The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characterization of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not immediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected.

Bioactive peptides (i.e., neuropeptides or peptide hormones) represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.

Neuropeptides are critical integrative elements within the central circadian clock in the suprachiasmatic nucleus (SCN), where they mediate both cell-to-cell synchronization and phase adjustments that cause light entrainment. Forward peptidomics identified little SAAS, derived from the proSAAS prohormone, among novel SCN peptides, but its role in the SCN is poorly understood.

Among songbirds, the zebra finch (Taeniopygia guttata) is an excellent model system for investigating the neural mechanisms underlying complex behaviours such as vocal communication, learning and social interactions. Neuropeptides and peptide hormones are cell-to-cell signalling molecules known to mediate similar behaviours in other animals. However, in the zebra finch, this information is limited. With the newly-released zebra finch genome as a foundation, we combined bioinformatics, mass-spectrometry (MS)-enabled peptidomics and molecular techniques to identify the complete suite of neuropeptide prohormones and final peptide products and their distributions.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

The visual selection of specific cells within an ex vivo brain slice, combined with whole-cell patch clamp recording and capillary electrophoresis (CE)-mass spectrometry (MS)-based metabolomics, yields high chemical information on the selected cells. By providing access to a cell's intracellular environment, the whole-cell patch clamp technique allows one to record the cell's physiological activity. A patch clamp pipet is used to withdraw ∼3 pL of cytoplasm for metabolomic analysis using CE-MS. Sampling the cytoplasm, rather than an intact isolated neuron, ensures that the sample arises from the cell of interest and that structures such as presynaptic terminals from surrounding, nontargeted neurons are not sampled. We sampled the rat thalamus, a well-defined system containing gamma-aminobutyric acid (GABA)-ergic and glutamatergic neurons. The approach was validated by recording and sampling from glutamatergic thalamocortical neurons, which receive major synaptic input from GABAergic thalamic reticular nucleus neurons, as well as neurons and astrocytes from the ventral basal nucleus and the dorsal lateral geniculate nucleus. From the analysis of the cytoplasm of glutamatergic cells, approximately 60 metabolites were detected, none of which corresponded to the compound GABA. However, GABA was successfully detected when sampling the cytoplasm of GABAergic neurons, demonstrating the exclusive nature of our cytoplasmic sampling approach. The combination of whole-cell patch clamp with single cell cytoplasm metabolomics provides the ability to link the physiological activity of neurons and astrocytes with their neurochemical state. The observed differences in the metabolome of these neurons underscore the striking cell to cell heterogeneity in the brain.

Neurochemical differences in the hypothalamic-pituitary axis between individuals and between ages may contribute to differential susceptibility to cocaine abuse. This study measured peptide levels in the pituitary gland (Pit) and lateral hypothalamus (LH) in adolescent (age 30 days) and adult (age 65 days) mice from four standard inbred strains, FVB/NJ, DBA/2J, C57BL/6J, and BALB/cByJ, which have previously been characterized for acute locomotor responses to cocaine. Individual peptide profiles were analyzed using mass spectrometric profiling and principal component analysis. Sequences of assigned peptides were verified by tandem mass spectrometry. Principal component analysis classified all strains according to their distinct peptide profiles in Pit samples from adolescent mice, but not adults. Select pro-opiomelanocortin-derived peptides were significantly higher in adolescent BALB/cByJ and DBA/2J mice than in FVB/NJ or C57BL/6J mice. A subset of peptides in the LH, but not in the Pit, was altered by cocaine in adolescents. A 15 mg/kg dose of cocaine induced greater peptide alterations than a 30 mg/kg dose, particularly in FVB/NJ animals, with larger differences in adolescents than adults. Neuropeptides in the LH affected by acute cocaine administration included pro-opiomelanocortin-, myelin basic protein-, and glutamate transporter-derived peptides. The observed peptide differences could contribute to differential behavioral sensitivity to cocaine among strains and ages. Peptides were measured using mass spectrometry (MALDI-TOF) in individual lateral hypothalamus and pituitary samples from four strains and two ages of inbred mice in response to acute cocaine administration. Principal component analyses (PCA) classified the strains according to their peptide profiles from adolescent mice, and a subset of peptides in the lateral hypothalamus was altered by cocaine in adolescents.

Neuropeptide identification in mass spectrometry experiments using database search programs developed for proteins is challenging. Unlike proteins, the detection of the complete sequence using a single spectrum is required to identify neuropeptides or prohormone peptides. This study compared the performance of three open-source programs used to identify proteins, OMSSA, X!Tandem and Crux, to identify prohormone peptides. From a target database of 7850 prohormone peptides, 23550 query spectra were simulated across different scenarios. Crux was the only program that correctly matched all peptides regardless of p-value and at p-value<1×10(-2), 33%, 64%, and >75%, of the 5, 6, and ≥7 amino acid-peptides were detected. Crux also had the best performance in the identification of peptides from chimera spectra and in a variety of missing ion scenarios. OMSSA, X!Tandem and Crux correctly detected 98.9% (99.9%), 93.9% (97.4%) and 88.7% (98.3%) of the peptides at E- or p-value<1×10(-6) (<1×10(-2)), respectively. OMSSA and X!Tandem outperformed the other programs in significance level and computational speed, respectively. A consensus approach is not recommended because some prohormone peptides were only identified by one program.

An increasing body of evidence suggests that endothelin-converting enzyme-2 (ECE-2) is a non-classical neuropeptide processing enzyme. Similar to other neuropeptide processing enzymes, ECE-2 exhibits restricted neuroendocrine distribution, intracellular localization, and an acidic pH optimum. However, unlike classical neuropeptide processing enzymes, ECE-2 exhibits a non-classical cleavage site preference for aliphatic and aromatic residues. We previously reported that ECE-2 cleaves a number of neuropeptides at non-classical sites in vitro; however its role in peptide processing in vivo is poorly understood. Given the recognized roles of neuropeptides in pain and opiate responses, we hypothesized that ECE-2 knockout (KO) mice might show altered pain and morphine responses compared with wild-type mice. We find that ECE-2 KO mice show decreased response to a single injection of morphine in hot-plate and tail-flick tests. ECE-2 KO mice also show more rapid development of tolerance with prolonged morphine treatment and fewer signs of naloxone-precipitated withdrawal. Peptidomic analyses revealed changes in the levels of a number of spinal cord peptides in ECE-2 KO as compared to wild-type mice. Taken together, our findings suggest a role for ECE-2 in the non-classical processing of spinal cord peptides and morphine responses; however, the precise mechanisms through which ECE-2 influences morphine tolerance and withdrawal remain unclear.

The pharmacological agent nitroglycerin (NTG) elicits hyperalgesia and allodynia in mice. This model has been used to study the neurological disorder of trigeminovascular pain or migraine, a debilitating form of hyperalgesia. The present study validates hyperalgesia in an established mouse model of chronic migraine triggered by NTG and advances the understanding of the associated molecular mechanisms. The RNA-seq profiles of two nervous system regions associated with pain, the trigeminal ganglia (TG) and the nucleus accumbens (NAc), were compared in mice receiving chronic NTG treatment relative to control (CON) mice. Among the 109 genes that exhibited an NTG treatment-by-region interaction, solute carrier family 32 (GABA vesicular transporter) member 1 (Slc32a1) and preproenkephalin (Penk) exhibited reversal of expression patterns between the NTG and CON groups. Erb-b2 receptor tyrosine kinase 4 (Erbb4) and solute carrier family 1 (glial high affinity glutamate transporter) member 2 (Slc1a2) exhibited consistent differential expression between treatments across regions albeit at different magnitude. Period circadian clock 1 (Per1) was among the 165 genes that exhibited significant NTG treatment effect. Biological processes disrupted by NTG in a region-specific manner included adaptive and innate immune responses; whereas glutamatergic and dopaminergic synapses and rhythmic process were disrupted in both regions. Regulatory network reconstruction highlighted the widespread role of several transcription factors (including Snrnp70, Smad1, Pax6, Cebpa, and Smpx) among the NTG-disrupted target genes. These results advance the understanding of the molecular mechanisms of hyperalgesia that can be applied to therapies to ameliorate chronic pain and migraine.

The Amphiprion (anemonefish or clownfish) family of teleost fish, which is not a common model species, exhibits multiple unique characteristics, including social control of body size and protandrous sex change. The social changes in sex and body size are modulated by neuropeptide signaling pathways. These neuropeptides are formed from complex processing from larger prohormone proteins; understanding the neuropeptide complement requires information on complete prohormones sequences. Genome and transcriptome information within and across 22 teleost fish species, including 11 Amphiprion species, were assembled and integrated to achieve the first comprehensive survey of their prohormone genes. This information enabled the identification of 175 prohormone isoforms from 159 prohormone proteins across all species. This included identification of 9 CART prepropeptide genes and the loss of insulin-like 5B and tachykinin precursor 1B genes in Pomacentridae species. Transcriptome assemblies generally detected most prohormone genes but provided fewer prohormone genes than genome assemblies due to the lack of expression of prohormone genes or specific isoforms and tissue sampled. Comparisons between duplicate genes indicated that subfunctionalization, degradation, and neofunctionalization may be occurring between all copies. Characterization of the prohormone complement lays the foundation for future peptidomic investigation of the molecular basis of social physiology and behavior in the teleost fish.

Recent research suggests that exercise may help facilitate abstinence from cocaine addiction, though the mechanisms are not well understood. In mice, wheel running accelerates the extinction of conditioned place preference (CPP) for cocaine, providing an animal model for evaluating potential neurological mechanisms. The objective of this study was to quantify dynamic changes in endogenous peptides in the amygdala and dentate gyrus of the hippocampus in mice exposed to a context paired with the effects of cocaine, and in response to exercise. Male C57BL/6J mice conditioned to cocaine were housed with or without running wheels for 30 days. Following a CPP test and final exposure to either a cocaine- or saline-associated context, peptides were measured in brain tissue extracts using label-free matrix-assisted laser desorption/ionization mass spectrometry (MS) and stable isotopic labeling with liquid chromatography and electrospray ionization MS. CPP in mice was significantly reduced with running, which correlated to decreased myelin basic protein derivatives in the dentate gyrus extracts, possibly reflecting increased unmyelinated granule neuron density. Exposure to a cocaine-paired context increased hemoglobin-derived peptides in runners and decreased an actin-derived peptide in sedentary animals. These results allowed us to characterize a novel set of biomarkers that are responsive to exercise in the hippocampus and in a cocaine-paired context in the amygdala.

Prolonged use of opioids can cause opioid-induced hyperalgesia (OIH). The impact of alternative splicing on OIH remains partially characterized. A study of the absolute and relative modes of action of alternative splicing further the understanding of the molecular mechanisms underlying OIH. Differential absolute and relative isoform profiles were detected in the trigeminal ganglia and nucleus accumbens of mice presenting OIH behaviors elicited by chronic morphine administration relative to control mice. Genes that participate in glutamatergic synapse (e.g., Grip1, Grin1, Wnk3), myelin protein processes (e.g., Mbp, Mpz), and axon guidance presented absolute and relative splicing associated with OIH. Splicing of genes in the gonadotropin-releasing hormone receptor pathway was detected in the nucleus accumbens while splicing in the vascular endothelial growth factor, endogenous cannabinoid signaling, circadian clock system, and metabotropic glutamate receptor pathways was detected in the trigeminal ganglia. A notable finding was the prevalence of alternatively spliced transcription factors and regulators (e.g., Ciart, Ablim2, Pbx1, Arntl2) in the trigeminal ganglia. Insights into the nociceptive and antinociceptive modulatory action of Hnrnpk were gained. The results from our study highlight the impact of alternative splicing and transcriptional regulators on OIH and expose the need for isoform-level research to advance the understanding of morphine-associated hyperalgesia.

Neuropeptides are a chemically diverse class of cell-to-cell signaling molecules that are widely expressed throughout the central nervous system, often in a cell-specific manner. While cell-to-cell differences in neuropeptides is expected, it is often unclear how exactly neuropeptide expression varies among neurons. Here we created a microscopy-guided, high-throughput single cell matrix-assisted laser desorption/ionization mass spectrometry approach to investigate the neuropeptide heterogeneity of individual neurons in the central nervous system of the neurobiological model Aplysia californica, the California sea hare. In all, we analyzed more than 26,000 neurons from 18 animals and assigned 866 peptides from 66 prohormones by mass matching against an in silico peptide library generated from known Aplysia prohormones retrieved from the UniProt database. Louvain-Jaccard (LJ) clustering of mass spectra from individual neurons revealed 40 unique neuronal populations, or LJ clusters, each with a distinct neuropeptide profile. Prohormones and their related peptides were generally found in single cells from ganglia consistent with the prohormones' previously known ganglion localizations. Several LJ clusters also revealed the cellular colocalization of behaviorally related prohormones, such as an LJ cluster exhibiting achatin and neuropeptide Y, which are involved in feeding, and another cluster characterized by urotensin II, small cardiac peptide, sensorin A, and FRFa, which have shown activity in the feeding network or are present in the feeding musculature. This mass spectrometry-based approach enables the robust categorization of large cell populations based on single cell neuropeptide content and is readily adaptable to the study of a range of animals and tissue types.

The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores (comb jellies) have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes, and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well recognized in ctenophores, many bilaterian neuron-specific genes and genes of 'classical' neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is used for the multiplex detection and characterization of diverse analytes over a wide mass range directly from tissues. However, analyte coverage with MALDI MSI is typically limited to the more abundant compounds, which have m/z values that are distinct from MALDI matrix-related ions. On-tissue analyte derivatization addresses these issues by selectively tagging functional groups specific to a class of analytes, while simultaneously changing their molecular masses and improving their desorption and ionization efficiency. We evaluated electrospray deposition of liquid-phase derivatization agents as a means of on-tissue analyte derivatization using 2-picolylamine; we were able to detect a range of endogenous fatty acids with MALDI MSI. When compared with airbrush application, electrospray led to a 3-fold improvement in detection limits and decreased analyte delocalization. Six fatty acids were detected and visualized from rat cerebrum tissue using a MALDI MSI instrument operating in positive mode. MALDI MSI of the hippocampal area allowed targeted fatty acid analysis of the dentate gyrus granule cell layer and the CA1 pyramidal layer with a 20-μm pixel width, without degrading the localization of other lipids during liquid-phase analyte derivatization.

Recent work demonstrates that processes of stress release in prestrained elastomeric substrates can guide the assembly of sophisticated 3D micro/nanostructures in advanced materials. Reported application examples include soft electronic components, tunable electromagnetic and optical devices, vibrational metrology platforms, and other unusual technologies, each enabled by uniquely engineered 3D architectures. A significant disadvantage of these systems is that the elastomeric substrates, while essential to the assembly process, can impose significant engineering constraints in terms of operating temperatures and levels of dimensional stability; they also prevent the realization of 3D structures in freestanding forms. Here, we introduce concepts in interfacial photopolymerization, nonlinear mechanics, and physical transfer that bypass these limitations. The results enable 3D mesostructures in fully or partially freestanding forms, with additional capabilities in integration onto nearly any class of substrate, from planar, hard inorganic materials to textured, soft biological tissues, all via mechanisms quantitatively described by theoretical modeling. Illustrations of these ideas include their use in 3D structures as frameworks for templated growth of organized lamellae from AgCl-KCl eutectics and of atomic layers of WSe2 from vapor-phase precursors, as open-architecture electronic scaffolds for formation of dorsal root ganglion (DRG) neural networks, and as catalyst supports for propulsive systems in 3D microswimmers with geometrically controlled dynamics. Taken together, these methodologies establish a set of enabling options in 3D micro/nanomanufacturing that lie outside of the scope of existing alternatives.

Peripheral immune challenge can elicit microglia activation and depression-related symptoms. The balance of inflammatory signals in the tryptophan pathway can skew the activity of indoleamine-pyrrole 2,3 dioxygenase (IDO1) towards the metabolization of tryptophan into kynurenine (rather than serotonin), and towards neuroprotective or neurotoxic metabolites. The proteome changes that accompany inflammation-associated depression-related behaviors are incompletely understood.

Metabolites are involved in a diverse range of intracellular processes, including a cell's response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment.

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.