specific. Recently, three sex-differentiated ERα subpopulations have been discovered in the VMHvl marked by co-expression with tachikinin1 (Tac1), reprimo (Rprm), or prodynorphin (Pdyn), that may subserve specific functions. These markers show sex differences in adulthood: females have many more Tac1/Esr1 and Rprm/Esr1 co-expressing cells, while males have more Pdyn/Esr1 cells. In this study, we sought to understand the development of these sex differences and pinpoint the sex-differentiating signal. We examined developmental changes in the number of Esr1 cells co-expressing Tac1, Rprm or Pdyn using single-molecule in situ hybridization. We found that both sexes have similarly high numbers of Tac1/Esr1 and Rprm/Esr1 cells at birth, but newborn males have many more Pdyn/Esr1 cells than females. However, the number of cells with Tac1/Esr1 and Rprm/Esr1 co-expression markedly decreases by weaning in males, but not females, leading to sex differences in neurochemical expression. Female mice administered testosterone at birth have expression patterns akin to male mice. Thus, a substantial neurochemical reorganization of the VMHvl occurs in males between birth and weaning that likely underlies the previously reported sex differences in behavioral and physiological responses to estrogens in adulthood.
Sleep restriction during opioid abstinence affects the hypothalamic-pituitary-adrenal (HPA) axis in male and female rats

Stress (Amsterdam, Netherlands)

2023 Jan 01

Raff, H;Glaeser, BL;Szabo, A;Olsen, CM;Everson, CA;
PMID: 36856367 | DOI: 10.1080/10253890.2023.2185864

Hypothalamic-pituitary-adrenal (HPA) axis dynamics are disrupted by opioids and may be involved in substance abuse; this persists during withdrawal and abstinence and is associated with co-morbid sleep disruption leading to vulnerability to relapse. We hypothesized that chronic sleep restriction (SR) alters the HPA axis diurnal rhythm and the sexually dimorphic response to acute stressor during opioid abstinence. We developed a rat model to evaluate the effect of persistent sleep loss during opioid abstinence on HPA axis dynamics in male and female rats. Plasma ACTH and corticosterone were measured diurnally and in response to acute restraint stress in rats Before (control) compared to During subsequent opioid abstinence without or with SR. Abstinence, regardless of sleep state, led to an increase in plasma ACTH and corticosterone in the morning in males. There was a tendency for higher PM plasma ACTH during abstinence in SR males (p = 0.076). ACTH and corticosterone responses to restraint were reduced in male SR rats whereas there was a failure to achieve the post-restraint nadir in female SR rats. There was no effect of the treatments or interventions on adrenal weight normalized to body weight. SR resulted in a dramatic increase in hypothalamic PVN AVP mRNA and plasma copeptin in male but not female rats. This corresponded to the attenuation of the HPA axis stress response in SR males during opioid abstinence. We have identified a potentially unique, sexually dimorphic role for magnocellular vasopressin in the control of the HPA axis during opioid abstinence and sleep restriction.
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.
Evidence that RXFP4 is located in enterochromaffin cells and can regulate production and release of serotonin

Bioscience reports

2023 Mar 22

Fernando, SJA;Wang, Q;Hay, DL;Bathgate, R;Shepherd, PR;Lee, KL;
PMID: 36947541 | DOI: 10.1042/BSR20221956

RXFP4 is a G protein-coupled receptor (GPCR) in the relaxin family. It has recently been recognised that this receptor and its cognate ligand INSL5 may have a role in the regulation of food intake, gut motility, and other functions relevant to metabolic health and disease.  Recent data from reporter-mice showed co-location of Rxfp4 and serotonin (5-HT) in the lower gut. We used human single cell RNA sequence data (scRNASeq) and immunohistochemistry to show that RXFP4 is in a subset of gut enterochromaffin cells that produce 5-HT in humans. We also used RNAScope to show co-location Rxfp4 mRNA and 5-HT in mouse colon, confirming prior findings. To understand how RXFP4 might regulate serotonin production we developed a cell model using Colo320 cells, a human gut-derived immortalised cell line that produces and releases serotonin.  Overexpression of RXFP4 in these cells resulted in a constitutive decrease in cAMP levels in both the basal state and in cells treated with forskolin.  Treatment of cells with two RXFP4 agonists, INSL5 derived peptide INSL5-A13 and small molecule compound-4, further reduced cAMP levels. This was paralleled by a reduction in expression of mRNA for TPH1, the enzyme controlling the rate limiting step in the production of serotonin.  Overexpression of RXFP4 also attenuated the cAMP-induced release of serotonin from Colo320 cells.  Together this demonstrates that serotonin producing enterochromaffin cells are the major site of RXFP4 expression in the gut and that RXFP4 can have inhibitory functional impacts on cAMP production as well as TPH1 expression and serotonin release.
Circulating miR-26b-5p and miR-451a as diagnostic biomarkers in medullary thyroid carcinoma patients

Journal of endocrinological investigation

2023 Jun 07

Besharat, ZM;Trocchianesi, S;Verrienti, A;Ciampi, R;Cantara, S;Romei, C;Sabato, C;Noviello, TMR;Po, A;Citarella, A;Caruso, FP;Panariello, I;Gianno, F;Carpino, G;Gaudio, E;Chiacchiarini, M;Masuelli, L;Sponziello, M;Pecce, V;Ramone, T;Maino, F;Dotta, F;Ceccarelli, M;Pezzullo, L;Durante, C;Castagna, MG;Elisei, R;Ferretti, E;
PMID: 37286863 | DOI: 10.1007/s40618-023-02115-2

The determination of tumour biomarkers is paramount to advancing personalized medicine, more so in rare tumours like medullary thyroid carcinoma (MTC), whose diagnosis is still challenging. The aim of this study was to identify non-invasive circulating biomarkers in MTC. To achieve this goal, paired MTC tissue and plasma extracellular vesicle samples were collected from multiple centres and microRNA (miRNA) expression levels were evaluated.The samples from a discovery cohort of 23 MTC patients were analysed using miRNA arrays. Lasso logistic regression analysis resulted in the identification of a set of circulating miRNAs as diagnostic biomarkers. Among them, miR-26b-5p and miR-451a, were highly expressed and their expression decreased during follow-up in disease-free patients in the discovery cohort. Circulating miR-26b-5p and miR-451a were validated using droplet digital PCR in a second independent cohort of 12 MTC patients.This study allowed the identification and validation of a signature of two circulating miRNAs, miR-26b-5p and miR-451a, in two independent cohorts reporting a significant diagnostic performance for MTC. The results of this study offer advancements in molecular diagnosis of MTC proposing a novel non-invasive tool to use in precision medicine.
Vasopressin V1a receptor and oxytocin receptor regulate murine sperm motility differently

Life science alliance

2023 Apr 01

Tsuchiya, H;Fujinoki, M;Azuma, M;Koshimizu, TA;
PMID: 36650057 | DOI: 10.26508/lsa.202201488

Specific receptors for the neurohypophyseal hormones, arginine vasopressin (AVP) and oxytocin, are present in the male reproductive organs. However, their exact roles remain unknown. To elucidate the physiological functions of pituitary hormones in male reproduction, this study first focused on the distribution and function of one of the AVP receptors, V1a. In situ hybridization analysis revealed high expression of the Avpr1a in Leydig cells of the testes and narrow/clear cells in the epididymis, with the expression pattern differing from that of the oxytocin receptor (OTR). Notably, persistent motility and highly proportional hyperactivation were observed in spermatozoa from V1a receptor-deficient mice. In contrast, OTR blocking by antagonist atosiban decreased hyperactivation rate. Furthermore, AVP stimulation could alter the extracellular pH mediated by the V1a receptor. The results highlight the crucial role of neurohypophyseal hormones in male reproductive physiology, with potential contradicting roles of V1a and OTR in sperm maturation. Our findings suggest that V1a receptor antagonists are potential therapeutic drugs for male infertility.

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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

Enabling research, drug development (CDx) and diagnostics

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