Adult sex change leads to extensive forebrain reorganization in clownfish.

BACKGROUND: Sexual differentiation of the brain occurs in all major vertebrate lineages but is not well understood at a molecular and cellular level. Unlike most vertebrates, sex-changing fishes have the remarkable ability to change reproductive sex during adulthood in response to social stimuli, offering a unique opportunity to understand mechanisms by which the nervous system can initiate and coordinate sexual differentiation. METHODS: This study explores sexual differentiation of the forebrain using single nucleus RNA-sequencing in the anemonefish Amphiprion ocellaris, producing the first cellular atlas of a sex-changing brain. RESULTS: We uncover extensive sex differences in cell type-specific gene expression, relative proportions of cells, baseline neuronal excitation, and predicted inter-neuronal communication. Additionally, we identify the cholecystokinin, galanin, and estrogen systems as central molecular axes of sexual differentiation. Supported by these findings, we propose a model of sexual differentiation in the conserved vertebrate social decision-making network spanning multiple subtypes of neurons and glia, including neuronal subpopulations within the preoptic area that are positioned to regulate gonadal differentiation. CONCLUSIONS: This work deepens our understanding of sexual differentiation in the vertebrate brain and defines a rich suite of molecular and cellular pathways that differentiate during adult sex change in anemonefish.

This study provides key insights into brain sex differences in sex-changing anemonefish (Amphiprion ocellaris), a species that changes sex in adulthood in response to the social environment. Using single nucleus RNA-sequencing, the study provides the first brain cellular atlas showing sex differences in two crucial reproductive areas: the preoptic area and telencephalon. The research identifies notable sex-differences in cell-type proportions and gene expression, particularly in radial glia and glutamatergic neurons that co-express the neuropeptide cholecystokinin. It also highlights differences in preoptic area neurons likely involved in gonadal regulation. This work deepens our understanding of sexual differentiation of the brain in vertebrates, especially those capable of adult sex change, and illuminates key molecular and cellular beginning and endpoints of the process.

Single-nucleus RNA-seq identifies one galanin neuronal subtype in mouse preoptic hypothalamus activated during recovery from sleep deprivation.

The preoptic area of the hypothalamus (POA) is essential for sleep regulation. However, the cellular makeup of the POA is heterogeneous, and the molecular identities of the sleep-promoting cells remain elusive. To address this question, this study compares mice during recovery sleep following sleep deprivation to mice allowed extended sleep. Single-nucleus RNA sequencing (single-nucleus RNA-seq) identifies one galanin inhibitory neuronal subtype that shows upregulation of rapid and delayed activity-regulated genes during recovery sleep. This cell type expresses higher levels of growth hormone receptor and lower levels of estrogen receptor compared to other galanin subtypes. single-nucleus RNA-seq also reveals cell-type-specific upregulation of purinergic receptor (P2ry14) and serotonin receptor (Htr2a) during recovery sleep in this neuronal subtype, suggesting possible mechanisms for sleep regulation. Studies with RNAscope validate the single-nucleus RNA-seq findings. Thus, the combined use of single-nucleus RNA-seq and activity-regulated genes identifies a neuronal subtype functionally involved in sleep regulation.

The parenting hub of the hypothalamus is a focus of imprinted gene action.

Imprinted genes are subject to germline epigenetic modification resulting in parental-specific allelic silencing. Although genomic imprinting is thought to be important for maternal behaviour, this idea is based on serendipitous findings from a small number of imprinted genes. Here, we undertook an unbiased systems biology approach, taking advantage of the recent delineation of specific neuronal populations responsible for controlling parental care, to test whether imprinted genes significantly converge to regulate parenting behaviour. Using single-cell RNA sequencing datasets, we identified a specific enrichment of imprinted gene expression in a recognised "parenting hub", the galanin-expressing neurons of the preoptic area. We tested the validity of linking enriched expression in these neurons to function by focusing on MAGE family member L2 (Magel2), an imprinted gene not previously linked to parenting behaviour. We confirmed expression of Magel2 in the preoptic area galanin expressing neurons. We then examined the parenting behaviour of Magel2-null(+/p) mice. Magel2-null mothers, fathers and virgin females demonstrated deficits in pup retrieval, nest building and pup-directed motivation, identifying a central role for this gene in parenting. Finally, we show that Magel2-null mothers and fathers have a significant reduction in POA galanin expressing cells, which in turn contributes to a reduced c-Fos response in the POA upon exposure to pups. Our findings identify a novel imprinted gene that impacts parenting behaviour and, moreover, demonstrates the utility of using single-cell RNA sequencing data to predict gene function from expression and in doing so here, have identified a purposeful role for genomic imprinting in mediating parental behaviour.

Detection of galanin receptors in the spinal cord in experimental autoimmune encephalomyelitis.

AIMS: The neuropeptide galanin is a widely distributed neurotransmitter/neuromodulator that regulates a variety of physiological processes and also participates in the regulation of stress responses. The aims of the present study were to investigate the expression of galanin receptors (GalR1, GalR2, GalR3) in the spinal cords in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) using qPCR analysis and to determine GalR1 cellular localization (oligodendrocytes, microglia, astrocytes, ependymal cells, and endothelial cells in the capillaries) by immunohistochemistry. METHODS: Twelve samples from the EAE group and 14 samples from the control group were analyzed. Spinal cords samples were obtained at the peak of the EAE disease. RESULTS: The GalR1 mRNA level was significantly decreased in the EAE mice compared with the controls (P=0.016), whereas the mRNA levels of GalR2 and GalR3 were not significantly different for the EAE and the control mice. No significant correlations were found between the severity of the EAE disease and the mRNA levels of GalR1, GalR2 and GalR3. Immunochemical detection of the GalR1 revealed its expression in the ependymal and endothelial cells. Additionally, a weak GalR1 immunoreactivity was occasionally detected in the oligodendrocytes. CONCLUSION: This study provides additional evidence of galanin involvement in EAE pathophysiology, but this has to be further investigated.

Cardioprotective effects of neuropeptide galanin: Focusing on its roles against diabetic heart.

Following an unprecedented rise in the number of the aged, the incidence of age-related diseases, such as diabetes and cardiovascular disease, is consequently increasing in the world. Type 2 diabetes mellitus (T2DM) is associated with excess cardiovascular morbidity and mortality. The diabetic heart is characterized by increased cardiomyocyte stiffness and fibrotic changes. Despite many factors resulting in cardiomyocyte injury and dysfunction in diabetes, insulin resistance is still a critical etiology of diabetic cardiomyopathy. Preclinical and clinical studies have revealed an intriguing role for galanin in the pathogenesis of insulin resistance and diabetic heart disease. A significant change in plasma galanin levels occurred in patients suffering from type 2 diabetes or cardiomyocyte injury. In turn, galanin may also distinctly mitigate hyperglycemia and insulin resistance in diabetes as well as increase glucose metabolism and mitochondrial biogenesis in cardiac muscle. Here, we critically review current data about the multivariate relationship among galanin, insulin resistance, and cardiac muscle to comprehensively evaluate the protective role of galanin and its receptors for the diabetic heart and to determine whether galanin receptor 2 agonists potentially represent a feasible way to treat diabetic cardiomyopathy in the future.

Characterization of spexin (SPX) in chickens: molecular cloning, functional analysis, tissue expression and its involvement in appetite regulation.

Spexin (SPX) is a conservative tetradecapeptide which has been proven to participate in multiple physiological processes, including anxiety, feed intake, and energy metabolism in fish and mammals. However, whether SPX exists and functions in birds remain largely unknown. Using chicken (c-) as a model, the full-length cDNA encoding cSPX precursor was cloned, and it was predicted to generate a mature peptide with 14 amino acids conserved across vertebrates. The pGL4-SRE-luciferase reporter system-based functional analysis demonstrated that cSPX was effective in activating chicken galanin type Ⅱ receptor (cGALR2), cGALR2-like receptor (cGALR2L) and galanin type Ⅲ receptor (cGALR3), thus to stimulate intracellular MAPK/ERK signaling pathway. Quantitative real-time PCR revealed that SPX was widely expressed in chicken tissues, especially abundant in the central nervous system, pituitary, testes, and pancreas. Interestingly, it was noted that chicken hypothalamic SPX mRNA could be up-regulated by 24-h and 36-h fasting, heralding its latent capacity in appetite regulation. In accordance with this speculation, peripheral injection of cSPX was proved to be functional in reducing feed intake of 3-wk-old chicks. Furthermore, we found that cSPX could reduce the expression of AgRP and MCH, with a concurrent rise in CART1 mRNA level in the hypothalamic of chicks. Collectively, our findings not only provide the evidences that SPX can act as a satiety factor by orchestrating the expression of key feeding regulators in the chicken hypothalamus but also help to facilitate a better understanding of its functional evolution across vertebrates.

Molecular Changes in the Dorsal Root Ganglion during the Late Phase of Peripheral Nerve Injury-induced Pain in Rodents: A Systematic Review.

The dorsal root ganglion is widely recognized as a potential target to treat chronic pain. A fundamental understanding of quantitative molecular and genomic changes during the late phase of pain is therefore indispensable. The authors performed a systematic literature review on injury-induced pain in rodent dorsal root ganglions at minimally 3 weeks after injury. So far, slightly more than 300 molecules were quantified on the protein or messenger RNA level, of which about 60 were in more than one study. Only nine individual sequencing studies were performed in which the most up- or downregulated genes varied due to heterogeneity in study design. Neuropeptide Y and galanin were found to be consistently upregulated on both the gene and protein levels. The current knowledge regarding molecular changes in the dorsal root ganglion during the late phase of pain is limited. General conclusions are difficult to draw, making it hard to select specific molecules as a focus for treatment.

Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is caused by allergic reaction to allergens such as pollen. Galanin (GAL), a neuropeptide that regulates inflammatory processes, is widely expressed in the central and peripheral nervous systems. Although neuropeptides are implicated in arthritis and chemically induced ileitis, their roles in AR remain unclear. METHODS: We developed a murine model of AR and generated control, systemic sensitization, mild AR, and severe AR groups. We examined GAL and GAL receptor (GALR) mRNA and protein levels and localization patterns in each group using reverse transcription PCR, western blotting, and immunohistochemical analyses. Additionally, we evaluated the effects of M871, a GALR2 antagonist, on mice with severe AR. RESULTS: Gal and Galr2 are expressed in nasal mucosa and brain (control) samples from control and AR mice. GAL and GALR2 were expressed at similar levels and localized to ciliated epithelial and submucosal gland cells of the nasal mucosa in all four groups. Intranasal M871 administration significantly reduced the incidence of nose rubbing behaviors and sneezing (p < 0.001 in 30 min, respectively) in severe AR mice relative to that in controls. Mechanistically, we postulate that GALR2 is expressed in B cells, and M871 administration reduces IgE production, as well as the number of B cells in tissues. CONCLUSIONS: GAL signaling may not change progressively with increasing nasal sensitization, suggesting that this signaling process exacerbates, rather than directly trigger, AR. GAL-GALR2 signaling likely mediates AR development, suggesting that its inhibition represents a novel therapeutic strategy for AR.

The Role of Galanin during Bacterial Infection in Larval Zebrafish.

Galanin is a peptide that is conserved among different species and plays various roles in an organism, although its entire role is not completely understood. For many years, galanin has been linked mainly with the neurotransmission in the nervous system; however, recent reports underline its role in immunity. Zebrafish (Danio rerio) is an intensively developing animal model to study infectious diseases. In this study, we used larval zebrafish to determine the role of galanin in bacterial infection. We showed that knockout of galanin in zebrafish leads to a higher bacterial burden and mortality during Mycobacterium marinum and Staphylococcus aureus infection, whereas administration of a galanin analogue, NAX 5055, improves the ability of fish to control the infection caused by both pathogens. Moreover, the transcriptomics data revealed that a lower number of genes were regulated in response to mycobacterial infection in gal-/- mutants compared with their gal+/+ wild-type counterparts. We also found that galanin deficiency led to significant changes in immune-related pathways, mostly connected with cytokine and chemokine functions. The results show that galanin acts not only as a neurotransmitter but is also involved in immune response to bacterial infections, demonstrating the complexity of the neuroendocrine system and its possible connection with immunity.

Parafacial neurons in the human brainstem express specific markers for neurons of the retrotrapezoid nucleus.

The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.

Galanin and prolactin expression in relation to parental care in two sympatric cichlid species from Lake Tanganyika.

Our understanding of the hormonal mechanisms underlying parental care mainly stems from research on species with uniparental care. Far less is known about the physiological changes underlying motherhood and fatherhood in biparental caring species. Here, using two biparental caring cichlid species (Neolamprologus caudopunctatus and Neolamprologus pulcher), we explored the relative gene-expression levels of two genes implicated in the control of parental care, galanin (gal) and prolactin (prl). We investigated whole brain gene expression levels in both, male and female caring parents, as well as in non-caring individuals of both species. Caring males had higher prl and gal mRNA levels compared to caring females in both fish species. Expression of gal was highest when young were mobile and the need for parental defense was greatest and gal was lowest during the more stationary egg tending phase in N. caudopunctatus. The onset of parenthood was associated with lower expression of prl and higher expression of gal in N. pulcher, but this pattern was not observed in N. caudopunctatus. Our study demonstrates that gal gene expression is correlated with changes in parental care in two biparental cichlid species and extends both knowledge and taxonomic coverage of the possible neurogenetic mechanisms underlying parental care.

Genomic and transcriptomic characterization of desmoplastic small round cell tumors.

Desmoplastic small round cell tumor (DSRCT) is a highly aggressive soft tissue tumor primarily affecting children and young adults. Most cases display a pathognomonic EWSR1-WT1 gene fusion, presumably constituting the primary driver event. Little is, however, known about secondary genetic changes that may affect tumor progression. We here studied 25 samples from 19 DSRCT patients using single nucleotide polymorphism arrays and found that all samples had copy number alterations. The most common imbalances were gain of chromosomes/chromosome arms 1/1q and 5/5p and loss of 6/6q and 16/16q, all occurring in at least eight of the patients. Five cases showed homozygous deletions, affecting a variety of known tumor suppressor genes, for example, CDKN2A and NF1. As almost all patients died of their disease, the impact of individual imbalances on survival could not be evaluated. Global gene expression analysis using mRNA sequencing on fresh-frozen samples from seven patients revealed a distinct transcriptomic profile, with enrichment of genes involved in neural differentiation. Two genes - GJB2 and GAL - that showed higher expression in DSRCT compared to control tumors could be further investigated for their potential as diagnostic markers at the protein level.

Co-released norepinephrine and galanin act on different timescales to promote stress-induced anxiety-like behavior.

Both the noradrenergic and galaninergic systems have been implicated in stress-related neuropsychiatric disorders, and these two neuromodulators are co-released from the stress-responsive locus coeruleus (LC); however, the individual contributions of LC-derived norepinephrine (NE) and galanin to behavioral stress responses are unclear. Here we aimed to disentangle the functional roles of co-released NE and galanin in stress-induced behavior. We used foot shock, optogenetics, and behavioral pharmacology in wild-type (WT) mice and mice lacking either NE (Dbh-/-) or galanin (GalcKO-Dbh) specifically in noradrenergic neurons to isolate the roles of these co-transmitters in regulating anxiety-like behavior in the elevated zero maze (EZM) either immediately or 24 h following stress. Foot shock and optogenetic LC stimulation produced immediate anxiety-like behavior in WT mice, and the effects of foot shock persisted for 24 h. NE-deficient mice were resistant to the anxiogenic effects of acute stress and optogenetic LC stimulation, while mice lacking noradrenergic-derived galanin displayed typical increases in anxiety-like behavior. However, when tested 24 h after foot shock, both Dbh-/- and GalcKO-Dbh mice lacked normal expression of anxiety-like behavior. Pharmacological rescue of NE, but not galanin, in knockout mice during EZM testing was anxiogenic. In contrast, restoring galanin, but not NE, signaling during foot shock normalized stress-induced anxiety-like behavior 24 h later. These results indicate that NE and noradrenergic-derived galanin play complementary, but distinguishable roles in behavioral responses to stress. NE is required for the expression of acute stress-induced anxiety, while noradrenergic-derived galanin mediates the development of more persistent responses following a stressor.

Galanin isoforms by alternative splicing: Structure, expression, and immunohistochemical location in the gonads of European sea bass.

Galanin (Gal) is a neuropeptide with multiple functions that is widely expressed in the central and peripheral nervous systems of vertebrates. Anatomical and functional evidence suggests a possible role in regulating reproduction in fishes. To test this possibility, we have isolated and characterized two gal alternative transcripts in European sea bass (Dicentrarchus labrax) that encode two prepropeptides, respectively of 29 (gal_MT853221) and 53 (gal_MT853222) amino acids. The two gal transcripts are highly expressed in brain, pituitary and gonads, and appear to be differentially regulated in males and females. In males, gal_MT853222 in the hypothalamus and gal_MT853221 in the pituitary were downregulated with the progression of spermatogenesis (stages I-III). Both transcripts are downregulated in testicles of 1-year (precocious) and 2-year spermiating males compared to immature fish of the same age. Gal peptides and receptors are expressed throughout ovarian development in the hypothalamic-pituitary-gonadal (HPG) axis of females. In the testis, immunoreactive Gal-29 and Gal-53 peptides were detected in blood vessels and Leydig cells during the spermatogenesis stages I-III but Gal immunostaining was barely undetected in more advanced stages. In the ovary, both peptides localized in interstitial cells and blood vessels and in theca cells surrounding the maturing oocytes. The immunolocalization of galanin in Leydig and theca cells suggests a possible role in steroid production regulation. The different pattern of gal expression and Gal localization in the testis and ovary may suggest the possibility that androgens and estrogens may also regulate Gal gene transcription and translation. Altogether, this study showed evidence for the possible involvement of locally produced Gal in gametogenesis and that its production is differentially regulated in male and female gonads.

Chronic Environmental or Genetic Elevation of Galanin in Noradrenergic Neurons Confers Stress Resilience in Mice.

The neuropeptide galanin has been implicated in stress-related neuropsychiatric disorders in humans and rodent models. While pharmacological treatments for these disorders are ineffective for many individuals, physical activity is beneficial for stress-related symptoms. Galanin is highly expressed in the noradrenergic system, particularly the locus coeruleus (LC), which is dysregulated in stress-related disorders and activated by exercise. Galanin expression is elevated in the LC by chronic exercise, and blockade of galanin transmission attenuates exercise-induced stress resilience. However, most research on this topic has been done in rats, so it is unclear whether the relationship between exercise and galanin is species specific. Moreover, use of intracerebroventricular (ICV) galanin receptor antagonists in prior studies precluded defining a causal role for LC-derived galanin specifically. Therefore, the goals of this study were twofold. First, we investigated whether physical activity (chronic wheel running) increases stress resilience and galanin expression in the LC of male and female mice. Next, we used transgenic mice that overexpress galanin in noradrenergic neurons (Gal OX) to determine how chronically elevated noradrenergic-derived galanin, alone, alters anxiogenic-like responses to stress. We found that three weeks of ad libitum access to a running wheel in their home cage increased galanin mRNA in the LC of mice, which was correlated with and conferred resilience to stress. The effects of exercise were phenocopied by galanin overexpression in noradrenergic neurons, and Gal OX mice were resistant to the anxiogenic effect of optogenetic LC activation. These findings support a role for chronically increased noradrenergic galanin in mediating resilience to stress.SIGNIFICANCE STATEMENT Understanding the neurobiological mechanisms underlying behavioral responses to stress is necessary to improve treatments for stress-related neuropsychiatric disorders. Increased physical activity is associated with stress resilience in humans, but the neurobiological mechanisms underlying this effect are not clear. Here, we investigate a potential causal mechanism of this effect driven by the neuropeptide galanin from the main noradrenergic nucleus, the locus coeruleus (LC). We show that chronic voluntary wheel running in mice increases stress resilience and increases galanin expression in the LC. Furthermore, we show that genetic overexpression of galanin in noradrenergic neurons causes resilience to a stressor and the anxiogenic effects of optogenetic LC activation. These findings support a role for chronically increased noradrenergic galanin in mediating resilience to stress.

RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake.

BACKGROUND: Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the transcriptional regulation of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time. RESULTS: RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(-) cells and to bulk VLPO samples. Gene set enrichment analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep. CONCLUSION: Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

Male-predominant galanin mediates androgen-dependent aggressive chases in medaka.

Recent studies in mice demonstrate that a subset of neurons in the medial preoptic area (MPOA) that express galanin play crucial roles in regulating parental behavior in both sexes. However, little information is available on the function of galanin in social behaviors in other species. Here, we report that, in medaka, a subset of MPOA galanin neurons occurred nearly exclusively in males, resulting from testicular androgen stimulation. Galanin-deficient medaka showed a greatly reduced incidence of male-male aggressive chases. Furthermore, while treatment of female medaka with androgen induced male-typical aggressive acts, galanin deficiency in these females attenuated the effect of androgen on chases. Given their male-biased and androgen-dependent nature, the subset of MPOA galanin neurons most likely mediate androgen-dependent male-male chases. Histological studies further suggested that variability in the projection targets of the MPOA galanin neurons may account for the species-dependent functional differences in these evolutionarily conserved neural substrates.

Epigenetic upregulation of galanin-like peptide mediates deoxynivalenol induced-growth inhibition in pituitary cells.

Deoxynivalenol (DON) is an unavoidable contaminant in human food, animal feeds, and agricultural products. Growth retardation in children caused by extensive DON pollution has become a global problem that cannot be ignored. Previous studies have shown that DON causes stunting in children through intestinal dysfunction, insulin-like growth factor-1 (IGF-1) axis disorder and peptide YY (PYY). Galanin-like peptide (GALP) is an important growth regulator, but its role in DON-induced growth retardation is unclear. In this study, we report the important role of GALP during DON-induced growth inhibition in the rat pituitary tumour cell line GH3. DON was found to increase the expression of GALP through hypomethylationin the promoter region of the GALP gene and upregulate the expression of proinflammatory factors, while downregulate the expression of growth hormone (GH). Furthermore, GALP overexpression promoted proinflammatory cytokines, including TNF-α, IL-1ß, IL-11 and IL-6, and further reduced cell viability and cell proliferation, while the inhibitory effect of GALP was the opposite. The expression of GALP and insulin like growth factor binding protein acid labile subunit (IGFALS) showed the opposite trend, which was the potential reason for the regulation of cell proliferation by GALP. In addition, GALP has anti-apoptotic effects, which could not eliminate the inflammatory damage of cells, thus aggravating cell growth inhibition. The present findings provide new mechanistic insights into the toxicity of DON-induced growth retardation and suggest a therapeutic potential of GALP in DON-related diseases.

Galanin System in Human Glioma and Pituitary Adenoma.

Expression of neuropeptides and their corresponding receptors has been demonstrated in different cancer types, where they can play a role in tumor cell growth, invasion, and migration. Human galanin (GAL) is a 30-amino-acid regulatory neuropeptide which acts through three G protein-coupled receptors, GAL1-R, GAL2-R, and GAL3-R that differ in their signal transduction pathways. GAL and galanin receptors (GALRs) are expressed by different tumors, and direct involvement of GAL in tumorigenesis has been shown. Despite its strong expression in the central nervous system (CNS), the role of GAL in CNS tumors has not been extensively studied. To date, GAL peptide expression, GAL receptor binding and mRNA expression have been reported in glioma, meningioma, and pituitary adenoma. However, data on the cellular distribution of GALRs are sparse. The aim of the present study was to examine the expression of GAL and GALRs in different brain tumors by immunohistochemistry. Anterior pituitary gland (n = 7), pituitary adenoma (n = 9) and glioma of different WHO grades I-IV (n = 55) were analyzed for the expression of GAL and the three GALRs with antibodies recently extensively validated for specificity. While high focal GAL immunoreactivity was detected in up to 40% of cells in the anterior pituitary gland samples, only one pituitary adenoma showed focal GAL expression, at a low level. In the anterior pituitary, GAL1-R and GAL3-R protein expression was observed in up to 15% of cells, whereas receptor expression was not detected in pituitary adenoma. In glioma, diffuse and focal GAL staining was noticed in the majority of cases. GAL1-R was observed in eight out of nine glioma subtypes. GAL2-R immunoreactivity was not detected in glioma and pituitary adenoma, while GAL3-R expression was significantly associated to high-grade glioma (WHO grade IV). Most interestingly, expression of GAL and GALRs was observed in tumor-infiltrating immune cells, including neutrophils and glioma-associated macrophages/microglia. The presence of GALRs on tumor-associated immune cells, especially macrophages, indicates that GAL signaling contributes to homeostasis of the tumor microenvironment. Thus, our data indicate that GAL signaling in tumor-supportive myeloid cells could be a novel therapeutic target.

Elimination of galanin synthesis in noradrenergic neurons reduces galanin in select brain areas and promotes active coping behaviors.

Accumulating evidence indicates that disruption of galanin signaling is associated with neuropsychiatric disease, but the precise functions of this neuropeptide remain largely unresolved due to lack of tools for experimentally disrupting its transmission in a cell type-specific manner. To examine the function of galanin in the noradrenergic system, we generated and crossed two novel knock-in mouse lines to create animals lacking galanin specifically in noradrenergic neurons (GalcKO-Dbh). We observed reduced levels of galanin peptide in pons, hippocampus, and prefrontal cortex of GalcKO-Dbh mice, indicating that noradrenergic neurons are a significant source of galanin to those brain regions, while midbrain and hypothalamic galanin levels were comparable to littermate controls. In these same brain regions, we observed no change in levels of norepinephrine or its major metabolite at baseline or after an acute stressor, suggesting that loss of galanin does not affect noradrenergic synthesis or turnover. GalcKO-Dbh mice had normal performance in tests of depression, learning, and motor-related behavior, but had an altered response in some anxiety-related tasks. Specifically, GalcKO-Dbh mice showed increased marble and shock probe burying and had a reduced latency to eat in a novel environment, indicative of a more proactive coping strategy. Together, these findings indicate that noradrenergic neurons provide a significant source of galanin to discrete brain areas, and noradrenergic-specific galanin opposes adaptive coping responses.