Sequences analysis and pituitary actions of tachykinins in Chinese sturgeon (Acipenser sinensis).

Tachykinins belong to a large, evolutionarily conserved family of brain/gut peptides that are involved in a variety of physiological functions in mammals, such as reproductive regulation. However, little information was available about tachykinins in ancient fish lineage. In the present study, we firstly identified three tachykinin genes (named tac1, tac3 and tac4) and three neurokinin receptors (named nk1r, nk2r and nk3r) from Chinese sturgeon brain and pituitary. Sequence analysis showed that tac1 encoded substance P (SP) and neurokinin A (NKA), tac3 encoded neurokinin B (NKB) and NKB-related peptide (NKBRP), and tac4 encoded hemokin 1 (HK-1) and hemokin 2 (HK-2), respectively. The luciferase reporter assay results showed that NK1R preferentially selected asSP, NK2R preferentially selected asNKA, and NK3R preferentially selected asNKB. Tissue expression analysis showed that the three tac genes were highly detected in the telencephalon and hypothalamus, whereas nkr genes were widely expressed in peripheral tissues. Spatio-temporal expression analysis showed that all three tac genes were highly expressed in unknown sex individuals. Intraperitoneal injection experiments showed that both asSP and asNKB could stimulate luteinizing hormone (LH) release in Chinese sturgeon serum. At the transcriptional level, asSP and asNKB could significantly reduce pituitary follicle-stimulating hormone beta (fshß) mRNA expression, but induce pituitary growth hormone (gh) mRNA expression. In addition, estradiol (E2) could stimulate tac3 mRNA expression in hypothalamus. Taken together, this study provided information on the tachykinin family in Chinese sturgeon and demonstrates that asNKB and asSP could be involved in reproductive and growth regulation in pituitary.

Female Infertility Is Associated with an Altered Expression Profile of Different Members of the Tachykinin Family in Human Granulosa Cells.

Neurokinin B (NKB) and its cognate receptor, NK3R, play a key role in the regulation of reproduction. NKB belongs to the family of tachykinins, which also includes substance P and neurokinin A, both encoded by the by the gene TAC1, and hemokinin-1, encoded by the TAC4 gene. In addition to NK3R, tachykinin effects are mediated by NK1R and NK2R, encoded by the genes TACR1 and TACR2, respectively. The role of these other tachykinins and receptors in the regulation of women infertility is mainly unknown. We have analyzed the expression profile of TAC1, TAC4, TACR1, and TACR2 in mural granulosa and cumulus cells from women presenting different infertility etiologies, including polycystic ovarian syndrome, advanced maternal age, low ovarian response, and endometriosis. We also studied the expression of MME, the gene encoding neprilysin, the most important enzyme involved in tachykinin degradation. Our data show that TAC1, TAC4, TACR1, TACR2, and MME expression is dysregulated in a different manner depending on the etiology of women infertility. The abnormal expression of these tachykinins and their receptors might be involved in the decreased fertility of these patients, offering a new insight regarding the diagnosis and treatment of women infertility.

Tachykinin-related peptides modulate immune-gene expression in the mealworm beetle Tenebrio molitor L.

Tachykinins (TKs) are a group of conserved neuropeptides. In insects, tachykinin-related peptides (TRPs) are important modulators of several functions such as nociception and lipid metabolism. Recently, it has become clear that TRPs also play a role in regulating the insect immune system. Here, we report a transcriptomic analysis of changes in the expression levels of immune-related genes in the storage pest Tenebrio molitor after treatment with Tenmo-TRP-7. We tested two concentrations (10-8 and 10-6 M) at two time points, 6 and 24 h post-injection. We found significant changes in the transcript levels of a wide spectrum of immune-related genes. Some changes were observed 6 h after the injection of Tenmo-TRP-7, especially in relation to its putative anti-apoptotic action. Interestingly, 24 h after the injection of 10-8 M Tenmo-TRP-7, most changes were related to the regulation of the cellular response. Applying 10-6 M Tenmo-TRP-7 resulted in the downregulation of genes associated with humoral responses. Injecting Tenmo-TRP-7 did not affect beetle survival but led to a reduction in haemolymph lysozyme-like antibacterial activity, consistent with the transcriptomic data. The results confirmed the immunomodulatory role of TRP and shed new light on the functional homology between TRPs and TKs.

The structural conformation of the tachykinin domain drives the anti-tumoural activity of an octopus peptide in melanoma BRAFV600E.

BACKGROUND AND PURPOSE: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma. EXPERIMENTAL APPROACH: We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish). KEY RESULTS: Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAFV600E -mutated cells with minimal effects on fibroblasts. In melanoma-treated cells, Octpep-1 increased ROS with unaltered mitochondrial membrane potential and promoted non-mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep-1 depends upon the α-helix and polyproline conformation in the C-terminal region of the peptide. A truncated form of the C-terminal end of Octpep-1 displayed enhanced potency and efficacy against melanoma. Octpep-1 reduced the progression of tumours in xenograft melanoma mice and zebrafish. CONCLUSION AND IMPLICATIONS: We unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

Developmental sex differences in the peri-pubertal pattern of hypothalamic reproductive gene expression, including Kiss1 and Tac2, may contribute to sex differences in puberty onset.

The mechanisms regulating puberty still remain elusive, as do the underlying causes for sex differences in puberty onset (girls before boys) and pubertal disorders. Neuroendocrine puberty onset is signified by increased pulsatile GnRH secretion, yet how and when various upstream reproductive neural circuits change developmentally to govern this process is poorly understood. We previously reported day-by-day peri-pubertal increases (Kiss1, Tac2) or decreases (Rfrp) in hypothalamic gene expression of female mice, with several brain mRNA changes preceding external pubertal markers. However, similar pubertal measures in males were not previously reported. Here, to identify possible neural sex differences underlying sex differences in puberty onset, we analyzed peri-pubertal males and directly compared them with female littermates. Kiss1 expression in male mice increased over the peri-pubertal period in both the AVPV and ARC nuclei but with lower levels than in females at several ages. Likewise, Tac2 expression in the male ARC increased between juvenile and older peri-pubertal stages but with levels lower than females at most ages. By contrast, both DMN Rfrp expressionand Rfrp neuronal activation strongly decreased in males between juvenile and peri-pubertal stages, but with similar levels as females. Neither ARC KNDy neuronal activation nor Kiss1r expression in GnRH neurons differed between males and females or changed with age. These findings delineate several peri-pubertal changes in neural populations in developing males, with notable sex differences in kisspeptin and NKB neuron developmental patterns. Whether these peri-pubertal hypothalamic sex differences underlie sex differences in puberty onset deserves future investigation.

Tachykinins, new players in the control of reproduction and food intake: A comparative review in mammals and teleosts.

In vertebrates, the tachykinin system includes tachykinin genes, which encode one or two peptides each, and tachykinin receptors. The complexity of this system is reinforced by the massive conservation of gene duplicates after the whole-genome duplication events that occurred in vertebrates and furthermore in teleosts. Added to this, the expression of the tachykinin system is more widespread than first thought, being found beyond the brain and gut. The discovery of the co-expression of neurokinin B, encoded by the tachykinin 3 gene, and kisspeptin/dynorphin in neurons involved in the generation of GnRH pulse, in mammals, put a spotlight on the tachykinin system in vertebrate reproductive physiology. As food intake and reproduction are linked processes, and considering that hypothalamic hormones classically involved in the control of reproduction are reported to regulate also appetite and energy homeostasis, it is of interest to look at the potential involvement of tachykinins in these two major physiological functions. The purpose of this review is thus to provide first a general overview of the tachykinin system in mammals and teleosts, before giving a state of the art on the different levels of action of tachykinins in the control of reproduction and food intake. This work has been conducted with a comparative point of view, highlighting the major similarities and differences of tachykinin systems and actions between mammals and teleosts.

Novel Pituitary Actions of TAC4 Gene Products in Teleost.

Tachykinin 4 (TAC4) is the latest member of the tachykinin family involved in several physiological functions in mammals. However, little information is available about TAC4 in teleost. In the present study, we firstly isolated TAC4 and six neurokinin receptors (NKRs) from grass carp brain and pituitary. Sequence analysis showed that grass carp TAC4 could encode two mature peptides (namely hemokinin 1 (HK1) and hemokinin 2 (HK2)), in which HK2 retained the typical FXGLM motif in C-terminal of tachyinin, while HK1 contained a mutant VFGLM motif. The ligand-receptor selectivity showed that HK2 could activate all 6 NKRs but with the highest activity for the neurokinin receptor 2 (NK2R). Interestingly, HK1 displayed a very weak activation for each NKR isoform. In grass carp pituitary cells, HK2 could induce prolactin (PRL), somatolactin α (SLα), urotensin 1 (UTS1), neuromedin-B 1 (NMB1), cocaine- and amphetamine-regulated transcript 2 (CART2) mRNA expression mediated by NK2R and neurokinin receptor 3 (NK3R) via activation cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC) and calcium2+ (Ca2+)/calmodulin (CaM)/calmodulin kinase-II (CaMK II) cascades. However, the corresponding stimulatory effects triggered by HK1 were found to be notably weaker. Furthermore, based on the structural base for HK1, our data suggested that a phenylalanine (F) to valine (V) substitution in the signature motif of HK1 might have contributed to its weak agonistic actions on NKRs and pituitary genes regulation.

Identification of hub genes associated with cognition in the hippocampus of Alzheimer's Disease.

Alzheimer's Disease (AD) is a neurodegenerative disease featured by cognitive impairment. This bioinformatic analysis was used to identify hub genes related to cognitive dysfunction in AD. The gene expression profile GSE48350 in the hippocampus of AD patients aged >70 years was obtained from the Gene Expression Omnibus (GEO) database. A total of 96 differentially expressed genes (DEGs) were identified, and subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses; a protein-protein interaction (PPI) network was constructed. The DEGs were enriched in synapse-related changes. A protein cluster was teased out of PPI. Furthermore, the cognition ranked the first among all the terms of biological process (BP). Next, 4 of 10 hub genes enriched in cognition were identified. The function of these genes was validated using APP/PS1 mice. Cognitive performance was validated by Morris Water Maze (MWM), and gene expression by RT-qPCR, Cholecystokinin (CCK), Tachykinin precursor 1 (TAC1), Calbindin 1 (CALB1) were downregulated in the hippocampus. These genes can provide new directions in the research of the molecular mechanism of AD.

Characterization of putative tachykinin peptides in Caenorhabditis elegans.

Tachykinin-like peptides, such as substance P, neurokinin A, and neurokinin B, are among the earliest discovered and best-studied neuropeptide families, and research on them has contributed greatly to our understanding of the endocrine control of many physiological processes. However, there are still many orphan tachykinin receptor homologs for which cognate ligands have not yet been identified, especially in small invertebrates, such as the nematode Caenorhabditis elegans (C. elegans). We here show that the C. elegans nlp-58 gene encodes putative ligands for the orphan G protein-coupled receptor (GPCR) TKR-1, which is a worm ortholog of tachykinin receptors. We first determine, through an unbiased biochemical screen, that a peptide derived from the NLP-58 preprotein stimulates TKR-1. Three mature peptides that are predicted to be generated from NLP-58 show potent agonist activity against TKR-1. We designate these peptides as C. elegans tachykinin (CeTK)-1, -2, and -3. The CeTK peptides contain the C-terminal sequence GLR-amide, which is shared by tachykinin-like peptides in other invertebrate species. nlp-58 exhibits a strongly restricted expression pattern in several neurons, implying that CeTKs behave as neuropeptides. The discovery of CeTKs provides important information to aid our understanding of tachykinin-like peptides and their functional interaction with GPCRs.

Characterization of the Action of Tachykinin Signaling on Pulsatile LH Secretion in Male Mice.

The alternation of the stimulatory action of the tachykinin neurokinin B (NKB) and the inhibitory action of dynorphin within arcuate (ARH) Kiss1 neurons has been proposed as the mechanism behind the generation of gonadotropin-releasing hormone (GnRH) pulses through the pulsatile release of kisspeptin. However, we have recently documented that GnRH pulses still exist in gonadectomized mice in the absence of tachykinin signaling. Here, we document an increase in basal frequency and amplitude of luteinizing hormone (LH) pulses in intact male mice deficient in substance P, neurokinin A (NKA) signaling (Tac1KO), and NKB signaling (Tac2KO and Tacr3KO). Moreover, we offer evidence that a single bolus of the NKB receptor agonist senktide to gonad-intact wild-type males increases the basal release of LH without changing its frequency. Altogether, these data support the dispensable role of the individual tachykinin systems in the generation of LH pulses. Moreover, the increased activity of the GnRH pulse generator in intact KO male mice suggests the existence of compensation by additional mechanisms in the generation of kisspeptin/GnRH pulses.

A spinoparabrachial circuit defined by Tacr1 expression drives pain.

Painful stimuli evoke a mixture of sensations, negative emotions and behaviors. These myriad effects are thought to be produced by parallel ascending circuits working in combination. Here, we describe a pathway from spinal cord to brain for ongoing pain. Activation of a subset of spinal neurons expressing Tacr1 evokes a full repertoire of somatotopically directed pain-related behaviors in the absence of noxious input. Tacr1 projection neurons (expressing NKR1) target a tiny cluster of neurons in the superior lateral parabrachial nucleus (PBN-SL). We show that these neurons, which also express Tacr1 (PBN-SLTacr1), are responsive to sustained but not acute noxious stimuli. Activation of PBN-SLTacr1 neurons alone did not trigger pain responses but instead served to dramatically heighten nocifensive behaviors and suppress itch. Remarkably, mice with silenced PBN-SLTacr1 neurons ignored long-lasting noxious stimuli. Together, these data reveal new details about this spinoparabrachial pathway and its key role in the sensation of ongoing pain.

Mass spectrometric studies on the peptide integrity of substance P and related human tachykinins in human biofluids.

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice-immunoassays-also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1-178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations.

Activation of Preoptic Tachykinin 1 Neurons Promotes Wakefulness over Sleep and Volatile Anesthetic-Induced Unconsciousness.

Endogenous sleep and general anesthesia are distinct states that share similar traits. Of particular interest to neuroscience is the loss of consciousness that accompanies both states. Multiple lines of evidence demonstrate that general anesthetics can co-opt the neural circuits regulating arousal to produce unconsciousness. However, controversy remains as to whether the neural circuits and, more specifically, the same neurons shaping sleep and wakefulness actually do influence the anesthetic state in vivo. Hypothalamic preoptic area (POA) neurons are intimately involved in modulating spontaneous and anesthetic-induced changes in arousal. Nevertheless, recent work suggests that POA GABAergic or glutamatergic neurons capable of regulating endogenous sleep fail to influence the onset or dissipation of anesthesia. We hypothesized that the POA's broad neuronal diversity could mask convergent roles of a subset of neurons in regulating both arousal and anesthesia. Contrary to a previously published report, we show that chemogenetic activation of POA Tac1 neurons obliterates both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep, strongly consolidating the waking state for hours, even during a period of elevated sleep drive. Moreover, chemogenetic activation of Tac1 POA neurons stabilizes the wake state against both isoflurane- and sevoflurane-induced unconsciousness. Tac1-activated mice display a partial resistance to entering isoflurane anesthesia and a more pronounced ability to exit both isoflurane- and sevoflurane-induced unconscious states. We conclude that POA Tac1 neurons can potently reinforce arousal both against endogenous and drug-induced unconscious states. POA Tac1 neurons thus add causal support for the involvement of arousal-regulating systems in the state of general anesthesia.

Androgen Suppresses In Vivo and In Vitro LH Pulse Secretion and Neural Kiss1 and Tac2 Gene Expression in Female Mice.

Androgens can affect the reproductive axis of both sexes. In healthy women, as in men, elevated exogenous androgens decrease gonad function and lower gonadotropin levels; such circumstances occur with anabolic steroid abuse or in transgender men (genetic XX individuals) taking androgen supplements. The neuroendocrine mechanisms by which endogenous or exogenous androgens regulate gonadotropin release, including aspects of pulsatile luteinizing hormone (LH) secretion, remain unknown. Because animal models are valuable for interrogating neural and pituitary mechanisms, we studied effects of androgens in the normal male physiological range on in vivo LH secretion parameters in female mice and in vitro LH secretion patterns from isolated female pituitaries. We also assessed androgen effects on hypothalamic and gonadotrope gene expression in female mice, which may contribute to altered LH secretion profiles. We used a nonaromatizable androgen, dihydrotestosterone (DHT), to isolate effects occurring specifically via androgen receptor (AR) signaling. Compared with control females, DHT-treated females exhibited markedly reduced in vivo LH pulsatility, with decreases in pulse frequency, amplitude, peak, and basal LH levels. Correlating with reduced LH pulsatility, DHT-treated females also exhibited suppressed arcuate nucleus Kiss1 and Tac2 expression. Separate from these neural effects, we determined in vitro that the female pituitary is directly inhibited by AR signaling, resulting in lower basal LH levels and reduced LH secretory responses to gonadotropin-releasing hormone pulses, along with lower gonadotropin gene expression. Thus, in normal adult females, male levels of androgen acting via AR can strongly inhibit the reproductive axis at both the neural and pituitary levels.

Transcriptomic approach predicts a major role for transforming growth factor beta type 1 pathway in L-Dopa-induced dyskinesia in parkinsonian rats.

Dyskinesia induced by long-term L-Dopa (LID) therapy in Parkinson disease is associated with altered striatal function whose molecular bases remain unclear. Here, a transcriptomic approach was applied for comprehensive analysis of distinctively regulated genes in striatal tissue, their specific pathways, and functional- and disease-associated networks in a rodent model of LID. This approach has identified transforming growth factor beta type 1 (TGFß1) as a highly upregulated gene in dyskinetic animals. TGFß1 pathway is a top aberrantly regulated pathway in the striatum following LID development based on differentially expressed genes (> 1.5 fold change and P < 0.05). The induction of TGFß1 pathway specific genes, TGFß1, INHBA, AMHR2 and PMEPA1 was also associated with regulation of NPTX2, PDP1, SCG2, SYNPR, TAC1, TH, TNNT1 genes. Transcriptional network and upstream regulator analyses have identified AKT-centered functional and ERK-centered disease networks revealing the association of TGFß1, IL-1ß and TNFα with LID development. Therefore, results support that TGFß1 pathway is a major contributor to the pathogenic mechanisms of LID.

Role of Nitric Oxide in the Change of 5-Hydroxytryptamine Synthesis in the Intestine by a Consecutive Administration of Methotrexate to Rats.

This study aimed to investigate whether the consecutive administration of methotrexate affects 5-hydroxytryptamine (5-HT) synthesis in the rat small intestine. Rats received methotrexate at a dose of 12.5 mg/kg intraperitoneally on 4 consecutive days. NG-nitro-L-arginine methyl ester (L-NAME) was given subcutaneously to inhibit nitric oxide (NO) synthase. Methotrexate moderately altered 5-HT synthesis, whereas the combined administration of methotrexate and L-NAME significantly changed 5-HT synthesis in the rat ileal tissue. These results suggest that endogenous NO has an antagonistic role in the induction of 5-HT synthesis in rats following the consecutive administration of methotrexate.

Detection of Promoter DNA Methylation in Urine and Plasma Aids the Detection of Non-Small Cell Lung Cancer.

PURPOSE: Low-dose CT screening can reduce lung cancer-related mortality. However, CT screening has an FDR of nearly 96%. We sought to assess whether urine samples can be a source for DNA methylation-based detection of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: This nested case-control study of subjects with suspicious nodules on CT imaging obtained plasma and urine samples preoperatively. Cases (n = 74) had pathologic confirmation of NSCLC. Controls (n = 27) had a noncancer diagnosis. We detected promoter methylation in plasma and urine samples using methylation on beads and quantitative methylation-specific real-time PCR for cancer-specific genes (CDO1, TAC1, HOXA7, HOXA9, SOX17, and ZFP42). RESULTS: DNA methylation at cancer-specific loci was detected in both plasma and urine, and was more frequent in patients with cancer compared with controls for all six genes in plasma and in CDO1, TAC1, HOXA9, and SOX17 in urine. Univariate and multivariate logistic regression analysis showed that methylation detection in each one of six genes in plasma and CDO1, TAC1, HOXA9, and SOX17 in urine were significantly associated with the diagnosis of NSCLC, independent of age, race, and smoking pack-years. When methylation was detected for three or more genes in both plasma and urine, the sensitivity and specificity for lung cancer diagnosis were 73% and 92%, respectively. CONCLUSIONS: DNA methylation-based biomarkers in plasma and urine could be useful as an adjunct to CT screening to guide decision-making regarding further invasive procedures in patients with pulmonary nodules.

Hemokinin-1 Gene Expression Is Upregulated in Trigeminal Ganglia in an Inflammatory Orofacial Pain Model: Potential Role in Peripheral Sensitization.

A large percentage of primary sensory neurons in the trigeminal ganglia (TG) contain neuropeptides such as tachykinins or calcitonin gene-related peptide. Neuropeptides released from the central terminals of primary afferents sensitize the secondary nociceptive neurons in the trigeminal nucleus caudalis (TNC), but also activate glial cells contributing to neuroinflammation and consequent sensitization in chronic orofacial pain and migraine. In the present study, we investigated the newest member of the tachykinin family, hemokinin-1 (HK-1) encoded by the Tac4 gene in the trigeminal system. HK-1 had been shown to participate in inflammation and hyperalgesia in various models, but its role has not been investigated in orofacial pain or headache. In the complete Freund's adjuvant (CFA)-induced inflammatory orofacial pain model, we showed that Tac4 expression increased in the TG in response to inflammation. Duration-dependent Tac4 upregulation was associated with the extent of the facial allodynia. Tac4 was detected in both TG neurons and satellite glial cells (SGC) by the ultrasensitive RNAscope in situ hybridization. We also compared gene expression changes of selected neuronal and glial sensitization and neuroinflammation markers between wild-type and Tac4-deficient (Tac4-/-) mice. Expression of the SGC/astrocyte marker in the TG and TNC was significantly lower in intact and saline/CFA-treated Tac4-/- mice. The procedural stress-related increase of the SGC/astrocyte marker was also strongly attenuated in Tac4-/- mice. Analysis of TG samples with a mouse neuroinflammation panel of 770 genes revealed that regulation of microglia and cytotoxic cell-related genes were significantly different in saline-treated Tac4-/- mice compared to their wild-types. It is concluded that HK-1 may participate in neuron-glia interactions both under physiological and inflammatory conditions and mediate pain in the trigeminal system.

GABAergic input through GABAB receptors is necessary during a perinatal window to shape gene expression of factors critical to reproduction such as Kiss1.

Lack of GABAB receptors in GABAB1 knockout mice decreases neonatal ARC kisspeptin 1 (Kiss1) expression in the arcuate nucleus of the hypothalamus (ARC) in females, which show impaired reproduction as adults. Our aim was to selectively impair GABAB signaling during a short postnatal period to evaluate its impact on the reproductive system. Neonatal male and female mice were injected with the GABAB antagonist CGP 55845 (CGP, 1 mg/kg body wt sc) or saline from postnatal day 2 (PND2) to PND6, three times per day (8 AM, 1 PM, and 6 PM). One group was killed on PND6 for collection of blood samples (hormones by radioimmunoassay), brains for gene expression in the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), and ARC micropunches [quantitative PCR (qPCR)] and gonads for qPCR, hormone contents, and histology. A second group of mice was injected with CGP (1 mg/kg body wt sc) or saline from PND2 to PND6, three times per day (8 AM, 1 PM, and 6 PM), and left to grow to adulthood. We measured body weight during development and parameters of sexual differentiation, puberty onset, and estrous cycles. Adult mice were killed, and trunk blood (hormones), brains for qPCR, and gonads for qPCR and hormone contents were obtained. Our most important findings on PND6 include the CGP-induced decrease in ARC Kiss1 and increase in neurokinin B (Tac2) in both sexes; the decrease in AVPV/PeN tyrosine hydroxylase (Th) only in females; the increase in gonad estradiol content in both sexes; and the increase in primordial follicles and decrease in primary and secondary follicles. Neonatally CGP-treated adults showed decreased ARC Kiss1 and ARC gonadotropin-releasing hormone (Gnrh1) and increased ARC glutamic acid decarboxylase 67 (Gad1) only in males; increased ARC GABAB receptor subunit 1 (Gabbr1) in both sexes; and decreased AVPV/PeN Th only in females. We demonstrate that ARC Kiss1 expression is chronically downregulated in males and that the normal sex difference in AVPV/PeN Th expression is abolished. In conclusion, neonatal GABAergic input through GABAB receptors shapes gene expression of factors critical to reproduction.