Phenotypic correlates between clock genes and phenology among populations of Diederik cuckoo, Chrysococcyx caprius.

The Diederik cuckoo, Chrysococcyx caprius, is a small Afrotropical bird in the family Cuculidae. It is taxonomically related to 13 other species within the genus Chrysococcyx and is migratory in sub-Saharan Africa. It has a unique breeding behaviour of being a brood parasite: Breeding pairs lay their eggs in the nests of a host species and hatchlings expel the eggs of the host species. The aim of the present study was to investigate diversity in two circadian clock genes, Clock and Adcyap1, to probe for a relationship between genetic polymorphisms and their role in circannual timing and habitat selection (phenology) in intra-African migrants. DNA extracted from blood was used for the PCR amplification and sequencing of clock genes in 30 Diederik cuckoos. Three alleles were detected for Clock with similar genotypes between individuals from the Northern and Southern breeding ranges while 10 alleles were detected for Adcyap1, having shorter alleles in the North and longer alleles in the South. Population genetic analyses, including allele frequency and zygosity analysis, showed distinctly higher frequencies for the most abundant Clock allele, containing 10 polyglutamine repeats, as well as a high degree of homozygosity. In contrast, all individuals were heterozygous for Adcyap1 and alleles from both regions showed distinct differences in abundance. Comparisons between both clock genes and phenology found several phenotypic correlations. This included evidence of a relationship between the shorter alleles and habitat selection as well as a relationship between longer alleles and timing. In both instances, evidence is provided that these effects may be sex-specific. Given that these genes drive some of the synchronicity between environments and the life cycles of birds, they provide valuable insight into the fitness of species facing global challenges including climate change, urbanisation and expanding agricultural practices.

Developmental adcyap1b loss leads to hemorrhage, disrupted hemostasis, and a blood coagulation cascade in zebrafish.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide is a neuropeptide with diverse roles in biological processes. Its involvement in the blood coagulation cascade is unclear. OBJECTIVES: This study unraveled adcyap1b's role in blood coagulation using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 in zebrafish. Effects were validated via adcyap1b knockdown. Gene expression changes in adcyap1b mutants were explored, linking them to clotting disorders. An analysis of proca gene splicing illuminated its role in adcyap1b-related anticoagulation deficiencies. METHODS: Zebrafish were genetically modified using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to induce adcyap1b knockout. Morpholino-mediated gene knockdown was employed for validation. Expression levels of coagulation factors, anticoagulant proteins, and fibrinolytic system genes were assessed in adcyap1b mutant zebrafish. Alternative splicing of proca gene was analyzed. RESULTS: Adcyap1b mutant zebrafish exhibited severe hemorrhage, clotting disorders, and disrupted blood coagulation. Morpholino-mediated knockdown replicated observed phenotypes. Downregulation in transcripts related to coagulation factors V and IX, anticoagulation protein C, and plasminogen was observed. Abnormal alternative splicing of the proca gene was identified, providing a mechanistic explanation for anticoagulation system deficiencies. CONCLUSION: Adcyap1b plays a crucial role in maintaining zebrafish blood coagulation and hemostasis. Its influence extends to the regulation of procoagulant and anticoagulant pathways, with abnormal alternative splicing contributing to observed deficiencies. These findings unveil a novel aspect of adcyap1b function, offering potential insights into similar processes in mammalian systems.

Genetic Contributors to PTSD: the Role of SNVs, Gene Interactions and Haplotypes for Developing PTSD Prevention Measures. A Comprehensive Review.

BACKGROUND: Post-traumatic stress disorder (PTSD) is a trauma- or stressor-related mental health condition with high socioeconomic burden. We aimed in this review to identify promising genetic markers predisposing for PTSD, which might serve in the design subsequent studies aiming to develop PTSD prevention and remediation measures. SUBJECTS AND METHODS: Our search queries in the PubMed database yielded 547 articles, of which 20 met our inclusion criteria for further analysis: published between 2018 and 2022, original research, containing molecular-genetic and statistical data, containing diagnosis verification methods, PTSD as a primary condition, and a sample of at least 60 patients. RESULTS: Among the 20 analyzed studies were reports of significant associations between PTSD and: FKBP5 variants rs9470080, regardless of the C or T allele; two FKBP5 haplotypes (A-G-C-C and A-G-C-T); gene-gene DRDхANNK1-COMT (rs1800497 × rs6269) and OXTR-DRD2 (rs2268498 × rs1801028); C-allele of CRHR1 (rs1724402). Other findings, such as the association of FKBP5 haplotypes (A-G-C-C, A-G-C-T) and the FKBP5-CRHR1 genotype, were of lesser statistical significance and less extensively studied. CONCLUSIONS: Although our literature analysis implicates certain genetic factors in PTSD, our understanding of the polygenic nature underlying the disorder remains limited, especially considering the hitherto underexplored epigenetic mechanisms. Future research endeavors should prioritize exploring these aspects to provide a more nuanced understanding of PTSD and its genetic underpinnings.

Candidate gene polymorphisms are linked to dispersive and migratory behaviour: Searching for a mechanism behind the "paradox of the great speciators".

The "paradox of the great speciators" has puzzled evolutionary biologists for over half a century. A great speciator requires excellent dispersal propensity to explain its occurrence on multiple islands, but reduced dispersal ability to explain its high number of subspecies. A rapid reduction in dispersal ability is often invoked to solve this apparent paradox, but a proximate mechanism has not been identified yet. Here, we explored the role of six genes linked to migration and animal personality differences (CREB1, CLOCK, ADCYAP1, NPAS2, DRD4, and SERT) in 20 South Pacific populations of silvereye (Zosterops lateralis) that range from highly sedentary to partially migratory, to determine if genetic variation is associated with dispersal propensity and migration. We detected genetic associations in three of the six genes: (i) in a partial migrant population, migrant individuals had longer microsatellite alleles at the CLOCK gene compared to resident individuals from the same population; (ii) CREB1 displayed longer average microsatellite allele lengths in recently colonized island populations (<200 years), compared to evolutionarily older populations. Bayesian broken stick regression models supported a reduction in CREB1 length with time since colonization; and (iii) like CREB1, DRD4 showed differences in polymorphisms between recent and old colonizations but a larger sample is needed to confirm. ADCYAP1, SERT, and NPAS2 were variable but that variation was not associated with dispersal propensity. The association of genetic variants at three genes with migration and dispersal ability in silvereyes provides the impetus for further exploration of genetic mechanisms underlying dispersal shifts, and the prospect of resolving a long-running evolutionary paradox through a genetic lens.

Does age matter in genetics? The role of ADCYAP1R1 in sex-specific risk for posttraumatic stress disorder in trauma-exposed preadolescent children.

Following trauma exposure, children are a vulnerable population and at risk for developing posttraumatic stress disorder (PTSD). A large body of research has demonstrated the impactful role of genetics in vulnerability for PTSD in adult samples; yet very little research has examined genetic risk for PTSD in children. It is unknown whether genetic associations identified in adults are true for children; replication of findings from adult samples is needed in child samples. This study investigated an estrogen-responsive variant (ADCYAP1R1) that has been well-established to confer sex-specific risk for PTSD in adult samples, but is hypothesized to function differently in children, potentially due to pubertal changes in the estrogen system. Participants were children (n = 87; 57% female) ages 7 to 11 exposed to a natural disaster. Participants were assessed for trauma exposure and symptoms of PTSD. Participants provided a saliva sample, which was genotyped for the ADCYAP1R1 rs2267735 variant. In girls, the ADCYAP1R1 CC genotype was associated with PTSD (OR = 7.30). In boys, evidence for the opposite effect emerged, with the CC genotype attenuating risk for PTSD (OR = 8.25). When investigating specific PTSD symptom clusters, an association between ADCYAP1R1 and arousal emerged. This study is the first to investigate the relationship between ADCYAP1R1 and PTSD in trauma-exposed children. Findings for girls mirrored prior research on adult women, whereas findings for boys diverged from prior research on adult men. These potential differences between children and adults in genetic vulnerability for PTSD underscore the need for more genetic studies in child samples.

Time trees and clock genes: a systematic review and comparative analysis of contemporary avian migration genetics.

Timing is a crucial aspect for survival and reproduction in seasonal environments leading to carefully scheduled annual programs of migration in many species. But what are the exact mechanisms through which birds (class: Aves) can keep track of time, anticipate seasonal changes, and adapt their behaviour? One proposed mechanism regulating annual behaviour is the circadian clock, controlled by a highly conserved set of genes, collectively called 'clock genes' which are well established in controlling the daily rhythmicity of physiology and behaviour. Due to diverse migration patterns observed within and among species, in a seemingly endogenously programmed manner, the field of migration genetics has sought and tested several candidate genes within the clock circuitry that may underlie the observed differences in breeding and migration behaviour. Among others, length polymorphisms within genes such as Clock and Adcyap1 have been hypothesised to play a putative role, although association and fitness studies in various species have yielded mixed results. To contextualise the existing body of data, here we conducted a systematic review of all published studies relating polymorphisms in clock genes to seasonality in a phylogenetically and taxonomically informed manner. This was complemented by a standardised comparative re-analysis of candidate gene polymorphisms of 76 bird species, of which 58 are migrants and 18 are residents, along with population genetics analyses for 40 species with available allele data. We tested genetic diversity estimates, used Mantel tests for spatial genetic analyses, and evaluated relationships between candidate gene allele length and population averages for geographic range (breeding- and non-breeding latitude), migration distance, timing of migration, taxonomic relationships, and divergence times. Our combined analysis provided evidence (i) of a putative association between Clock gene variation and autumn migration as well as a putative association between Adcyap1 gene variation and spring migration in migratory species; (ii) that these candidate genes are not diagnostic markers to distinguish migratory from sedentary birds; and (iii) of correlated variability in both genes with divergence time, potentially reflecting ancestrally inherited genotypes rather than contemporary changes driven by selection. These findings highlight a tentative association between these candidate genes and migration attributes as well as genetic constraints on evolutionary adaptation.

The main effect and gene-environment interaction effect of the ADCYAP1R1 polymorphism rs2267735 on the course of posttraumatic stress disorder symptoms-A longitudinal analysis.

Background: Many studies have been performed to investigate the association between the ADCYAP1R1 polymorphism rs2267735 and posttraumatic stress disorder (PTSD), but the results have been inconsistent, and the way in which this gene affects the course of PTSD has not been widely investigated. Thus, a longitudinal study of the course (development trajectory) of PTSD is needed. Methods: In this study, we performed a longitudinal analysis of rs2267735 in 1017 young, trauma-exposed Chinese people (549 females and 468 males, ranging from 7 to 11 years old). At four time points after trauma exposure (2.5, 3.5, 4.5, and 5.5 years), we measured PTSD symptoms with the University of California, Los Angeles PTSD Reaction Index (PTSD-RI) for DSM-IV (Child Version). We employed a latent growth model (LGM) for the longitudinal data to test the association between rs2267735 (main and gene-environment interaction effects) and the course of PTSD symptoms. Results: The results of LGM showed that the gene-environment interaction (rs2267735 × trauma exposure) effects were associated with PTSD symptoms in girls at 2.5 years (ß = -0.291 and P = 0.013 for LGM intercept). The gene-environment interaction (rs2267735 × trauma exposure) effect was also correlated with PTSD symptoms in girls at 3.5 and 4.5 years (ß = -0.264 and P = 0.005; ß = -0.217 and P = 0.013). Conclusion: Our study revealed that the gene-environment interaction of the ADCYAP1R1 polymorphism rs2267735 is associated with PTSD symptoms in girls at 2.5 years and that the effects may be stable over time and not related to the PTSD symptom recovery rate. This is the first study to detect the how the ADCYAP1R1 gene affects the course of PTSD after trauma exposure in a longitudinal view.

PACAP-expressing neurons in the lateral habenula diminish negative emotional valence.

The lateral habenula (LHb) is a small, bilateral, epithalamic nucleus which processes aversive information. While primarily glutamatergic, LHb neurons express genes coding for many neuropeptides, such as Adcyap1 the gene encoding pituitary adenylate cyclase-activating polypeptide (PACAP), which itself has been associated with anxiety and stress disorders. Using Cre-dependent viral vectors, we targeted and characterized these neurons based on their anatomical projections and found that they projected to both the raphe and rostromedial tegmentum but only weakly to ventral tegmental area. Using RiboTag to capture ribosomal-associated mRNA from these neurons and reanalysis of existing single cell RNA sequencing data, we did not identify a unique molecular phenotype that characterized these PACAP-expressing neurons in LHb. In order to understand the function of these neurons, we conditionally expressed hM3 Dq DREADD selectively in LHb PACAP-expressing neurons and chemogenetically excited these neurons during behavioral testing in the open field test, contextual fear conditioning, sucrose preference, novelty suppressed feeding, and conditioned place preference. We found that Gq activation of these neurons produce behaviors opposite to what is expected from the LHb as a whole-they decreased anxiety-like and fear behavior and produced a conditioned place preference. In conclusion, PACAP-expressing neurons in LHb represents a molecularly diverse population of cells that oppose the actions of the remainder of LHb neurons by being rewarding or diminishing the negative consequences of aversive events.

At the heart of the interoception network: Influence of the parasubthalamic nucleus on autonomic functions and motivated behaviors.

The parasubthalamic nucleus (PSTN), a small nucleus located on the lateral edge of the posterior hypothalamus, has emerged in recent years as a highly interconnected node within the network of brain regions sensing and regulating autonomic function and homeostatic needs. Furthermore, the strong integration of the PSTN with extended amygdala circuits makes it ideally positioned to serve as an interface between interoception and emotions. While PSTN neurons are mostly glutamatergic, some of them also express neuropeptides that have been associated with stress-related affective and motivational dysfunction, including substance P, corticotropin-releasing factor, and pituitary adenylate-cyclase activating polypeptide. PSTN neurons respond to food ingestion and anorectic signals, as well as to arousing and distressing stimuli. Functional manipulation of defined pathways demonstrated that the PSTN serves as a central hub in multiple physiologically relevant networks and is notably implicated in appetite suppression, conditioned taste aversion, place avoidance, impulsive action, and fear-induced thermoregulation. We also discuss the putative role of the PSTN in interoceptive dysfunction and negative urgency. This review aims to synthesize the burgeoning preclinical literature dedicated to the PSTN and to stimulate interest in further investigating its influence on physiology and behavior.

Targeted deletion of PAC1 receptors in retinal neurons enhances neuron loss and axonopathy in a model of multiple sclerosis and optic neuritis.

Chronic inflammation drives synaptic loss in multiple sclerosis (MS) and is also commonly observed in other neurodegenerative diseases. Clinically approved treatments for MS provide symptomatic relief but fail to halt neurodegeneration and neurological decline. Studies in animal disease models have demonstrated that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) exhibits anti-inflammatory, neuroprotective and regenerative properties. Anti-inflammatory actions appear to be mediated primarily by two receptors, VPAC1 and VPAC2, which also bind vasoactive intestinal peptide (VIP). Pharmacological experiments indicate that another receptor, PAC1 (ADCYAP1R1), which is highly selective for PACAP, provides protection to neurons, although genetic evidence and other mechanistic information is lacking. To determine if PAC1 receptors protect neurons in a cell-autonomous manner, we used adeno-associated virus (AAV2) to deliver Cre recombinase to the retina of mice harboring floxed PAC1 alleles. Mice were then subjected to chronic experimental autoimmune encephalomyelitis (EAE), a disease model that recapitulates major clinical and pathological features of MS and associated optic neuritis. Unexpectedly, deletion of PAC1 in naïve mice resulted in a deficit of retinal ganglionic neurons (RGNs) and their dendrites, suggesting a homeostatic role of PAC1. Moreover, deletion of PAC1 resulted in increased EAE-induced loss of a subpopulation of RGNs purported to be vulnerable in animal models of glaucoma. Increased axonal pathology and increased secondary presence of microglia/macrophages was also prominently seen in the optic nerve. These findings demonstrate that neuronal PAC1 receptors play a homeostatic role in protecting RGNs and directly protects neurons and their axons against neuroinflammatory challenge. SIGNIFICANCE STATEMENT: Chronic inflammation is a major component of neurodegenerative diseases and plays a central role in multiple sclerosis (MS). Current treatments for MS do not prevent neurodegeneration and/or neurological decline. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to have anti-inflammatory, neuroprotective and regenerative properties but the cell type- and receptor-specific mechanisms are not clear. To test whether the protective effects of PACAP are direct on the PAC1 receptor subtype on neurons, we delete PAC1 receptors from neurons and investigate neuropathologigical changes in an animal model of MS. The findings demonstrate that PAC1 receptors on neurons play a homeostatic role in maintaining neuron health and can directly protect neurons and their axons during neuroinflammatory disease.

The ADCYAP1R1 Gene Is Correlated With Posttraumatic Stress Disorder Symptoms Through Diverse Epistases in a Traumatized Chinese Population.

The adenylate cyclase activating polypeptide 1 (pituitary) receptor (ADCYAP1R1) gene is associated with the hypothalamic-pituitary-adrenal (HPA) axis, which controls stress responses. The single-nucleotide polymorphism of ADCYAP1R1, rs2267735, has been investigated in many studies to test its association with posttraumatic stress disorder (PTSD), but the results have not been consistent. It is worth systematically exploring the role of rs2267735 in PTSD development. In this study, we analyzed rs2267735 in 1,132 trauma-exposed Chinese individuals (772 females and 360 males). We utilized the PTSD checklist for DSM-5 (PCL-5) to measure the PTSD symptoms. Then, we analyzed the main, G × E (rs2267735 × trauma exposure), and G × G (with other HPA axis gene polymorphisms) effects of rs2267735 on PTSD severity (total symptoms). There were no significant main or G × E effects (P > 0.05). The G × G ADCYAP1R1-FKBP5 interaction (rs2267735 × rs1360780) was associated with PTSD severity (beta = -1.31 and P = 0.049) based on all subjects, and the G × G ADCYAP1R1-CRHR1 interaction (rs2267735 × rs242924) was correlated with PTSD severity in men (beta = -4.72 and P = 0.023). Our study indicated that the ADCYAP1R1 polymorphism rs2267735 may affect PTSD development through diverse gene-gene interactions.

Shorebirds' Longer Migratory Distances Are Associated With Larger ADCYAP1 Microsatellites and Greater Morphological Complexity of Hippocampal Astrocytes.

For the epic journey of autumn migration, long-distance migratory birds use innate and learned information and follow strict schedules imposed by genetic and epigenetic mechanisms, the details of which remain largely unknown. In addition, bird migration requires integrated action of different multisensory systems for learning and memory, and the hippocampus appears to be the integration center for this task. In previous studies we found that contrasting long-distance migratory flights differentially affected the morphological complexity of two types of hippocampus astrocytes. Recently, a significant association was found between the latitude of the reproductive site and the size of the ADCYAP1 allele in long distance migratory birds. We tested for correlations between astrocyte morphological complexity, migratory distances, and size of the ADCYAP1 allele in three long-distance migrant species of shorebird and one non-migrant. Significant differences among species were found in the number and morphological complexity of the astrocytes, as well as in the size of the microsatellites of the ADCYAP1 gene. We found significant associations between the size of the ADCYAP1 microsatellites, the migratory distances, and the degree of morphological complexity of the astrocytes. We suggest that associations between astrocyte number and morphological complexity, ADCYAP1 microsatellite size, and migratory behavior may be part of the adaptive response to the migratory process of shorebirds.

Attenuation of relaxing response induced by pituitary adenylate cyclase-activating polypeptide in bronchial smooth muscle of experimental asthma.

Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1 receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1 receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1 receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.

Molecular and cellular correlates of human nerve regeneration: ADCYAP1/PACAP enhance nerve outgrowth.

We only have a rudimentary understanding of the molecular and cellular determinants of nerve regeneration and neuropathic pain in humans. This cohort study uses the most common entrapment neuropathy (carpal tunnel syndrome) as a human model system to prospectively evaluate the cellular and molecular correlates of neural regeneration and its relationship with clinical recovery. In 60 patients undergoing carpal tunnel surgery [36 female, mean age 62.5 (standard deviation 12.2) years], we used quantitative sensory testing and nerve conduction studies to evaluate the function of large and small fibres before and 6 months after surgery. Clinical recovery was assessed with the global rating of change scale and Boston Carpal Tunnel Questionnaire. Twenty healthy participants provided normative data [14 female, mean age 58.0 (standard deviation 12.9) years]. At 6 months post-surgery, we noted significant recovery of median nerve neurophysiological parameters (P < 0.0001) and improvements in quantitative sensory testing measures of both small and large nerve fibre function (P < 0.002). Serial biopsies revealed a partial recovery of intraepidermal nerve fibre density [fibres/mm epidermis pre: 4.20 (2.83), post: 5.35 (3.34), P = 0.001], whose extent correlated with symptom improvement (r = 0.389, P = 0.001). In myelinated afferents, nodal length increased postoperatively [pre: 2.03 (0.82), post: 3.03 (1.23), P < 0.0001] suggesting that this is an adaptive phenomenon. Transcriptional profiling of the skin revealed 31 differentially expressed genes following decompression, with ADCYAP1 (encoding pituitary adenylate cyclase activating peptide, PACAP) being the most strongly upregulated (log2 fold-change 1.87, P = 0.0001) and its expression was associated with recovery of intraepidermal nerve fibres. We found that human induced pluripotent stem cell-derived sensory neurons expressed the receptor for PACAP and that this peptide could significantly enhance axon outgrowth in a dose-dependent manner in vitro [neurite length PACAP 1065.0 µm (285.5), vehicle 570.9 µm (181.8), P = 0.003]. In conclusion, carpal tunnel release is associated with significant cutaneous reinnervation, which correlates with the degree of functional improvement and is associated with a transcriptional programme relating to morphogenesis and inflammatory processes. The most highly dysregulated gene ADCYAP1 (encoding PACAP) was associated with reinnervation and, given that this peptide signals through G-protein coupled receptors, this signalling pathway provides an interesting therapeutic target for human sensory nerve regeneration.

Localization of area prostriata and its connections with primary visual cortex in rodent.

Area prostriata (Pro) has been found to play important roles in the rapid processing of moving stimuli in the far peripheral visual field. However, the specific neural substrates responsible for these functions remain unknown. In this study, we first examined the location, extent, and topography of the rodent equivalent of the primate Pro based on cytoarchitecture and molecular markers. We then identified its intimate connections with the primary visual cortex (V1) using retrograde and anterograde tracers. Our main finding is that medial V1, which receives peripheral visual information, has strong reciprocal connections with the Pro in both rat and mouse while lateral V1 has significantly fewer such connections. The direct V1 inputs to the Pro provide at least one of the shortest pathways for visual information to reach the Pro, and may be crucial to the fast processing of unexpected stimuli in the peripheral visual field.

Length polymorphisms at two candidate genes explain variation of migratory behaviors in blackpoll warblers (Setophaga striata).

Migratory behaviors such as the timing and duration of migration are genetically inherited and can be under strong natural selection, yet we still know very little about the specific genes or molecular pathways that control these behaviors. Studies in candidate genes Clock and Adcyap1 have revealed that both of these loci can be significantly correlated with migratory behaviors in birds, though observed relationships appear to vary across species. We investigated geographic genetic structure of Clock and Adcyap1 in four populations of blackpoll warblers (Setophaga striata), a Neotropical-Nearctic migrant that exhibits geographic variation in migratory timing and duration across its boreal breeding distribution. Further, we used data on migratory timing and duration, obtained from light-level geolocator trackers to investigate candidate genotype-phenotype relationships at the individual level. While we found no geographic structure in either candidate gene, we did find evidence that candidate gene lengths are correlated with five of the six migratory traits. Maximum Clock allele length was significantly and negatively associated with spring arrival date. Minimum Adcyap1 allele length was significantly and negatively associated with spring departure date and positively associated with fall arrival date at the wintering grounds. Additionally, we found a significant interaction between Clock and Adcyap1 allele lengths on both spring and fall migratory duration. Adcyap1 heterozygotes also had significantly shorter migration duration in both spring and fall compared to homozygotes. Our results support the growing body of evidence that Clock and Adcyap1 allele lengths are correlated with migratory behaviors in birds.

Estrogen-Dependent Upregulation of Adcyap1r1 Expression in Nucleus Accumbens Is Associated With Genetic Predisposition of Sex-Specific QTL for Alcohol Consumption on Rat Chromosome 4.

Humans show sex differences related to alcohol use disorders (AUD). Animal model research has the potential to provide important insight into how sex differences affect alcohol consumption, particularly because female animals frequently drink more than males. In previous work, inbred strains of the selectively bred alcohol-preferring (P) and non-preferring (NP) rat lines revealed a highly significant quantitative trait locus (QTL) on rat chromosome 4, with a logarithm of the odds score of 9.2 for alcohol consumption. Recently, interval-specific congenic strains (ISCS) were developed by backcrossing the congenic P.NP line to inbred P (iP) rats to further refine the chromosome 4 QTL region. Two ISCS sub-strains, ISCS-A and ISCS-B, were obtained with a narrowed QTL, where the smallest region of overlap consisted of 8.9 Mb in ISCS-B. Interestingly, we found that females from both ISCS lines consumed significantly less alcohol than female iP controls (p < 0.05), while no differences in alcohol consumption were observed between male ISCS and iP controls. RNA-sequencing was performed on the nucleus accumbens of alcohol-naïve female ISCS-B and iP rats, which revealed differentially expressed genes (DEG) with greater than 2-fold change and that were functionally relevant to behavior. These DEGs included down-regulation of Oxt, Asb4, Gabre, Gabrq, Chat, Slc5a7, Slc18a8, Slc10a4, and Ngfr, and up-regulation of Ttr, Msln, Mpzl2, Wnt6, Slc17a7, Aldh1a2, and Gstm2. Pathway analysis identified significant alterations in gene networks controlling nervous system development and function, as well as cell signaling, GABA and serotonin receptor signaling and G-protein coupled receptor signaling. In addition, ß-estradiol was identified as the most significant upstream regulator. The expression levels of estrogen-responsive genes that mapped to the QTL interval and have been previously associated with alcohol consumption were measured using RT-qPCR. We found that expression of the Adcyap1r1 gene, encoding the pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor, was upregulated in female ISCS-B compared to female iP controls, while no differences were exhibited in males. In addition, sequence variants in the Adcyap1r1 promoter region showed a differential response to estrogen stimulation in vitro. These findings demonstrate that rat chromosome 4 QTL contains genetic variants that respond to estrogen and are associated with female alcohol consumption.

Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells.

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.

Hypoxia and nicotine effects on Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor 1 (PAC1) in the developing piglet brainstem.

Pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate receptor 1 (PAC1), have been implicated in the pathophysiology of the Sudden Infant Death Syndrome (SIDS). Two main risk factors for SIDS are prone sleeping and cigarette smoke exposure. Using piglet models of these risk factors, intermittent hypercapnic hypoxia (IHH-mimicking rebreathing in prone position) and nicotine (main reinforcing element of cigarettes), this study aimed to determine their effects on PACAP and PAC1 protein expression in the medulla. IHH was delivered for 1 (n=7), 2 (n=6), 3 (n=6) and 4 (n=7) days prior to euthanasia at 13-14days of age, while nicotine (n=7) was continuous for the first 14days of life. An additional group of combined nicotine and 1day IHH (1DIHH) was studied to determine the combined effects of the risk factors. Changes in expression were seen after the acute 1DIHH exposure (none after repeated daily exposures) and included a decrease in PACAP in the dorsal motor nucleus of vagus (DMNV; p=0.024), nucleus of the solitary tract (NTS; p=0.024) and the gracile nucleus (GRAC; p=0.001), and a decrease in PAC1 in the NTS (p=0.01). No PACAP change was noted in the nicotine-exposed piglets, however, a decrease in PAC1 was found in the DMNV (p=0.02). IHH exposure in piglets with pre-exposure to nicotine led to a significant decrease in PACAP in the Grac (p=0.04) but had no effect on PAC1. These findings show for the first time, the vulnerability of PACAP in the brainstem during early development to an acute hypercapnic hypoxic exposure and that those effects are greater than from nicotine exposure.

Candidate genes have sex-specific effects on timing of spring migration and moult speed in a long-distance migratory bird.

The timing of major life-history events, such as migration and moult, is set by endogenous circadian and circannual clocks, that have been well characterized at the molecular level. Conversely, the genetic sources of variation in phenology and in other behavioral traits have been sparsely addressed. It has been proposed that inter-individual variability in the timing of seasonal events may arise from allelic polymorphism at phenological candidate genes involved in the signaling cascade of the endogenous clocks. In this study of a long-distance migratory passerine bird, the willow warbler Phylloscopus trochilus, we investigated whether allelic variation at 5 polymorphic loci of 4 candidate genes (Adcyap1, Clock, Creb1, and Npas2), predicted 2 major components of the annual schedule, namely timing of spring migration across the central Mediterranean sea and moult speed, the latter gauged from ptilochronological analyses of tail feathers moulted in the African winter quarters. We identified a novel Clock gene locus (Clock region 3) showing polyQ polymorphism, which was however not significantly associated with any phenotypic trait. Npas2 allele size predicted male (but not female) spring migration date, with males bearing longer alleles migrating significantly earlier than those bearing shorter alleles. Creb1 allele size significantly predicted male (but not female) moult speed, longer alleles being associated with faster moult. All other genotype-phenotype associations were statistically non-significant. These findings provide new evidence for a role of candidate genes in modulating the phenology of different circannual activities in long-distance migratory birds, and for the occurrence of sex-specific candidate gene effects.