Neuropeptide Receptor Expression Research Articles

FRI278 Kisspeptin Neuron Projections And Expression Patterns Are Unaltered In Prenatally Androgenized Female Mice Modeling PCOS

Abstract Disclosure: M. Prescott: None. Y. Watanabe: None. A. Ruddenklau: None. L. Fisher: None. C.L. Jasoni: None. R.E. Campbell: Consulting Fee; Self; Bayer, Inc. Polycystic ovary syndrome (PCOS) is associated with impaired gonadal steroid hormone feedback and a hyperactive central reproductive axis, implicating the brain in PCOS pathophysiology. Excess prenatal androgen (PNA) exposure is linked with the development of PCOS, and preclinical evidence suggests that PNA can program altered connections within the gonadotropin-releasing hormone (GnRH) neuronal network. Kisspeptin neurons are critical upstream regulators of GnRH neurons, relaying hormone feedback and potently activating GnRH secretion, but their role in PCOS remains incompletely understood. This study tested the hypothesis that prenatal androgen excess programmes altered neuroanatomical connections between kisspeptin neurons and GnRH neurons and modified neuropeptide and steroid hormone receptor expression in kisspeptin neurons. To investigate this, we labelled, imaged and assessed kisspeptin fibers, GnRH neurons, and gonadal steroid hormone receptors in adult female transgenic kisspeptin reporter mice (Kiss1(Cre-GFP)Coll; ROSA26tdTomato) exposed prenatally to di-hydrotestosterone or an oil vehicle (VEH) on gestational days 16-18 (n=7-10/group). PNA mice were acyclic and all tissue was collected in diestrus. Fixed coronal brain sections were immunofluorescently labelled for GnRH and kisspeptin reporter tdTomato and imaged on a confocal microscope to assess close fiber contacts to the soma and proximal dendrite of GnRH neurons. Neither the proportion of GnRH neurons with kisspeptin fiber contacts (VEH: 86.3±3.4%, PNA: 87.3±4.3%), nor the mean number of kisspeptin fiber close appositions per GnRH neuron (VEH: 6.1±0.6, PNA: 5.7±0.8) was different between groups. Kisspeptin neuron number and kisspeptin neuron co-expression with estrogen and progesterone receptors was evaluated in immunolabelled sections throughout the rostral periventricular nucleus of the 3rd ventricle (RP3V) and the arcuate nucleus (ARN). PNA had no significant impact on kisspeptin neuron number or the proportion of neurons with gonadal hormone co-expression in either population. Expression of neuropeptides was quantified by RT-qPCR in micro-dissected RP3V and ARN tissue from a separate cohort of adult PNA and VEH mice (n=5-8/group). PNA did not affect Kiss1, Tac2 or Pdyn expression in the ARN or Kiss1 expression in the RP3V. These data indicate that PNA does not modify anatomical connections between RP3V kisspeptin neurons and GnRH neurons, nor does it have a robust impact on steroid hormone responsiveness or neuropeptide expression of kisspeptin neurons. While the neuroanatomical relationship between ARN kisspeptin neurons and the GnRH neuron distal dendron in PNA mice remains to be determined, these data suggest that PNA-mediated neuroendocrine dysfunction is not due to programmed changes in the neuroanatomical projections or gonadal hormone sensitivity of kisspeptin neurons. Presentation: Friday, June 16, 2023

Reference gene recommendations and PACAP receptor expression in murine sympathetic ganglia of the autonomic nervous system that innervate adipose tissues after chronic cold exposure.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important regulator of the stress response in mammals, influencing both the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). PACAP has been reported to influence energy homeostasis, including adaptive thermogenesis, an energy burning process in adipose tissue regulated by the SNS in response to cold stress and overfeeding. While research suggests PACAP acts centrally at the level of the hypothalamus, knowledge of PACAP's role within the sympathetic nerves innervating adipose tissues in response to metabolic stressors is limited. This work shows, for the first time, gene expression of PACAP receptors in stellate ganglia and highlights some differential expression with housing temperature. Additionally, we present our dissection protocol, analysis of tyrosine hydroxylase gene expression as a molecular biomarker for catecholamine producing tissue and recommend three stable reference genes for the normalization of quantitative real time-polymerase chain reaction (qRT-PCR) data when working with this tissue. This study adds to information about neuropeptide receptor expression in peripheral ganglia of the sympathetic nervous system innervating adipose tissue and provides insight into PACAP's role in the regulation of energy metabolism.

Hypo-Osmoregulatory Roles of Vasotocinergic and Isotocinergic Systems in the Intestines of Two European Sea Bass Lineages.

European sea bass (Dicentrarchus labrax) are a major aquaculture species that live in habitats with fluctuating salinities that are sometimes higher than in seawater (SW). Atlantic and West-Mediterranean genetic lineages were compared regarding intestinal neuropeptide receptor expression in SW (36%) and following a two-week transfer to hypersalinity (HW, 55%). Phylogenetic analysis revealed seven neuropeptide receptors belonging to the arginine vasotocine (AVTR) family and two isotocin receptors (ITR). Among AVTR paralogs, the highest mRNA levels were recorded for v1a2, with a two- to fourfold upregulation in the European sea bass intestinal sections after transfer of fish to HW. Principal component analysis in posterior intestines showed that v1a2 expression grouped together with the expression and activity of main ion transporters and channels involved in solute-coupled water uptake, indicating a possible role of this receptor in triggering water absorption. v1a1 expression, however, was decreased or did not change after transfer to hypersaline water. Among ITR paralogs, itr1 was the most expressed paralog in the intestine and opposite expression patterns were observed following salinity transfer, comparing intestinal sections. Overall, different expression profiles were observed between genetic lineages for several analyzed genes which could contribute to different osmotic stress-related responses in D. labrax lineages.

Prolactin promotes parental responses and alters reproductive axis gene expression, but not courtship behaviors, in both sexes of a biparental bird

Prolactin, a hormone involved in vertebrate parental care, is hypothesized to inhibit reproductive hypothalamic-pituitary-gonadal (HPG) axis activity during parenting, thus maintaining investment in the current brood as opposed to new reproductive efforts. While prolactin underlies many parental behaviors in birds, its effects on other reproductive behaviors, such as courtship, remain unstudied. How prolactin affects neuropeptide and hormone receptor expression across the avian HPG axis also remains unknown. To address these questions, we administered ovine prolactin (oPRL) or a vehicle control to both sexes in experienced pairs of the biparental rock dove (Columba livia), after nest removal at the end of incubation. We found that oPRL promoted parental responses to novel chicks and stimulated crop growth compared to controls, consistent with other studies. However, we found that neither courtship behaviors, copulation rates nor pair maintenance differed with oPRL treatment. Across the HPG, we found oPRL had little effect on gene expression in hypothalamic nuclei, but increased expression of FSHB and hypothalamic hormone receptor genes in the pituitary. In the gonads, oPRL increased testes size and gonadotropin receptor expression, but did not affect ovarian state or small white follicle gene expression. However, the oviducts of oPRL-treated females were smaller and had lower estrogen receptor expression compared with controls. Our results highlight that some species, especially those that show multiple brooding, may continue to express mating behavior despite elevated prolactin. Thus, mechanisms may exist for prolactin to promote investment in parental care without concurrent inhibition of reproductive function or HPG axis activity.

Differential regulation of NPY and SP receptor expression in STRO-1+ve PDLSCs by inflammatory cytokines.

The aims of this study were to investigate neuropeptide receptor expression regulation on STRO-1 +ve periodontal ligament stem cells (PDLSCs) in response to inflammatory cytokines and to investigate a potential osteogenic effect of neuropeptides. Nerve fibres innervating the periodontal tissues in humans contain several neuropeptides including neuropeptide Y and substance P. The role of neuropeptide receptors on PDLSCs, including their response to the local inflammatory environment of periodontitis, is currently unknown. A homogenous population of STRO-1 +ve PDLSCs was prepared by immunomagnetic separation of cells obtained by the tissue out-growth method from healthy premolar teeth from a single donor. Regulation of gene expression of the neuropeptide Y Y1 receptor and substance P receptor tachykinin receptor 1 was investigated. A potential osteogenic effect of neuropeptide Y and substance P was also investigated by measuring alkaline phosphatase (ALP) activity, Alizarin red staining and quantifying osteogenic gene expression. Treatment of STRO-1 +ve PDLSCs with tumour necrosis factor-alpha or interleukin 1-beta up-regulated the expression of the neuropeptide Y's Y1 receptor, but down-regulated substance P's receptor. Significantly increased ALP activity was observed in STRO-1 +ve PDLSCs treated with neuropeptide Y but not substance P. Further studies showed that neuropeptide Y had a modest osteogenic effect on cells at both a functional level and a gene level. Expression of the neuropeptide Y Y1 receptor gene on STRO-1 +ve PDLSCs was sensitive to local inflammatory cytokines. Treatment of cells with neuropeptide Y was found to produce a modest enhanced osteogenic effect.

A conserved behavioral role for a nematode interneuron neuropeptide receptor.

Neuropeptides are evolutionarily conserved modulators of many aspects of animal behavior and physiology, and expand the repertoire of processes that can be controlled by a limited number of neurons. Deciphering the neuropeptidergic codes that govern distinct processes requires systematic functional analyses of neuropeptides and their cognate receptors. Even in well-studied model organisms like Caenorhabditis elegans, however, such efforts have been precluded by a lack of mutant reagents. Here, we generated and screened 21 C. elegans neuropeptide G-protein coupled receptor mutants with no pre-existing reagents for the touch-evoked escape response, and implicated six receptors expressed in diverse neuron classes representing multiple circuit levels in this behavior. We further characterized the mutant with the most severe phenotype, frpr-14, which was defective in multiple behavioral paradigms. We leveraged this range of phenotypes to reveal that FRPR-14 modulation of different precommand interneuron classes, AVH and AIB, can drive distinct behavioral subsets, demonstrating cellular context-dependent roles for FRPR-14 signaling. We then show that Caenorhabditis briggsae CBR-FRPR-14 modulates an AVH-like interneuron pair to regulate the same behaviors as C. elegans but to a smaller extent. Our results also suggest that differences in touch-evoked escape circuit architecture between closely related species results from changes in neuropeptide receptor expression pattern, as opposed to ligand-receptor pairing. This study provides insights into the principles utilized by a compact, multiplexed nervous system to generate intraspecific behavioral complexity and interspecific variation.

Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. Invivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

Highly redundant neuropeptide volume co-transmission underlying episodic activation of the GnRH neuron dendron.

The necessity and functional significance of neurotransmitter co-transmission remains unclear. The glutamatergic 'KNDy' neurons co-express kisspeptin, neurokinin B (NKB), and dynorphin and exhibit a highly stereotyped synchronized behavior that reads out to the gonadotropin-releasing hormone (GnRH) neuron dendrons to drive episodic hormone secretion. Using expansion microscopy, we show that KNDy neurons make abundant close, non-synaptic appositions with the GnRH neuron dendron. Electrophysiology and confocal GCaMP6 imaging demonstrated that, despite all three neuropeptides being released from KNDy terminals, only kisspeptin was able to activate the GnRH neuron dendron. Mice with a selective deletion of kisspeptin from KNDy neurons failed to exhibit pulsatile hormone secretion but maintained synchronized episodic KNDy neuron behavior that is thought to depend on recurrent NKB and dynorphin transmission. This indicates that KNDy neurons drive episodic hormone secretion through highly redundant neuropeptide co-transmission orchestrated by differential post-synaptic neuropeptide receptor expression at the GnRH neuron dendron and KNDy neuron.

Bombesin-Like Peptide Recruits Disinhibitory Cortical Circuits and Enhances Fear Memories

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory and excitatory neurons suggests that each circuit motif is controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a newly-developed genetically-encoded GRP sensor and trans-synaptic tracing we reveal that GRP regulates VIP cells via extrasynaptic diffusion from several putative local and long-range sources. In vivo photometry and CRISPR/Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and that GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

Acute exposure to high‐induction electromagnetic field affects activity of model peripheral sensory neurons

Exposure to repetitive low‐frequency electromagnetic field (LF‐EMF) shows promise as a non‐invasive approach to treat various sensory and neurological disorders. Despite considerable progress in the development of modern stimulation devices, there is a limited understanding of the mechanisms underlying their biological effects and potential targets at the cellular level. A significant impact of electromagnetic field on voltage‐gated calcium channels and downstream signaling pathways has been convincingly demonstrated in many distinct cell types. However, evidence for clear effects on primary sensory neurons that particularly may be responsible for the analgesic actions of LF‐EMF is still lacking.Here, we used F11 cells derived from a fusion between mouse embryonic neuroblastoma and rat dorsal root ganglion (DRG) neurons as an authentic in vitro model of peripheral sensory neurons. These cells retain many features of native DRG neurons, including expression of neuropeptide receptors and various voltage‐gated channels (Vetter a Lewis, 2010, Yin et al., 2016). Using calcium‐imaging, we examined the effects of three different protocols of high induction electromagnetic stimulation on F11 chemical responsiveness and spontaneous activity. We show that short‐term (< 180 s) exposure to LF‐EMF reduces calcium transients in response to bradykinin, a potent pain‐producing inflammatory agent formed at sites of injury. Moreover, we characterize an immediate and reversible potentiating effect of LF‐EMF on neuronal spontaneous activity. Our results provide new evidence that electromagnetic field may directly modulate the activity of sensory neurons and highlight the potential of sensory neuron‐derived cell line as a physiologically relevant tool for studying the effects of LF‐EMF, and the underlying mechanisms at the cellular and molecular level.Support or Funding InformationThis work was supported by the Ministry of Health of the Czech Republic (NV16‐28784A)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sex- and sub region-dependent modulation of arcuate kisspeptin neurones by vasopressin and vasoactive intestinal peptide.

A population of kisspeptin neurones located in the hypothalamic arcuate nucleus (ARN) very likely represent the gonadotrophin-releasing hormone pulse generator responsible for driving pulsatile luteinising hormone secretion in mammals. As such, it has become important to understand the neural inputs that modulate the activity of ARN kisspeptin (ARNKISS ) neurones. Using a transgenic GCaMP6 mouse model allowing the intracellular calcium levels ([Ca2+ ]i ) of individual ARNKISS neurones to be assessed simultaneously, we examined whether the circadian neuropeptides vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) modulated the activity of ARNKISS neurones directly. To validate this methodology, we initially evaluated the effects of neurokinin B (NKB) on [Ca2+ ]i in kisspeptin neurones residing within the rostral, middle and caudal ARN subregions of adult male and female mice. All experiments were undertaken in the presence of tetrodotoxin and ionotropic amino acid antagonists. NKB was found to evoke an abrupt increase in [Ca2+ ]i in 95%-100% of kisspeptin neurones throughout the ARN of both sexes. By contrast, both VIP and AVP were found to primarily activate kisspeptin neurones located in the caudal ARN of female mice. Although 58% and 59% of caudal ARN kisspeptin neurones responded to AVP and VIP, respectively, in female mice, only 0%-8% of kisspeptin neurones located in other ARN subregions responded in females and 0%-12% of cells in any subregion in males (P<0.05). These observations demonstrate unexpected sex differences and marked heterogeneity in functional neuropeptide receptor expression amongst ARNKISS neurones organised on a rostro-caudal basis. The functional significance of this unexpected influence of VIP and AVP on ARNKISS neurones remains to be established.

Transcriptomic analysis of crustacean neuropeptide signaling during the moult cycle in the green shore crab, Carcinus maenas

BackgroundEcdysis is an innate behaviour programme by which all arthropods moult their exoskeletons. The complex suite of interacting neuropeptides that orchestrate ecdysis is well studied in insects, but details of the crustacean ecdysis cassette are fragmented and our understanding of this process is comparatively crude, preventing a meaningful evolutionary comparison. To begin to address this issue we identified transcripts coding for neuropeptides and their putative receptors in the central nervous system (CNS) and Y-organs (YO) within the crab, Carcinus maenas, and mapped their expression profiles across accurately defined stages of the moult cycle using RNA-sequencing. We also studied gene expression within the epidermally-derived YO, the only defined role for which is the synthesis of ecdysteroid moulting hormones, to elucidate peptides and G protein-coupled receptors (GPCRs) that might have a function in ecdysis.ResultsTranscriptome mining of the CNS transcriptome yielded neuropeptide transcripts representing 47 neuropeptide families and 66 putative GPCRs. Neuropeptide transcripts that were differentially expressed across the moult cycle included carcikinin, crustacean hyperglycemic hormone-2, and crustacean cardioactive peptide, whilst a single putative neuropeptide receptor, proctolin R1, was differentially expressed. Carcikinin mRNA in particular exhibited dramatic increases in expression pre-moult, suggesting a role in ecdysis regulation. Crustacean hyperglycemic hormone-2 mRNA expression was elevated post- and pre-moult whilst that for crustacean cardioactive peptide, which regulates insect ecdysis and plays a role in stereotyped motor activity during crustacean ecdysis, was elevated in pre-moult.In the YO, several putative neuropeptide receptor transcripts were differentially expressed across the moult cycle, as was the mRNA for the neuropeptide, neuroparsin-1. Whilst differential gene expression of putative neuropeptide receptors was expected, the discovery and differential expression of neuropeptide transcripts was surprising. Analysis of GPCR transcript expression between YO and epidermis revealed 11 to be upregulated in the YO and thus are now candidates for peptide control of ecdysis.ConclusionsThe data presented represent a comprehensive survey of the deduced C. maenas neuropeptidome and putative GPCRs. Importantly, we have described the differential expression profiles of these transcripts across accurately staged moult cycles in tissues key to the ecdysis programme. This study provides important avenues for the future exploration of functionality of receptor-ligand pairs in crustaceans.

Anxiety-like behavior and neuropeptide receptor expression in male and female prairie voles: The effects of stress and social buffering

Strong social support can negate negative health outcomes – an effect defined as ‘social buffering’. In the present study, using the socially monogamous prairie vole (Microtus ochrogaster), we examined whether the presence of a bonded partner during a stressful event can reduce stress responses. Adult, pair-bonded female and male voles were assigned into experimental groups that were either handled (Control), experienced a 1-h immobilization (IMO) stress alone (IMO-Alone), or experienced IMO with their partner (IMO-Partner). Thereafter, subjects were tested for anxiety-like behavior, and brain sections were subsequently processed for oxytocin receptor (OTR) and vasopressin V1a-type receptor (V1aR) binding. Our data indicate that while IMO stress significantly decreased the time that subjects spent in the open arms of an elevated plus maze, partner’s presence prevented this behavioral change – this social buffering on anxiety-like behavior was the same for both male and female subjects. Further, IMO stress decreased OTR binding in the nucleus accumbens (NAcc), but a partner’s presence dampened this effect. No effects were found in V1aR binding. These data suggest that the neuropeptide- and brain region-specific OTR alterations in the NAcc may be involved in both the mediation and social buffering of stress responses. Some sex differences in the OTR and V1aR binding were also found in selected brain regions, offering new insights into the sexually dimorphic roles of the two neuropeptides. Overall, our results suggest a potential preventative approach in which the presence of social interactions during a stressor may buffer typical negative outcomes.

Altered Expression of Neuropeptide Receptors in the Bleomycin (Bleo)‐Induced Mouse Model of Scleroderma (SSc)

Study ObjectiveBackgroundSSc is a chronic collagen‐vascular disease that initially presents with dermal fibrosis, later progressing to multiple organ fibrosis. There is no treatment to arrest SSc. Recently, a mouse model of SSc was reported, in which Bleo is injected intradermally (ID) for 21–28 days. Two groups showed that reactive oxygen species (ROS) cause this dermal fibrosis. We previously observed that ROS trigger GRP‐mediated pulmonary fibrosis induced by hyperoxia or radiation. We first verified that ROS trigger this dermal fibrosis, and now test the hypothesis that gastrin‐releasing peptide (GRP) from cutaneous nerves has a role in this process by acting on myofibroblasts [alpha‐smooth muscle actin, SMA+] and pericytes [SMA+ and neural/glial antigen 2, NG2+]. We tested expression of the two GRP receptors, GRPR and neuromedin B receptor (NMBR), by using immunohistochemistry.Methods10‐wk old C3H/HeJ females were injected ID on the flank with Bleo (100‐μg) 5d/wk for 3‐wks. Some Bleo‐injected mice also received antioxidant N‐acetylcysteine (NAC) IP, or GRP blocking mAb‐2A11. After 21d, sections of skin lesions were immunostained for SMA, NG2, GRPR, or NMBR. Relative extent of immunostaining in dermis and epidermis was scored on a scale from 0–3, comparing prevalence of (+) cells (0, 1=detected in few cells, 2=many cells (+), &amp; 3=most cells positive. ImageJ analysis (immunopositive area as a percentage of tissue volume) was used to confirm scoring results.ResultsPericytes &amp; myofibroblasts were significantly increased in areas of dermal thickening, with NG2 and SMA immunostaining linearly correlated (R2 = .87. P&lt;0.05). Dermal SMA &amp; NG2 were reduced by NAC (~80% decrease, P&lt;0.001) or mAb2A11 (~50% decrease, P&lt;0.01), similar to measurements of dermal thickness. Epidermal GRPR was decreased by over 50% in Bleo+2A11 mice compared to Bleo alone (P &lt; 0.005). There was no other difference in GRPR between study groups. NMBR expression was generally unchanged between the groups.ConclusionIn the Bleo mouse model of SSc, increased pericytes and myofibroblasts occur in dermal fibrosis. Although either GRPR &amp;/or NMBR could contribute to Bleo‐induced dermal fibrosis their expression in dermis is unchanged between groups. Sustained epidermal expression of both receptors would be consistent with potential GRP signaling in epidermis as a mechanism leading to epidermal hyperplasia and dermal fibrosis, possibly via epithelial‐mesenchymal transformation.Support or Funding InformationThis work was supported by a P30 NIH Pilot Grant Award to M. Sunday by the Duke Skin Disease Research Center [Dr. Russell Hall, Program Director].

Impact of Orexin-A Treatment on Food Intake, Energy Metabolism and Body Weight in Mice.

Orexin-A and -B are hypothalamic neuropeptides of 33 and 28-amino acids, which regulate many homeostatic systems including sleep/wakefulness states, energy balance, energy homeostasis, reward seeking and drug addiction. Orexin-A treatment was also shown to reduce tumor development in xenografted nude mice and is thus a potential treatment for carcinogenesis. The aim of this work was to explore in healthy mice the consequences on energy expenditure components of an orexin-A treatment at a dose previously shown to be efficient to reduce tumor development. Physiological approaches were used to evaluate the effect of orexin-A on food intake pattern, energy metabolism body weight and body adiposity. Modulation of the expression of brain neuropeptides and receptors including NPY, POMC, AgRP, cocaine- and amphetamine related transcript (CART), corticotropin-releasing hormone (CRH) and prepro-orexin (HCRT), and Y2 and Y5 neuropeptide Y, MC4 (melanocortin), OX1 and OX2 orexin receptors (Y2R, Y5R, MC4R, OX1R and OX2R, respectively) was also explored. Our results show that orexin-A treatment does not significantly affect the components of energy expenditure, and glucose metabolism but reduces intraperitoneal fat deposit, adiposity and the expression of several brain neuropeptide receptors suggesting that peripheral orexin-A was able to reach the central nervous system. These findings establish that orexin-A treatment which is known for its activity as an inducer of tumor cell death, do have minor parallel consequence on energy homeostasis control.

Increase of Mast Cell-Nerve Association and Neuropeptide Receptor Expression on Mast Cells in Perennial Allergic Rhinitis

Objectives: Mast cells (MCs) and nerves play an important role in allergic rhinitis (AR), but little is known about their crosstalk in AR. The aim of this study was to investigate MC-nerve interaction in the human nasal mucosa during AR. Methods: The association between MCs and nerves, the expression of neuropeptide receptors (neurokinin 1 receptor [NK1R], neurokinin 2 receptor [NK2R], calcitonin gene-related peptide receptor [CGRPR], and MrgX2) on MCs, and protease-activated receptor 2 (PAR2) and tyrosine receptor kinase A (TrkA) on nerve fibres in the human nasal mucosa were investigated with immunofluorescence and real-time PCR. Results: The association between MCs and nerves was found to be significantly increased, although the numbers of MCs and nerve fibres were unchanged during AR. MCs expressing tryptase-chymase (MC_tc) were frequently associated with nerve fibres and these contacts increased significantly in AR. Neuropeptide receptors NK1R, NK2R, and CGRPR were firstly found to be largely localised on MCs. The number of MCs expressing NK1R and NK2R, but not CGRPR, was significantly increased in AR. Interestingly, MC_tc mostly expressed these neuropeptide receptors. The newly discovered tachykinin receptor MrgX2 was not expressed on nasal MCs, but was expressed on gland cells and increased in AR. Additionally, tachykinergic nerve fibres were found to express PAR2 or TrkA as receptors for MCs. Conclusions: This study revealed for the first time an increase of MC-nerve association and neuropeptide receptor expression on MCs during AR as well as nerve fibres containing receptors for MCs. These results suggest that targeting or controlling airway sensory nerve function as a modulator of MCs may prevent allergic airway inflammation such as AR.

Postnatal light alters hypothalamic-pituitary-adrenal axis function and induces a depressive-like phenotype in adult mice.

The postnatal light environment that a mouse experiences during the critical first three postnatal weeks has long-term effects on both its circadian rhythm output and clock gene expression. Furthermore, data from our lab suggest that postnatal light may also impact the hypothalamic-pituitary-adrenal (HPA) axis, which is a key regulator of stress. To test the effect of postnatal light exposure on adult stress responses and circadian rhythmicity, we raised mice under either 24-h light-dark cycles (LD), constant light (LL) or constant dark (DD) during the first three postnatal weeks. After weaning we then exposed all animals to LD cycles (basal conditions), followed by LL (stressed conditions) environments. We examined brain neuropeptide and glucocorticoid receptor (GR) expression, plasma corticosterone concentration rhythm and body temperature rhythm, together with depression- and anxiety-related behaviour. Results showed that LL- and DD-raised mice exhibited decreased GR expression in the hippocampus, increased plasma corticosterone concentration at the onset of the dark phase and a depressive phenotype when exposed to LD cycles later in life. Furthermore, LL-raised mice showed increased corticotrophin-releasing hormone mRNA expression in the paraventricular nucleus of the hypothalamus. When exposed to LL as adults, LL-raised mice showed a significant circadian rhythm of plasma corticosterone concentration, together with a shorter period and stronger circadian rhythm of body temperature compared to DD-raised mice. Taken together, these data suggest that altered postnatal light environments have long-term effects on the HPA axis and the circadian system, which can lead to altered stress responses and a depressive phenotype in adulthood.

TRPV1 and neuropeptide receptor immunoreactivity and expression in the rat lung and brainstem after lung ischemia-reperfusion injury

BackgroundActivation of capsaicin-sensitive sensory nerves and TRPV1 present on them can ameliorate the ischemia-reperfusion injury in vital organs by evoking the release of neuropeptides including calcitonin gene-related peptide (CGRP) and substance P. However, the underlying changes in TRPV1 and neuropeptide receptor expressions, including calcitonin receptor-like receptor (CRLR) and neurokinin 1 receptor (NK1R), after lung ischemia-reperfusion injury (LIRI) remain uncharacterized. MethodsThirty-two male Sprague–Dawley rats were randomly and equally divided into sham (sham thoracotomy) and ischemia-reperfusion (occlusion of the left pulmonary hilus for 1 h followed by reperfusion for 4 h) groups. Blood gas levels were measured and histopathologic examination was performed. Left lung lobes and brainstem tissue samples were harvested for use in quantitative real-time PCR, Western blot, and immunohistochemistry to measure TRPV1, CRLR, and NK1R transcript and protein levels. Additionally, CGRP and substance P protein levels were quantified in the lungs using enzyme-linked immunosorbent assay. ResultsLIRI exacerbated blood gas exchange and increased the pulmonary tissue injury score. Furthermore, LIRI increased CGRP levels in the lung, TRPV1–immunoreactivity (ir) in the bronchiolar epithelium and smooth muscle of the pulmonary artery, and the intensity of neuronal CRLR-ir and NK1R-ir in the commissural nucleus of the solitary tract. Similarly, LIRI significantly elevated both transcription and translation of TRPV1 in the lungs and CRLR and NK1R in the brainstem. ConclusionsBoth transcription and translation of TRPV1 in the lungs and CRLR and NK1R in the brainstem of rats can be upregulated by LIRI in a rapid manner (within 5 h).

Effects of reproductive experience on central expression of progesterone, oestrogen α, oxytocin and vasopressin receptor mRNA in male California mice (Peromyscus californicus).

Fatherhood in biparental mammals is accompanied by distinct neuroendocrine changes in males, involving some of the same hormones involved in maternal care. In the monogamous, biparental California mouse (Peromyscus californicus), paternal care has been linked to changes in the central and/or peripheral availability of oestrogen, progesterone, vasopressin and oxytocin, although it is not known whether these endocrine fluctuations are associated with changes in receptor availability in the brain. Thus, we compared mRNA expression of oestrogen receptor (ER)α, progesterone receptor (PR), vasopressin receptor (V1a) and oxytocin receptor (OTR) in brain regions implicated in paternal care [i.e. medial preoptic area (MPOA)], fear [i.e. medial amygdala (MeA)] and anxiety [i.e. bed nucleus of the stria terminalis (BNST)] between first-time fathers (n = 8) and age-matched virgin males (n = 7). Males from both reproductive conditions behaved paternally towards unrelated pups, whereas fathers showed significantly shorter latencies to behave paternally and less time investigating pups. Furthermore, fathers showed significantly lower PR, OTR and V1a receptor mRNA expression in the BNST compared to virgins. Fathers also showed a marginally significant (P = 0.07) reduction in progesterone receptor mRNA expression in the MPOA, although fatherhood was not associated with any other changes in receptor mRNA in the MPOA or MeA. The results of the present study indicate that behavioural and endocrine changes associated with the onset of fatherhood, and/or with cohabitation with a (breeding) female, are accompanied by changes in mRNA expression of hormone and neuropeptide receptors in the brain.

Keratinocytes express cytokines and nerve growth factor in response to neuropeptide activation of the ERK1/2 and JNK MAPK transcription pathways

Sensory neurons innervating the skin can release neuropeptides that are believed to modulate cellular proliferation, wound healing, pigmentation, and keratinocyte innate immune responses. While the ability of neuropeptides to stimulate keratinocyte production of inflammatory mediators has been demonstrated, there is no information concerning the mechanisms by which neuropeptide activation of keratinocyte cell surface receptors ultimately leads to the up-regulation of mediator production. In this study we used a keratinocyte cell line to identify the presence of substance P (SP) and calcitonin gene-related peptide (CGRP) receptors on keratinocytes and examined the effects of SP and CGRP stimulation on keratinocyte neuropeptide signaling, cell proliferation, and interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and nerve growth factor (NGF) expression. Neuropeptide stimulation caused an up-regulation of neuropeptide receptor expression in keratinocytes and a dramatic increase in keratinocyte secretion of SP and CGRP, suggesting possible autocrine or paracrine stimulatory effects and amplification of neuropeptide signaling. Both SP and CGRP concentration-dependently stimulated cellular proliferation and the expression and secretion of inflammatory cytokines and NGF in keratinocytes. SP also activated all 3 families of mitogen activated protein kinase (MAPK) and nuclear factor κB (NFκB) in keratinocytes, while CGRP only activated p38 and extracellular signal related kinase1/2 (ERK1/2) MAPKs. Neuropeptide stimulated inflammatory mediatory production in keratinocytes was reversed by ERK1/2 and JNK inhibitors. The current study is the first to observe; 1) that CGRP stimulates keratinocyte expression of CGRP and its receptor complex, 2) that SP and CGRP stimulate IL-6 and TNF-α secretion in keratinocytes, 3) that SP activated all three MAPK families and the NFκB transcriptional signaling pathway in keratinocytes, and 4) that SP and CGRP stimulated inflammatory mediator production in keratinocytes is dependent on ERK1/2 and JNK activation. These studies provide evidence suggesting that disruption of ERK1/2 and JNK signaling may potentially be an effective therapy for inflammatory skin diseases and pain syndromes mediated by exaggerated sensory neuron–keratinocyte signaling.