Neurotransmitter systems have been long recognized as important targets of the developmental actions of alcohol . Short- and long-term effects of ethanol on amino acid  and biogenic amine  neurotransmitters have been demonstrated in animal models of fetal alcohol spectrum disorders (FASD). Researchers have detected ethanol effects after exposure during developmental periods equivalent to the first, second, and third trimesters of human pregnancy. Results support the recommendation that pregnant women should abstain from drinking—even small quantities—as effects of ethanol on neurotransmitter systems have been detected at low levels of exposure. Recent studies have elucidated new mechanisms and/or consequences of the actions of ethanol on amino acid and biogenic amine neurotransmitter systems. Alterations in these neurotransmitter systems could, in part, be responsible for many of the conditions associated with FASD, including (1) learning, memory, and attention deficits; (2) motor coordination impairments; (3) abnormal responsiveness to stress; and (4) increased susceptibility to neuropsychiatric disorders, such as substance abuse and depression, and also neurological disorders, such as epilepsy and sudden infant death syndrome. However, future research is needed to conclusively establish a causal relationship between these conditions and developmental dysfunctions in neurotransmitter systems. This article reviews recent research on the short- and long-term effects of developmental ethanolEfficient communication among large numbers of brain cells  is necessary for the normal functioning of the nervous system. A central mechanism of neuronal communication involves the release of neurotransmitters that bind to specialized receptors on the target cell, changing its activity. Although neuropeptides are an important category of neurotransmitters involved in neuronal communication and the developmental actions of ethanol, they are beyond the scope of this article. This article will focus on chemical neurotransmitters, which can be divided into three classes: (1) amino acids ; (2) biogenic amines ; and (3) other . This review focuses on the actions of ethanol on the GABA, glutamate, serotonin, and dopamine neurotransmitter systems in the developing brain. Neurons synthesize these neurotransmitters and package them in vesicles that typically are localized at the ends of projections known as axons. Neuronal activation causes the release of neurotransmitters from the axonal terminals onto branch-like projections in adjacent neurons, which are called dendrites. Dendrites contain small thorn-like protrusions known as dendritic spines. Axonal terminals and dendritic spines meet at specialized points of contact, called synapses, which mediate a significant portion of information exchange between neurons see . NeurotrNeurotransmission in the mature central nervous system relies on the proper assembly of synapses during development, a process that requires multiple steps. Development of the nervous system begins with the recruitment of progenitor cells  into a specialized structure called the neural plate. The neural plate then folds to form the neural tube, which subdivides in a complex manner. The anterior and posterior regions of the neural tube ultimately give rise to the brain and spinal cord, respectively. Within the neural tube, progenitor cells are transformed into neuronal and glial cells. Immature neurons migrate to their final locations and gradually acquire axons, dendrites, and synapses. Information exchange between axons and dendrites determines whether synapses are maintained or eliminated, and chemical neurotransmitters play important roles in each of these neuronal developmental steps. Even before synapses are formed, GABA and glutamate regulate progenitor cell proliferation, migration, and differentiation . These nMultiple studies have identified neurotransmitter systems as major substrates of neurodevelopmental disorders, including autism, Down syndrome, and fetal alcohol spectrum disorders (FASD). Although these conditions have different causes, they are all characterized by altered neuronal communication that can be explained by underlying deficiencies in synapse development. Ethanol exposure during development has been shown to cause long-lasting defects in both the structure and function of synapses. Several mechanisms could underlie these persistent defects. Death, abnormal migration, or arrested maturation in a population of neurons will deprive their targets from receiving appropriate synaptic inputs, allowing for abnormal synaptic connections to be formed.Even if synapses are formed properly, ethanol can affect the normal progression of their developmental program, which involves stabilization of functional synapses and pruning of unneeded synapses. Importantly, these synaptic refinement processes are regulated by chemical neurotransmission and require equilibrium  between inhibitory and excitatory influences . This eqA and GABAB receptors—mediate the actions of this neurotransmitter. Most studies related to FASD have focused on GABAA receptors, which are GABA-activated ion channels that are permeable to chloride ions (Cl−). When these receptors are activated by GABA in mature neurons, Cl− flows into the cell making the membrane potential more negative and thereby decreasing excitability of the neuron .GABA is synthesized from glutamate by the enzyme glutamate decarboxylase and is the main inhibitory transmitter in the mature mammalian brain. Two classes of receptors—the GABAuron see . Howeverrons see   that stabilize neuronal connections  channels that trigger the dendritic release of BDNF. This effect also was observed after repeated in vivo exposures to low doses of ethanol. Inhibition of this BDNF-dependent form of synaptic potentiation is likely to have a deleterious effect on the maturation of inhibitory circuits in the CA3 hippocampal region, causing an imbalance between excitatory and inhibitory synaptic transmission, and ultimately resulting in alterations in learning, memory, and other cognitive processes.A recent study by A receptor function and the researchers hypothesized that ethanol triggered cell death by inducing excessive inhibition of neuronal activity via GABAA receptor potentiation. However, A receptor–mediated inhibition of cortical neurons in slices from neonatal rats, which is surprising given that these neurons were shown to be particularly sensitive to ethanol-induced cell death  during the third trimester–equivalent period was shown to cause widespread neuronal death in rats . This efll death .A receptor subunits (α1 and/or β2/3) in the cerebral cortex and hippocampus of adult offspring   . GABAA rarea see  and exerarea see .A study of adolescent monkeys exposed to ethanol in utero found reduced numbers of GABA neurons in the cerebral cortex see  Miller . PersistN-methyl-d-aspartate (NMDA) receptor, (2) the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor, and (3) the kainate receptor (AMPA and kainate receptors are collectively known as non-NMDA receptors). There also are three families of G protein–coupled glutamate receptors, and these are denoted as group I, II, and III metabotropic glutamate receptors (mGluR I–III), based on their functional properties and effects on neuronal function.Glutamate is the main excitatory neurotransmitter in the mammalian brain and is locally synthesized from glucose. It binds to two classes of receptors: glutamate-gated ion channels and G protein–coupled receptors, which regulate a variety of intracellular signaling pathways via activation of G proteins . There are three families of glutamate-gated ion channels, named for the compounds that were used to initially identify these channels: (1) the A receptors and ethanol produced a similar effect  can be impaired by certain patterns of developmental ethanol exposure, and this also could be a consequence of persistent alterations in NMDA receptor levels and/or function, including deficits in activation of NMDA receptor–dependent intracellular signaling pathways . For insLong-lasting changes in AMPA receptor function also have been detected in a few recent studies. Increases in AMPA receptor function were detected in medial septum/diagonal band neurons from juvenile rats exposed to ethanol in a binge-like fashion during the third trimester–equivalent period see  Hsiao a. DecreasMetabotropic GluRs are powerful modulators of synaptic transmission in many brain regions, including the regulation of synaptic potentiation. Metabotropic GluRs have been implicated in learning and memory processes, as well as a human intellectual disabilities (Fragile X syndrome), where the absence of a protein encoded by the Fragile X mental retardation 1 (FMR1) gene causes dysregulation of mGluR-dependent signalling . Metabot3 receptors) or G protein–coupled receptors . The body of most serotonin neurons is located in a series of nuclei—known as the raphe nuclei—that are shaped like a seam and are located in the brain stem (see ®). Serotonin neurons are expressed early in development (embryonic day 12 in rodents), and serotonin released from these neurons has been shown to control progenitor cell proliferation, differentiation, migration, and synapse formation , is synthesized from tryptophan by the enzymes tryptophan hydroxylase and aromatic amino acid decarboxylase. It binds to two types of receptors: serotonin-gated ion channels  that feed-back onto serotonin neurons to nurture them. Activators of 5-HT1A receptors and some neuroprotective peptides have been shown to ameliorate the effects of ethanol on serotonin neuron development  and D2-like receptors . The action of dopamine is terminated by reuptake into axonal terminals via dopamine transporters. The cell bodies of dopaminergic neurons are located in the hypothalamus and in brain stem regions known as the substantia nigra pars compacta and the ventral tegmental area , a drug used to treat attention deficit disorders, restores normal dopaminergic neuronal activity for a prolonged period of time in rats exposed to ethanol prenatally (FASD is associated with attention deficits and increased susceptibility to substance abuse, and this could be the result of alterations in the dopaminergic neurotransmitter system. Studies carried out in the 1980s and 1990s demonstrated that repeated prenatal ethanol exposure decreases dopamine content and turnover, reduces D1 receptor levels, distorts the shape of dopaminergic neurons, and affects sensitivity of dopamine receptors to pharmacological agents see  Schneid. Other senatally . The pre1 and/or dopamine transporter levels were detected in the striatum of these rats. 2 receptor gene (2 receptor expression and changes in olfactory ethanol sensitivity.In addition to mediating FASD-related attention deficits, alterations in the dopaminergic neurotransmitter system also could underlie the increased incidence of ethanol abuse and other substance abuse disorders that has been observed in individuals exposed to ethanol in utero as well as in animal models of FASD . Barbiertor gene . Future Research on the effects of developmental ethanol exposure on chemical neurotransmitter systems has significantly increased over the past decade. Studies have convincingly demonstrated that neurotransmission in the developing brain does not always respond to ethanol as in the adult brain and that components of developing neurotransmitter systems have unique properties that make them particularly sensitive to the adverse actions of ethanol, even at low levels of exposure. Ethanol-induced abnormalities in the formation and refinement of developing neuronal circuits are likely to be, in part, responsible for the persistent structural and functional brain deficits that characterize FASD. These deficits are ultimately responsible for the behavioral and cognitive alterations present in patients with this disorder and for their increased propensity to have comorbid neuropsychiatric diseases and some neurolgical disorders see  and 4. F


A prevailing view regarding the systems neurobiology of memory formation is that different types of memory are mediated by relatively independent brain systems. In particular, the early phase of processing of declarative memories is thought to rely mostly on the dorsal hippocampus and related temporal lobe areas, while the dorsal striatum is proposed to mediate the formation of memories for procedures and habits. For example, pharmacological stimulation of the dorsal hippocampus enhances memory in water maze and radial maze tasks when training protocols based on spatial memory are used, whereas stimulation of the dorsal striatum enhances procedural versions of these tasks that use visible cues during training  glutamate receptors in the dorsal hippocampus, and (2) context-footshock association, which is impaired by NMDA receptor blockade in the basolateral amygdala (BLA) but not in the hippocampus , a widely used model of fear-motivated conditioning, crucially depends on neurotransmitter and protein kinase signaling, protein synthesis, and gene expression in the dorsal hippocampus , Quirarte and colleagues (Sánchez-Resendis et al., In a study published in Some aspects of the findings reported by Sánchez-Resendis et al.  can be hSecond, this study reveals a novel interaction between GRs and mAchRs within the striatum in modulating memory. A requirement of cholinergic receptors for GR-induced enhancement of memory consolidation had been previously shown in the BLA (Power et al.,


FoxP2 gene, underpinning vocal behavior and sensorimotor coordination, has undergone diversification in bats, while several genes associated with audition show parallel amino acid substitutions in unrelated lineages of echolocating bats and, in some cases, in echolocating dolphins, representing a classic case of convergent molecular evolution. Vision genes encoding the photopigments rhodopsin and the long-wave sensitive opsin are functional in bats, while that encoding the short-wave sensitive opsin has lost functionality in rhinolophoid bats using high-duty cycle laryngeal echolocation, suggesting a sensory trade-off between investment in vision and echolocation. In terms of olfaction, bats appear to have a distinctive OR repertoire compared with other mammals, and a gene involved in signal transduction in the vomeronasal system has become non-functional in most bat species. Bitter taste receptors appear to have undergone a “birth-and death” evolution involving extensive gene duplication and loss, unlike genes coding for sweet and umami tastes that show conservation across most lineages but loss in vampire bats. Common vampire bats have also undergone adaptations for thermoperception, via alternative splicing resulting in the evolution of a novel heat-sensitive channel. The future for understanding the molecular basis of sensory biology is promising, with great potential for comparative genomic analyses, studies on gene regulation and expression, exploration of the role of alternative splicing in the generation of proteomic diversity, and linking genetic mechanisms to behavioral consequences.Great advances have been made recently in understanding the genetic basis of the sensory biology of bats. Research has focused on the molecular evolution of candidate sensory genes, genes with known functions  and genes identified from mutations associated with sensory deficits . For example, the  We consider echolocation at the levels of both signal production and reception. We then describe how sequencing studies of genes associated with vision, olfaction, taste and thermoperception have revealed remarkable cases of convergent evolution, sensory trade-offs and novel adaptations. Gene symbol nomenclature is dynamic, and in this review we have followed the symbols used by the authors of the research papers on bats, though always presenting the symbols in lower case as is recommended for non-human homologues. Some of these gene symbols differ from those in the official nomenclature (see Rousettus—echolocate by tongue clicking), but belong to the suborder Yinpterochiroptera that also includes laryngeal echolocators from the families Megadermatidae, Craseonycteridae, Rhinopomatidae, Hipposideridae, and Rhinolophidae  are paraphyletic. Bats in the family Pteropodidae do not use laryngeal echolocation  of the brain, an area involved in motor control and important in vocalization , whereas in species without laryngeal echolocation (Rousettus leschenaultii and Cynopterus sphinx) expression was stronger in the olfactory tubercles  on the evolutionary branch leading to mammals: subsequently Prestin has been under strong purifying selection in many mammalian lineages  of the cochlea. Prestin functions by directly converting voltage to displacement and consequently acts several orders of magnitude faster than enzymatically-driven proteins , from the New World has independently evolved an echolocation system that uses long CF signals and Doppler shift compensation (DSC), it shares most amino acid changes in Prestin with its congeners and with phyllostomid bats that do not use DSC rather than with rhinolophoid bats , and in toothed whales, and the relationship remains even after accounting for phylogenetic relatedness  result in non-syndromic hearing loss in mammals. Tmc1 encodes a transmembrane protein found in inner and OHCs in the cochlea, and may function in moving molecules to the plasma membrane, or may provide intracellular regulatory signals during hair cell development  associated with hair bundle motility in OHCs, and Otof, which encodes a protein that may trigger membrane fusion in ribbon synapses in inner hair cells and potentially functions in transmitting auditory signals to the brain. Mutations in all these genes are again associated with deafness in humans. Otof shows strong expression in the auditory cortex of adult bats that use laryngeal echolocation (Miniopterus schreibersii) compared with Rousettus leschenaultii that echolocates by tongue clicking [which is a sophisticated but non-laryngeal form of echolocation , Ush1g, Strc, Tecta, Tectb, Otog, Col11a2, Gjb2, Cldn14, Kcnq4 [which was reported as showing parallel evolution by Liu et al. (Pou3f4) and found good support for the paraphyly of echolocating bats across these loci as well as a high level of evolutionary conservation. Consequently it is apparent that as expected, only some hearing genes have been modified in bats during the evolution of echolocation, with others being subjected to purifying selection and perhaps being involved in more general aspects of audition rather than in specialized adaptations associated with echolocation. There is no evidence for positive selection acting on Myo6 in echolocating bats  would also have evolved convergent genetic mechanisms for auditory processing similar to those of laryngeal echolocators given the apparent sophistication of their biosonar  gene from 15 bat species, and found that the gene was intact in all species studied. The authors determined the spectral tuning of rhodopsin from its amino acid structure. Wavelengths of maximum absorbance (λmax) were inferred as 497–501 nm, with most species having values at the upper extreme of this range (501 nm), fitting with the bats possessing the mammalian consensus compliment of critical amino acids. Rhodopsin has been under purifying selection during mammalian diversification, although rhinolophoid bats using high-duty cycle echolocation  showed higher ratios of non-synonymous relative to synonymous mutations compared with other bats, perhaps as a consequence of relaxed selection  between pteropodid (Yinpterochiroptera) and emballonurid (Yangochiroptera) bats. The authors argued that the relatively large eyes found in both these groups of bats might utilize specialized rod-based visual mechanisms that resulted in convergent amino acid substitutions.Shen et al.  amplifieSws1, which has become pseudogenized, rendering color vision impossible , and in some Old World fruit bats, especially in taxa that roost in caves  and Scotophilus kuhlii (uses low duty cycle echolocation) than in Rousettus leschenaultii (a cave roosting pteropodid) and Hipposideros armiger (uses high duty cycle echolocation)  species. One phyllostomid (Glossophaga soricina) is indeed able to see UV stimuli, and UV signals may reflect strongly from flowers at low light levels  and Pteronotus (Mormoopidae) was the result of multiple gains, however, we suggest that multiple losses of an AOS is equally or more plausible.Tetrapods possess two olfactory systems that have distinctive anatomical and neurophysiological bases . All vertebrates studied to date, with the exception of some cetaceans  are expressed in the cell membranes of olfactory sensory neurons located mainly in a small region of the upper nasal epithelium and initiate signal transduction cascades that send nerve impulses to the brain. They belong to the class A rhodopsin-like family of G protein-coupled receptors  from aquatic mammals, semi-aquatic mammals, twelve bat species, and coupled these data with whole genome data from terrestrial mammals, resulting in ~50,000 OR gene sequences from 50 phylogenetically and ecologically diverse species. They analysed these data using a combination of phylogenetic, principal component, and Bayesian assignment tests, and identified unique signatures of OR gene family usage in bats. They uncovered spectacular examples of OR gene losses in three independent lineages of aquatic and semi-aquatic mammals, yet convergent, selective retention of similar functional OR families.Given that sensory trade-offs may have resulted in high rates of pseudogenization in other mammals that use specialized senses including electrolocation, echolocation, and trichromatic color vision, it is pertinent to ask whether high rates of pseudogenization are also apparent in echolocating bats. To address this question and explore the evolution of olfaction in bats Hayden et al.  generateOR genes—bats appear to show similar percentages of pseudogenes  regardless of whether they use laryngeal echolocation or not , yet at the same time has a relatively small olfactory bulb , Carollia perspicillata and Desmodus rotundus (Phyllostomidae) showed intact exon 2 ORFs and the sequence was under purifying selection  found on the cell membranes of taste buds.Tas1r genes have been described in mammals, with the Tas1r2 and Tas1r3 heterodimer functioning in the detection of sweetness, and the Tas1r1 and Tas1r3 heterodimer functioning as the umami taste receptor. Hence Tas1r2 is thought to be the only taste receptor specific to sweetness, and Tas1r2 knockout mice show disrupted responses to sweet taste  genome of the insectivorous little brown bat Myotis lucifugus. Twenty-eight T2R genes were detected in the bat genome, of which nine appeared intact, eight partial but perhaps still functional, and nine were pseudogenes. This compared with 37 functional genes and 11 pseudogenes in humans, and 37 functional genes and five pseudogenes in the rat. One clade of bat-specific genes was identified, implying that bitter tastants specific to bats may have evolved. Strong positive selection had shaped the evolution of the T2R gene repertoire in bats  and hearing . Advances in transcriptomics and whole genome sequencing will allow genomic comparisons between mammals with different sensory abilities to be performed at a much larger scale and potentially identify novel genomic regions under sensory selection in bats. Next generation sequencing is making it increasingly possible to identify genetic loci responsible for adaptive evolution in non-model organisms, and the field of adaptation genomics holds great promise , the hypothesis that rhinolophoid and yangochiropteran bats show different abilities in their detection and discrimination between different wavelengths of light seems ripe for testing. Given that bats with intact vomeronasal signal transduction genes are indeed those species known to have functional vomeronasal systems, and that bats with pseudogenized sweet and umami taste receptors are unable to learn taste aversions suggests that linking the genetic basis of sensory behavior to sensory performance has great promise. Research on the sense of touch might also be illuminating. The recent discovery that tactile receptors on bat wings are sensitive to airflow (Sterbing-D'Angelo et al., Studies on molecular evolution suggest major differences in the sensory performance of different bat lineages, and set a platform for exciting behavioral experiments. For example, the loss of function of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plastoglobules are globular compartments found in plastids. Before initial proteomic studies were published, these particles were often viewed as passive lipid droplets whose unique role was to store lipids coming from the thylakoid turn-over, or to accumulate carotenoids in the chromoplasts. Yet, two proteomic studies, published concomitantly, suggested for the first time that plastoglobules are more than “junk cupboards” for lipids. Indeed, both studies demonstrated that plastoglobules do not only include structural proteins belonging to the plastoglobulin/fibrillin family, but also contain active enzymes. The specific plastoglobule localization of these enzymes has been confirmed by different approaches such as immunogold localization and GFP protein fusions, thus providing evidence that plastoglobules actively participate in diverse pathways of plastid metabolism. These proteomic studies have been the basis for numerous recent works investigating plastoglobule function. However, a lot still needs to be discovered about the molecular composition and the role of plastoglobules. In this chapter, we will describe how the proteomic approaches have launched new perspectives on plastoglobule functions. In addition to the network of thylakoid membranes which are the site of photosynthesis, plastids contain in their soluble phase, the stroma, some enigmatic lipoprotein bodies named the plastoglobules , and harbor proteins  represent more than 50% of the protein mass of the plastoglobule core proteome in 2-9), another prenyl quinone, into plastochromanol (PC-8)  and products (γ-tocopherol and PC-8) are present at least partially in plastoglobules  story illustrates how proteomics can sometimes prompt us to reconsider accepted models. VTE1 catalyzes the second to last step of α-tocopherol (vitamin E) synthesis consisting of the cyclization of 2,3-dimethyl-5-phytyl-1,4-hydroquinol (DMPQ) to γ-tocopherol , suggesting that the enzyme plays a part in the phylloquinone production.When the first plastoglobule proteomes were published, the function of NDC1 was unknown, and its localization was believed to be mitochondrial   has been reported to occur in the plastoglobule proteomes  kinases have been identified in plastoglobules, representing the second most abundant protein family of the plastoglobule proteome  under stress conditions or during senescence , and the allene oxide synthase (AOS) were identified in the two first published proteomes (Vidi et al., Some additional low abundant proteins were described in plastoglobules. The majority was represented by proteins with unknown function, such as two proteins with methyltransferase domains, a SOUL heme binding protein, or newly identified proteins (Lundquist et al., Arabidopsis chloroplasts by spectral-counting proteomics (Joyard et al., The specialized structures wherein carotenoids accumulate during chromoplastogenesis define the morphology of chromoplasts (reviewed in Egea et al., Chlamydomonas reinhardtii eyespot. The eyespot apparatus is believed to play the role of a directional light sensor (reviewed in Kreimer, Chlamydomonas reinhardtii eyespot and plastoglobules contain common homologous proteins, such as proteins with plastoglobulin domain, ABC1 kinases, or FBPA (Schmidt et al., Finally, other proteomic data about plastoglobules could be taken from the proteome of While the plastoglobule proteomes from different plastid types share proteins in common, they also contain specific proteins depending on the plastid type. This variation in the plastoglobule protein composition may indicate a possible specialization of the plastoglobule function depending on the tissues considered.Proteomic approaches have brought substantial insight into our understanding of plastoglobule functions. Our conception of the plastoglobules has dramatically changed from insipid passive lipid droplets inside plastids to particles with active role at the crossroad of diverse metabolic pathways, for example in vitamin biosynthesis. Notably, the common denominator of the vast majority of plastoglobule proteins is a possible involvement in the response to stress. Recently, our understanding of the plastoglobule function has reached an upper level with the combination of proteomics and co-expression analysis (Lundquist et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These studies have particularly highlighted the significance of regulatory mechanisms to proper functioning of the serotonin system. This article will review the findings of FSCV investigations of serotonergic neurotransmission and discuss this technique's potential in future studies of the serotonin system.Serotonin modulates a variety of processes throughout the brain, but it is perhaps best known for its involvement in the etiology and treatment of depressive disorders. Microdialysis studies have provided a clear picture of how ambient serotonin levels fluctuate with regard to behavioral states and pharmacological manipulation, and anatomical and electrophysiological studies describe the location and activity of serotonin and its targets. However, few techniques combine the temporal resolution, spatial precision, and chemical selectivity to directly evaluate serotonin release and uptake. Fast-scan cyclic voltammetry (FSCV) is an electrochemical method that can detect minute changes in neurotransmitter concentration on the same temporal and spatial dimensions as extrasynaptic neurotransmission. Subsecond measurements both  The neurotransmitter serotonin [also called 5-hydroxytryptamine (5-HT)] can be found in nearly every region of the central nervous system. Its functions are as diverse as the areas they innervate, and it is a complex component of many psychiatric disorders. This pervasive involvement in brain-wide neurocircuitry is supported by an exceptionally large family of receptors whose collective functional scope enables the multifarious actions of serotonin throughout the brain , a networked relay region that includes the densest serotonergic projections from the DRN to any forebrain region (9 × 106 sites per mm3) , a tiny hub in the core of the medulla, contains the majority of serotonin-producing neurons that send ascending projections into the brain. Voltammetric measurements detect serotonin efflux from both axonal and somatodendritic sites in this region because a subset of serotonergic neurons synapse locally. Although axonal serotonin release is prevalent throughout the central nervous system, experiments employing FSCV are typically constrained to brain regions dense with serotonergic terminals and limited interference from other neurotransmitters and metabolites. These studies predominantly take place in the substantia nigra, a midbrain region composed of the in vivo FSCV experiments have taken place exclusively in the SNr. Thick vasculature and meninges above the DRN make targeting this region in the intact brain with a fragile carbon-fiber microelectrode difficult. Other serotonergic regions of interest, such as the hippocampus and prefrontal cortex, have not been explored due to significant chemical interference from other monoamines. However, recent advancements in neuronal stimulation technology may help circumvent this problem, and these potential future directions will be discussed in more detail in the conclusion of this article.Serotonin measurements in Electrochemical methods, including FSCV, lack absolute chemical specificity. Some chemical species, particularly those with similar structure, can interfere with detection of the desired substance by oxidizing at similar or identical potentials. Therefore, voltammetric measurements rely on five criteria for identification of endogenously released substances: First, cyclic voltammograms obtained under experimental conditions must have high correlation with cyclic voltammograms of the authentic compound. Second, presence of the neurotransmitter must be validated by independent chemical identification, such as microdialysis, tissue content analysis, or radioligand binding in the targeted brain region. The third criterion requires precise anatomical positioning of the sensor into the brain region of interest. Fourth, observed release should follow known physiological properties for the neurotransmitter and target brain region. Finally, identification of the released substance is dependent on pharmacological validation.O'Connor and Kruk  systematBunin and Wightman  later inSince O'Connor and Kruk's first report of voltammetric detection of serotonin, several modifications have been implemented to adapt and improve the use of FSCV for novel applications. The voltage potential waveform (−1 to +1.4 to −1) used by the Stamford and Kruk labs, as well as others, in serotonin studies cited throughout this review was adjusted by Jackson et al.  to improin vivo voltammetric measurements of endogenous serotonin concentrations in a rat owe their success to the enhanced sensitivity and temporal resolution facilitated by Nafion-coated sensors and the modified voltage potential waveform  stimulations consist of 5–10 pulses applied at 100–200 Hz and are shorter than 100 ms in duration. They are designed to approximate a single electrical impulse but evoke more consistent efflux. In brain slice experiments, POP stimulations are often used to avoid creating an endogenous “tone” at receptors, which facilitates more direct investigation of selective agonists and antagonist's effects on autoreceptor-mediated modulation of release . A subset of serotonergic neurons that project to the SNr also send axon collaterals to forebrain structures via the MFB  uptake and (ii) release and representative signals predicted for stimulations of varying frequency. In brain slice preparations, the concentration evoked per stimulation pulse ([5-HT]pulse) was found to be 100 ± 20 nM in DRN, and significantly lower in the SNr, at 55 ± 7 nM. Differences in [5-HT]pulse are proportional to differences in tissue content between the two brain regions, indicating that local stores of serotonin may influence the concentration that can be evoked by electrical stimulation  considerably decreased the concentration of evoked dopamine to 18% and 6% of control amplitudes, respectively, but affected serotonin to a much lesser extent . Serotonin efflux was also resistant to short term depression after repeated stimulation pulse trains, while dopamine efflux was attenuated by 38% after 20 stimulations. This suggests that a relatively small proportion of the available vesicular serotonin is mobilized for release by each electrical stimulation train, a finding which may partially explain the low concentrations observed in vivo.Hashemi et al.  investigi-coupled mechanism . 5-HT1B and 1D autoreceptors negatively couple to adenylyl cyclase  by 150–700%. This wide spread of responses may be attributable to experimental variability between studies, particularly differences in stimulation parameters. Indeed, in vivo studies of SSRI effects in the SNr using identical stimulation parameters report comparable changes in t1/2 using MFB and DRN stimulation sites   in the hypothalamus increased after 21 days of SSRI treatment (Schoups et al., t1/2 was not altered in any voltammetric investigation of long-term SSRI treatment. Therefore, increases in evoked concentrations induced by chronic treatment must rely on another mechanism. Changes in other aspects of release may contribute to this effect, for example: the quantity or composition of serotonin stored in vesicles, regulation of intracellular calcium, or excitability of the synaptic membrane. In-depth exploration of these mechanisms has not yet been explored using voltammetric methods.Long-term SSRI treatment increases stimulation-evoked serotonin concentrations in the DRN and other brain regions by 20–100%, depending on the experiment and brain region studied (O'Connor and Kruk, less sensitive, and release amplitude more sensitive, to this manipulation. Contradictory findings of 5-HT1A receptor sensitivity was not a total surprise: prior studies found similar desensitization of 5-HT1A receptors in the control of firing rate after chronic paroxetine treatment (Chaput et al., in vivo.Alterations in 5-HT1A autoreceptors contribute to the effects of chronic SSRIs on serotonin signaling. Under normal conditions, activated 5-HT1A receptors inhibit serotonin release and neuronal firing rates, and chronic SSRI treatment may modify this activity. Selective suppression of 5-HT1A autoreceptors can produce antidepressant behavioral effects in the absence of SSRIs (Bortolozzi et al., 5-HT1B and 1D receptors also desensitize after chronic SERT inhibition, although the extent to which this occurs appears to vary between brain regions. O'Connor and Kruk  reportedIn vivo work supports this idea, as a recent study has shown that MAO inhibitors dramatically increase serotonin efflux in the SNr (Hashemi et al., in vivo, suggesting a unique role for metabolic degradation in the regulation of serotonin transmission compared to other monoaminergic systems.Metabolic degradation of serotonin by the enzyme MAO also contributes to serotonin clearance, especially in the developing brain (Cases et al., Most of the studies reviewed in this article focused on describing the role of autoreceptors and transporters in modulating serotonin signaling throughout the brain. However, signaling is also considerably influenced by many other neurotransmitter systems, including norepinephrine, glutamate, GABA, and a number of neuroendocrine modulators. These external influences are highly implicated in serotonin's involvement in a number of psychiatric disorders and, while they have been investigated by other techniques, their functions have not been fully described using subsecond voltammetric measurements. Evaluating the effects of external modulatory mechanisms on the subsecond dynamics of serotonin signaling could provide important clues about their role in neurological disorders.in vivo. FSCV can also be paired with concurrent electrophysiological measurements to couple information about neurotransmitter release to single-unit responses of post-synaptic neurons. Iontophoretic and electrophysiological methods have already been applied to voltammetric studies of dopamine release in anaesthetized and freely-moving animals (Takmakov et al., Ongoing methodological developments continue to progress voltammetric measurements beyond the current experimental limits. While electrochemical techniques have been optimized for serotonin detection (Lama et al., in vivo because homologous receptors are expressed throughout the brain. However, many novel drug-delivery and transgenic methods have been developed to avoid this type of complication. DREADDs, designer receptors with exogenous ligands, have been used to target specific G-protein-activated cascades in serotonergic neurons (Dong et al., in vivo preparation. Many conditional knockout mouse models, which avoid confounding developmental effects, are now available for serotonin's transporter and receptors. These techniques could lead to more selective targeting and better characterization of serotonin's receptors and their downstream effectors in combination with voltammetric measurements.It has previously been challenging to selectively study serotonin's autoreceptors Voltammetric measurements have, until recently, been limited to brain regions with high levels of the neurotransmitter of interest and limited presence of other electroactive compounds. This is because electrical stimulations indiscriminately excite all proximal nerve terminals. Use of optogenetic stimulation circumvents this barrier by enabling selective excitation of a specific population of neurons. Channelrhodopsin-2-mediated serotonin efflux has been measured in fly larvae using FSCV in a technique developed by the Venton group. The light-evoked efflux is vesicular and subject to regulation by synthesis and uptake transport in a manner that is similar to mammalian serotonin release (Borue et al., Finally, while voltammetric measurements of serotonin have presently only occurred in brain slices and anaesthetized animals, an exciting future direction for research will be monitoring serotonin signaling in an awake, freely moving animal. FSCV has been used to measure endogenous dopamine and norepinephrine release in freely moving animals, and this research has led to groundbreaking information coupling real-time neurotransmission to specific facets of behaviors. Many questions remain about serotonin's role in both basic and complex nervous system processes, and coupling FSCV to relevant behavioral paradigms may yield important clues about its function.in vivo measurements suggest that intact brain circuitry supports the involvement of multiple modulatory mechanisms in the control of serotonin signaling. New developments in a variety of techniques present potential for more intricate assessment of regulation within and external to the serotonin system. Future studies using FSCV in combination with new technologies will likely elucidate many of the mysteries of the serotonin system.Serotonin signaling is an important component in the etiology and treatment of many neurological disorders. By combining subsecond temporal resolution with nanomolar sensitivity to concentration changes, FSCV has revealed a great deal about dynamic serotonin transmission. These findings are summarized by the illustration in Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Central vestibular neurons form identifiable subgroups within the boundaries of classically outlined octavolateral nuclei in primitive vertebrates that are distinct from those processing lateral line, electrosensory, and auditory signals. Each vestibular subgroup exhibits a particular morpho-physiological property that receives origin-specific sensory inputs from semicircular canal and otolith organs. Behaviorally characterized phenotypes send discrete axonal projections to extraocular, spinal, and cerebellar targets including other ipsi- and contralateral vestibular nuclei. The anatomical locations of vestibuloocular and vestibulospinal neurons correlate with genetically defined hindbrain compartments that are well conserved throughout vertebrate evolution though some variability exists in fossil and extant vertebrate species. The different vestibular subgroups exhibit a robust sensorimotor signal processing complemented with a high degree of vestibular and visual adaptive plasticity. Carassius auratus), the octavolateral nuclei occupy a considerable part of the dorso-lateral hindbrain between the fifth and tenth cranial nerves . Functionally, the octavolateral nuclei are heterogeneous subgroups of neurons subserving four different sensory modalities: lateral line for detection of either water motion (mechanoreceptive) or objects (electroreceptive), auditory for acoustic pressure and vestibular for body motion . Based on their evolutionary-conserved neuronal projections to brainstem and spinal regions responsible for the control of balance and locomotion, the lateral portions of the octavolateral nuclei can be clearly distinguished from the more medial, and dorsal nuclei that process acoustic and lateral line sensory signals .Vestibular sensory function in the somewhat inappropriately called “inner ear” of early vertebrates consists of separate semicircular canals attached to a single sac-like structure, the utricle with additional sac-like structures, the saccule, and the lagena, utilized for audition and/or graviception  and magnFigure 1B). Each subdivision can be further divided into distinct functional groups involved in the control of eye and body motion based on (1) regionally restricted terminations of first order afferent fibers from the different peripheral vestibular endorgans, (2) anatomical tract tracing from efferent projection areas in the midbrain, hindbrain and spinal cord (SC), and (3) electrophysiological correlates of second order neurons during different behavioral paradigms .The most comprehensive developmental and topographical mapping of overall vestibular organization has been carried out in cyprinids (goldfish and zebrafish). Based on the cellular organization, the vestibular nuclei are generally illustrated as five major subdivisions of second order neurons related to balance, postural control, and motion . Distinct subsets of vestibuloocular neurons in r1-3 and r5-6 and of vestibulospinal neurons in r4-6 occupy unique segmental compartments, respectively . Each hindbrain segment is characterized by a combinatorial expression of distinct families of transcription factors that play a dominant patterning role as, e.g., Hox1-5 genes  1-8 . The vestibular subdivision of the octavolateral nuclei was modified in jawed fishes after a third canal, the horizontal (HC), was added to the ontogenetic blueprint. Notably, the structural redesign in vestibular subgroups, in particular the unique central horizontal eye movement circuitry, has been conserved ever since it evolved in fishes to present day mammals . Accordingly, anterior semicircular canal-related neuronal subgroups along with the first major cerebellar nucleus , originate from r1-3 . The vestibulocerebellum (VCB) is therefore the phylogenetically oldest part of the cerebellum. This embryological blueprint schematically illustrates the vestibulocerebellar circuitry showing the closely linked evolutionary history of neuronal processing co-adapted for the control of eye and body motion in a three dimensional environment that was little modified during the subsequent transition of vertebrates from water to land , and caudal, posterior (PON) semicircular canal-related subdivisions separated by an intermediate (ION) otolith-related group of neurons . However to land .Figures 4). In addition, first order vestibular afferents likely exhibited the ability to contact neurons in any of the r2-7 derived subnuclei. As a result, in the most primitive jawless and jawed fishes the vestibular system was quickly converted from point-to-point, hard-wired canal/otolith specific subgroups relaying purely sensory signals to remarkably plastic subsets of neurons that effectively could both generate and relay on-line motor signals as illustrated by the specific visuo-vestibular behavioral repertoire of horizontal eye movement signals in goldfish second order neurons .Since the earliest jawless vertebrates, the vestibular nuclei appear to align with the location of the peripheral anterior and posterior semicircular canals surrounding a central otolith . The hindbrain commissural innovations appeared to evolve largely, if not exclusively, in the direction of anterior canal second order inhibitory neurons contacting contralateral (plane-specific) posterior canal-related inhibitory and excitatory vestibular neurons. This arrangement had a significant impact on subsequent evolution of vestibulocerebellar circuitry in which, for example, efferent neurons , only terminate directly onto either excitatory or inhibitory second order neurons originally associated with the anterior canal system . While the asymmetry of the fish pathways appears more complicated than necessary, especially from the perspective of just aligning paired canals across the midline , evolution of inhibitory canal-specific connections across the midline was a parsimonious solution to both structural and functional constraints imposed by an original two canal sensory system that endured for around 100 million years.From a morpho-physiological perspective, interconnections between the bilateral vestibular nuclei may have had the most significant effect for vestibular function . A midlil system . Interesl system . ConsequFigure 2; Figures 2 and 3). In contrast, the horizontal canal afferent termination coincides with the parvocellular part of the magnocellular octavolateral nucleus (MO) that originates from r3-4 as well as more uniformly throughout DO and T .The distribution of first order afferent fibers from individual canals (AC-PC-HC) and the utricle allows central vestibular nuclei to be recognized in respect to head/body motion. Anterior and posterior semicircular canal afferents in fish terminate predominantly, even though not exclusively, in the anterior octavolateral nucleus (AO) that originates from r1-3 and in the r5 tangential nucleus (T) ; however they also extend ventromedially outside the octavolateral nuclei toward the horizontal eye movement nuclei . This wide spread vestibular pattern is thus clearly different from topological maps of other sensory systems such as the closely related visual system. The absence of a sensory point-to-point map is consistent with the finding that signal processing in central vestibular nuclei is more spatially organized according to motor targets rather than to topological hindbrain location  formed by vestibuloocular neurons illustrate the essential link that evolved between sensory and extraocular reference frames that could in many early vertebrate species nearly perfectly stabilize gaze in response to either retinal image drift or eye/head body motion . The vestibular neuronal subgroups critical for sensorimotor transformation of linear and angular acceleration signals are located laterally within the borders of r1-3 and r5-7 . Afferent input from the two vertical semicircular canals comprise the major input for vestibular subgroups activating extraocular muscles  with corresponding spatially aligned pulling directions . The subgroup of vestibular neurons that excites contralateral IR and SO motoneurons is located more caudally in the DO/PO . The caudal r5-7 position of these neurons is expected from the observed PON location in jawless fish and lamprey . Axons from both the rostral and caudal vestibuloocular subgroups reach their respective extraocular motor subdivisions in the oculomotor/trochlear nuclei by axonal pathways in the ipsi- or contralateral MLF .Different populations of second order vestibular neurons form spatially, non-overlapping subgroups within the AO in which neuronal phenotypes can be functionally distinguished with respect to their respective extraocular motor target . OculomoFigures 2 and 7; Figure 9; Figure 6). However, conjugacy of eye movements in fish is much more apparent than real. Foveae are absent in the majority of fish species, and even when present would not likely produce stereoscopic vision by fusion of targets between the two eyes. Moreover, from early larval onset to adults, monocular horizontal eye movements can be elicited with suitable visual/vestibular stimuli thus demonstrating separate r5-6 populations of vestibular neurons to selectively target either Abd or AI neurons . This neuronal correlate is observed during either head rotation or optokinetic visual stimulation about the vertical axis. By contrast, the r5-6 vestibular neurons do not exhibit any activity during scanning horizontal eye motion , suggesting a different embryonic origin and evolution of the hindbrain saccadic/fixation system . The separate vestibular sites in r3 and r5-6 likely arose during the speciation event that gave rise to the horizontal semicircular canal during the transition from jawless to jawed fish . While the head velocity signal on the two subpopulations might be considered as redundant, it likely is not, as the r3 neurons subserve an essential high frequency vestibular role not provided by AI neurons. Moreover, in contrast to the r5-6 excitatory vestibular neurons projecting to Abd motoneurons, cerebellar Purkinje cells, according to their phylogenetic AON linkage in lamprey, directly inhibit the r3 vestibular neurons that project to MR motoneurons . The r3 vestibular subgroup is maintained through mammals giving rise to a well-recognized pathway (Ascending Tract of Deiter’s) to the oculomotor nucleus. The highly conserved locations of horizontal semicircular canal related second order vestibuloocular neurons within the vertebrate hindbrain clearly indicate an organizational principle centered on motor target rather than topography of spatially segregated inputs from the sensory periphery  in early developing fish before semicircular canals become operational  nucleus just caudal to the eighth nerve entry  . The most caudal subgroup is clustered within a narrow band in the ventrolateral part of the PO. Given the dense projection and termination of anterior and posterior canal afferent fibers in AO, DO, and PO regions where commissural neurons are located, these latter cell groups likely establish a “push-pull” connectivity to increase the sensitivity for detection of vertical coplanar canal signals, as demonstrated earlier with morpho-physiological and pharmacological methods in frog  . The cau in frog  and cat  in frog . In cont in frog .Figure 3). Both first and second order vestibular projections convey angular head/body acceleration as mossy fiber input to the granule cell layer of the VCB. In turn, Purkinje cells from the caudal lobe of the VCB provide a direct output to particular vestibular subgroups that project to extraocular motor nuclei .Based on an intimate phylogenetic linkage between the cerebellum and the octavolateral nuclei, the vestibulolateral lobe is a major efferent target for central vestibular neurons (r nuclei . Functior nuclei , 1997. Tr nuclei  that, alFigure 9; Comparison of the spatial distribution of individual vestibular endorgan terminations in different species of fish, such as damselfish , goby T, toadfisA comparison of vestibular system anatomy and physiology of fish with tetrapods also reveals a remarkably conserved pattern . In factThe manuscript was written and illustrated by Hans Straka and Robert Baker.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


COMP); however, AD-MED is genetically heterogenous and can also result from mutations in matrilin-3 (MATN3) and type IX collagen . In contrast, autosomal recessive MED (rMED) appears to result exclusively from mutations in sulphate transporter solute carrier family 26 (SLC26A2). The diagnosis of PSACH and MED can be difficult for the nonexpert due to various complications and similarities with other related diseases and often mutation analysis is requested to either confirm or exclude the diagnosis. Since 2003, the European Skeletal Dysplasia Network (ESDN) has used an on-line review system to efficiently diagnose cases referred to the network prior to mutation analysis. In this study, we present the molecular findings in 130 patients referred to ESDN, which includes the identification of novel and recurrent mutations in over 100 patients. Furthermore, this study provides the first indication of the relative contribution of each gene and confirms that they account for the majority of PSACH and MED. Hum Mutat 33:144–157, 2012. © 2011 Wiley Periodicals, Inc.Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias resulting in short-limbed dwarfism, joint pain, and stiffness. PSACH and the largest proportion of autosomal dominant MED (AD-MED) results from mutations in cartilage oligomeric matrix protein ( Pseudoachondroplasia  and multiple epiphyseal dysplasia  are relatively common skeletal dysplasias that can be inherited as either autosomal dominant (PSACH and AD-MED) or recessive  conditions ); SMD (1); PPRD (2); mild DTD (3), or without any formal diagnosis (4).COMP mutation was subsequently found  patients with these mutations had a correct diagnosis upon referral to ESDN; see In the other 56 patients in whom we identified a s of MED , which wDTDST mutation, 69% (11/16 patients) had originally been referred with a diagnosis of MED or rMED; with PPRD (2) and mild DTD/SED/SEMD (3) being proposed as a diagnosis in five cases. This observation would therefore suggest that rMED is slightly harder to diagnose than classical AD-MED. This may be explained partly by the fact that unlike AD-MED where family history is often positive, most cases of rMED lack a family history and therefore physicians may be less inclined to think of a possible genetic cause of the disorder. Likewise, all the referrals came from geneticists within clinical genetics departments and also included three referrals from members of the ESDN panel.Finally, in those patients in which we identified a DTDST mutation in the European population . Importantly, the p.Gly501Asp mutation in ESDN-00422 was recently confirmed as de novo in this family , confirming that it is the cause of MED in ESDN-00594. Nevertheless, the intracellular retention of a significant proportion of p.Val245Met suggests the intriguing possibility that it might be a genetic modifier of phenotypic severity.ESDN-00594 had four changes; three in reported . The COMCOMP and COL2A1 in the remaining PSACH patient and in three MED patients. In PSACH patient ESDN-01040, we identified a heterozygous p.Pro234Ser substitution in the fourth EGF-like repeat of COMP and MED patient ESDN-00521 was heterozygous for p.Gly167Glu in the second EGF-like repeat of COMP. Both of these residues are conserved in murine COMP and the substitution of glycine and proline residues in the EGF-like repeats of fibrilin-1 has been shown to cause Marfan Syndrome [Arbustini et al., COMP EGF-like mutation in a patient with PSACH, p.Gly258Arg, which is in the fourth repeat and again conserved across species (unpublished data), but the precise affect of these COMP mutations remains undetermined and will require extensive studies in vitro.By extending our standard screening protocol, we identified mutations in COL2A1 mutations that we identified  were in suspected MED patients (ESDN-00283 and ESDN-00050) in whom there was limited clinical information and radiographic images in which to make an unambiguous diagnosis (Both of the iagnosis . HoweverMATN3, or type IX collagen. The genetic cause of these diseases remains underdetermined, but the careful use exome sequencing may help identify potential candidate genes. Finally, it is interesting to note that like the mutation positive cases, the majority of mutation negative patients had also been referred by geneticists within clinical genetics departments including six from members of the ESDN panel. This would indicate that in these patients there are clear difficulties in making a correct diagnosis rather than a lack of relevant expertise.Finally, in those patients in whom we could not identify a mutation in the core exons of our screening protocol, the predominant diagnosis on referral had been MED or rMED (Supp. COMP mutation should be identified, however, we have additional evidence to confirm that a PSACH-like phenotype is distinct from classical PSACH and does not result from a COMP mutation [Spranger et al., COMP, MATN3, and type IX collagen genes account for the vast majority of classical AD-MED.In summary, we have shown that in the context of PSACH and the MED disease spectrum, the classical form of PSACH is relatively straightforward to diagnose provided there is sufficient clinical and radiographic information. In cases of PSACH, a


New pharmacological tools such as highly selective peptide CRF1 or CRF2 agonists or antagonists along with genetic knock-in or knock-out models have allowed delineating the primary role of CRF2 involved in the anorexic response to exogenous administration of CRF and Ucns. Several stressors trigger behavioral changes including suppression of feeding behavior which are mediated by brain CRF receptor activation. The present review will highlight the state-of-knowledge on the effects and mechanisms of action of CRF/Ucns-CRF1/2 signaling under basal conditions and the role in the alterations of food intake in response to stress.Early on, corticotropin-releasing factor (CRF), a hallmark brain peptide mediating many components of the stress response, was shown to affect food intake inducing a robust anorexigenic response when injected into the rodent brain. Subsequently, other members of the CRF signaling family have been identified, namely urocortin (Ucn) 1, Ucn 2, and Ucn 3 which were also shown to decrease food intake upon central or peripheral injection. However, the kinetics of feeding suppression was different with an early decrease following intracerebroventricular injection of CRF and a delayed action of Ucns contrasting with the early onset after systemic injection. CRF and Ucns bind to two distinct G-protein coupled membrane receptors, the CRF This contrasts with icv Ucn 2 that does not elicit behavioral arousal or anxiogenic effects  and the rate of ingestion, whereas meal frequency was not altered in rats  injection of Ucns reduced food intake in several species , whereas the size of the meal (as a characteristic of satiation) was less robustly altered during the re-feeding period following a 24-h fast in rats -induced decreased food intake and increased grooming while astressin2-B had no effect  was associated with an early onset of severe obesity  and body mass index physiological relevance is the demonstration that mutation in the CRF2 by Ucns reduced feeding after central as well as peripheral injection without provoking behavioral arousal or anxiogenic effects as observed for the anorexigenic action induced by the activation CRF-CRF1 signaling pathways. Brain sites of action of Ucns involve the lateral septum, PVN, VMH, medial amygdala, dorsal raphe and nucleus tractus solitarius. Both, CRF1 and CRF2 receptor activation contributes to the reduction of food intake associated with exposure to various stressors. Their respective involvement is stressors and brain sites specific mostly in relation with endogenous CRF ligands and receptors recruited under the condition of stress. Still little is known whether peripheral Ucn-CRF2 signaling plays a role in the food intake response to visceral stressors. More research is needed using site specific knockout or overexpression of CRF receptors in order to address this issue and investigate the impact on food intake and body weight.In summary, activation of CRFThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Furthermore, the last analyses addressed the effect of meditation experience . We found that frontal activation was present for short-term, as compared with long-term experience meditators, confirming that experts are better enabled to sustain attentional focus, rather recruiting the right SMG and concentrating on aspects involving disembodiment.The brain network governing meditation has been studied using a variety of meditation practices and techniques practices eliciting different cognitive processes . It is very possible that different practices of meditation are subserved by largely, if not entirely, disparate brain networks. This assumption was tested by conducting an activation likelihood estimation (ALE) meta-analysis of meditation neuroimaging studies, which assessed 150 activation foci from 24 experiments. Different ALE meta-analyses were carried out. One involved the subsets of studies involving meditation induced through exercising focused attention (FA). The network included clusters bilaterally in the medial gyrus, the left superior parietal lobe, the left insula and the right supramarginal gyrus (SMG). A second analysis addressed the studies involving meditation states induced by chanting or by repetition of words or phrases, known as “ Meditation is a complex cognitive task aiming at self-regulating the body and mind and is often associated with neurophysiological and psychological modifications , which is exercising the regulation of attention and executive frontal functions  on neuroimaging experiments. The literature cited in the obtained papers was also assessed to identify additional neuroimaging studies pertaining to meditation.N = 8) have been excluded  . Differences in coordinate spaces  were accounted for by transforming coordinates reported in Talairach space into MNI coordinates using a linear transformation  and (2) meditation induced through FA . In addition, in a last analysis (“long- and short-term meditation experience”), we divided studies according to experience  were designated as suitable for the first general meta-analysis , more superiorly the left superior medial gyrus (cluster 5), medially the left paracentral lobule (cluster 10) and the right supplementary motor area (SMA) , the left superior  and inferior parietal lobe , and left insula (cluster 8). Right lateralized activation was found in the supramarginal gyrus (SMG) (cluster 2)  included bilaterally the precuneus (clusters 3 and 5) and the superior medial gyrus (clusters 2 and 7). In addition, a right lateralized network of deactivated areas included the middle temporal gyrus cluster 1), the angular gyrus (cluster 4), and the fusiform gyrus (cluster 6) , the right SMA and paracentral lobule cluster 2), and the left postcentral gyrus . The network supporting the forms of meditation induced through FA, included clusters bilaterally in the medial gyrus , the left superior parietal lobe (cluster 2), the left insula (cluster 4) and the right SMG (cluster 4) , the SMA and the paracentral lobule bilaterally clusters 2 and 4), the middle cingulated cortex and the postcentral gyrus (clusters 5 and 6). Short experience included clusters of activity in the superior medial gyrus and the inferior parietal lobule . This activation can be related to egocentric (body- and body part-centered) coordinates coding  is similar to activation found during disembodiment , which supports a representation of somatic and visceral responses accessible to consciousness . This area has been related to emotional processing  was found in the general analysis. Meditation on the body parts demonstrated strong activation of the SMA  Raichle, , which hx = 2, y = 52, z = 0) , an area known to be involved in the implicit representation of the self and in linking subjective experiences across time . This activation might be related to disembodiment . Aside being related to egocentric (body- and body part-centered) coordinates coding  was also found activated during FA-based meditation. Particularly relevant for the present study is the role of the insular cortex in corporeal awareness . This parietal area is responsible for touch representation. This activation could be related to the technique used in several meditation practices where participants touched fingers two to five to the thumb, according to a particular rhythm . Activation in this area was found both in the sub-analysis on FA induced meditation and in that on mantra induced meditation, meaning that activation in this area is shared by both practices. This activation might be related to disembodiment  was found in the mantra-induced meditation analysis. Aside the role of this area in internal simulation of kinesthetic sensation during motor imagery  and structural changes (in the morphometric studies) particularly in two key areas, corresponding to the anterior part of the righ insula (Lazar et al., The pattern of our meta-analysis highlights how meditation consistently triggers changes in executive attentive networks, in body representations and in areas processing interoceptive awareness information, as well as negative signal changes in the default brain network. Furthermore, the present study evidenced how activation in some areas are specifically driven by a subgroup of studies, dependent on the expertise and on the practices needed to obtain it.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The lymphatic fluid originates from the interstitial fluid which bathes every parenchymal organ and reflects the “omic” composition of the tissue from which it originates in its physiological or pathological signature. Several recent proteomic analyses have mapped the proteome-degradome and peptidome of this immunologically relevant fluid pointing to the lymph as an important source of tissue-derived self-antigens. A vast array of lymph-circulating peptides have been mapped deriving from a variety of processing pathways including caspases, cathepsins, MMPs, ADAMs, kallikreins, calpains, and granzymes, among others. These self peptides can be directly loaded on circulatory dendritic cells and expand the self-antigenic repertoire available for central and peripheral tolerance. The lymphatic fluid originates from the interstitial fluid which bathes every parenchymal organ and it is generated through a process of ultrafiltration of the plasma, circulating through the blood capillaries, as well as by the addition of metabolic and catabolic products collected from the tissue of origin –4.Once the proteins have been filtered into the extracellular space, they will not re-enter the blood circulatory system by uptake into the venous capillaries as previously thought. Indeed, what was known as the Starling principle has been recently revisited and it is now apparent that almost all the extravasated fluid will be drained into the lymphatics .In addition to the proteins and molecules originating from plasma ultrafiltration, the interstitial fluid will be further enriched with products derived from tissue/organ catabolism/metabolism –13.The interstitial fluid will then drain into open end lymphatic capillaries and hence forth be called lymph , 15. TheDuring the last two decades there has been an increasing interest in the analysis of the protein composition of human and rodent lymph under physiological and pathological conditions and in the comparative analysis with plasma samples , 16–20. Proteomic profiles have been reported for human, rodent, bovine, and ovine lymph, and two major conclusions can be drawn from the compilation of these: , 16–20.(i)The lymph proteomic profile is not merely overlapping with the one from the plasma but qualitative and quantitative differences can be appreciated; indeed the lymph proteome appears to be enriched in products deriving from tissue and cellular metabolism/catabolism, organ remodeling, extracellular matrix processing, and cellular apoptosis.(ii)The proteomic molecular signature reflects the tissue from where the lymph is collected and the organ’s physiological or pathological condition. Indeed tissue-specific proteins have been mapped in the lymph collected from capillaries draining specific organs and infectious or inflammatory tissue conditions are reflected in proteomic changes in lymph more so than in plasma.The proteomic profile of the lymph also revealed the presence of several low molecular weight products composed by fragments, derived from protein processing, and short peptides . A simil14N/15N labeling and amino acid sequencing from 2D-DIGE spots indicated that many peptides were present in human lymph in at least nanomolar concentrations  to the draining lymph nodes to convey a snapshot of each parenchymal organ in physiological and pathological conditions.Two major advances have improved our capability in identifying the lymph and plasma degradome and peptidome’s processing pathways; (i) improved mass spectrometric techniques, which allow high confidence peptide identification and correct amino acid assignment and (ii) increased representation of proteins in databases , which facilitated prediction of the processing enzymes involved in peptide cleavage. Analysis of the human lymph- and plasma-carried peptidome identified peptides derived from both intra and extracellular sources and mapped several proteases likely involved in peptide processing including caspases, cathepsins, MMPs, plasmin, kallikreins, calpains, and granzymes , 29–31. (i)there is a great variety of processing pathways involved in the formation of the degradome/peptidome present in different biological fluids and(ii)the degradome/peptidome profile changes according to the physiological or pathological state of the organ from where the lymph/plasma are collected.Several other studies mapped the peptidome present in plasma, serum, and other biological fluids in physiological and pathological conditions including various types of cancer, inflammatory, and degenerative pathologies , 39, 40.Indeed, in pathological conditions, the mapped peptidome/degradome is highly enriched with new peptides as compared to the peptidome/degradome found in healthy physiological conditions , 41, 42.MHC II peptide complexes can be loaded in late endosomal MIIC compartments, early endosomes and at the plasma membrane –53. In tHaving distinct MHC class II loading compartments allows presentation of a larger array of peptides. Indeed many of the peptides loaded in recycling/early endosomes and at the cell surface are low affinity and are eliminated by HLA-DM in endosomal compartments . Thus, iThese two different pathways are exploited by the antigen-presenting cells (APC) to control immunogenicity , 66.Indeed the early endosomes/plasma membrane MHC II loading pathway is active in immature dendritic cells (DC) and down-regulated upon DC maturation –52, 64. Different mechanisms ensure that tissue-derived self antigens are constantly presented to the immune system for the maintenance of central and peripheral self tolerance –70.+ CD11c+ CD8−), CD8+ DC (Sirpα− CD11c+ CD8+), and plasmacytoid DC delete immature thymocytes with high affinity for the self MHC II peptidome , conventional dendritic cells (cDC)  and macrophages (MΦ) which continuously process and present the self proteome of parenchymal organs. Additionally, AIRE-independent mechanisms have also been described in the periphery which mediate expression of tissue-specific self antigens by lymphatic endothelial cells, further expanding the presentation of the self proteome .All the above described mechanisms depend on self-antigen delivery to endosomal compartments, through endocytosis or autophagy. Thus, they generate an MHCII peptidome mostly restricted by endosomal processing enzymes .+, CCR7+, CD103+, and express high levels of MHC II and intermediate expression of co-stimulatory molecules  is much higher then what previously known, it is broader in repertoire and has a long half life –31, 39. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Disease gene prioritization aims to suggest potential implications of genes in disease susceptibility. Often accomplished in a guilt-by-association scheme, promising candidates are sorted according to their relatedness to known disease genes. Network-based methods have been successfully exploiting this concept by capturing the interaction of genes or proteins into a score. Nonetheless, most current approaches yield at least some of the following limitations: (1) networks comprise only curated physical interactions leading to poor genome coverage and density, and bias toward a particular source; (2) scores focus on adjacencies (direct links) or the most direct paths (shortest paths) within a constrained neighborhood around the disease genes, ignoring potentially informative indirect paths; (3) global clustering is widely applied to partition the network in an unsupervised manner, attributing little importance to prior knowledge; (4) confidence weights and their contribution to edge differentiation and ranking reliability are often disregarded. We hypothesize that network-based prioritization related to local clustering on graphs and considering full topology of weighted gene association networks integrating heterogeneous sources should overcome the above challenges. We term such a strategy Interactogeneous. We conducted cross-validation tests to assess the impact of network sources, alternative path inclusion and confidence weights on the prioritization of putative genes for 29 diseases. Heat diffusion ranking proved the best prioritization method overall, increasing the gap to neighborhood and shortest paths scores mostly on single source networks. Heterogeneous associations consistently delivered superior performance over single source data across the majority of methods. Results on the contribution of confidence weights were inconclusive. Finally, the best Interactogeneous strategy, heat diffusion ranking and associations from the STRING database, was used to prioritize genes for Parkinson’s disease. This method effectively recovered known genes and uncovered interesting candidates which could be linked to pathogenic mechanisms of the disease. Biomarkers play a crucial role in modern medicine as a means to improve accuracy in diagnosis, prognosis and treatment. In recent years, large-scale studies on the involvement of genes in disease have been empowered both by advances in high-throughput techniques and the proliferation of accessible resources of biological data. Manually inspecting the outstanding amounts of available ‘omics’ data is infeasible and can only be achieved by narrowing down the search space, often through a pre-selection of genes yielding higher potential using automatic recognition methods. Disease gene prioritization addresses this problem by generating ordered lists of candidates pertaining a particular gene-disease relatedness score. Most computational strategies follow a guilt-by-association approach, where new genes are implicated based on a measure of relatedness with a set of other genes known to be involved in the occurrence of a particular phenotype. Relatedness can be defined based on a number of properties, including intrinsic and functional Many studies A key feature in network prioritization with known genes is the set of paths considered when calculating disease-relatedness. Popular approaches use neighborhood measures based on direct links between the candidates and the known genes Random walk or diffusion kernel methods arise as natural choices to devise full topology scores and their application to gene prioritization has been proven effective vs neighborhood and shortest paths; (2) network - heterogeneous associations vs single source interactions; (3) confidence weights and other parameters. Finally, we test the effectiveness of the best prioritization strategy to recover prior knowledge and suggest genes likely to play a role in Parkinson’s disease (PD).In this work, we advocate for a particular category of network-based prioritization strategies, which we term Interactogeneous. These combine full topology scores computed using local clustering on graphs or diffusion kernels over confidence weighted gene association networks integrating evidence from heterogeneous sources. We formulate the hypothesis that Interactogeneous strategies should outperform approaches missing at least one of the described features. We investigate the validity of this statement in three case studies concerning the prioritization of genes for 29 diseases: (1) gene-disease score - full topology V and edge in E correspond to a gene and an association between two genes, respectively. Let A and D denote the adjacency and diagonal matrices of G, respectively. V given a set In this section, we first outline formal concepts and the prioritization problem. We then describe the prioritization methods and networks included in this study. An association or interaction network can be described as a weighted undirected graph One of our goals is to evaluate whether a full topology measure is more informative than a neighborhood or shortest paths score . In thisp, containing as many entries as genes in the network. Distinct schemes can be used to initialize p, whose instantiation is denoted by N steps and take Full topology scores were obtained with PageRank Heat diffusion is a discrete approximation of the heat kernel t, the diffusion coefficient. The iterative score update for gene v is given byPageRank with priors is an extension of the original PageRank to consider a custom initial distribution of the scores Disease neighbor weightingv, the weight of every edge linking v to a disease gene. Disease neighbor countingv, the number of direct neighbors known to be involved in the disease. Computing any of these two scores for all genes takes Disease neighborhood overlapv as the relative overlap between two sets containing: (i) disease genes and their direct neighbors; and (ii) v and its direct neighbors. This is the score computed by the Endeavour tool Three neighborhood scores were calculated. v to be the average of the lengths of the shortest paths between v and the disease genes. Computation of the shortest paths scores for all genes takes Different versions of shortest paths can be defined Absent genes and missing links affect the neighborhood of genes, and thus the overall network topology on which network-based methods rely to compute gene-disease scores, thus hampering the discovery of novel genes . EssentiSTRING database The HEFalMp is a network of functional gene associations supported by physical, genetic, sequence, or co-expression evidence We parsed a network of physical protein-protein interactions (PPI) from the NCBI Entrez Gene repository An additional network was extracted from STRING, ST-Text mining, containing associations based on the co-occurrence of gene pairs in literature abstracts. We prefer to classify ST-Text mining as a heterogeneous network, as it implicitly includes information from alternative sources . Upon separation of the distinct sources from STRING, it was necessary to convert the resulting protein-protein networks into gene-gene networks. However, choosing the edge of maximum weight between every pair of genes before or after the integration (as described for STRINGv8.2 above) would not yield identical integrated confidence weights. In order to preserve consistency between the interaction weights in the single source and integrated networks, eventually perturbed by the protein to gene conversion, we also built an integrated network from the single source networks, which we name ST-Integrated. We first performed the protein to gene network conversion per source, and then integrated all sources for every edge following the integration approach in the original STRING. Subfigure B shows the intersection between ST-Integrated and each of the extracted single source networks. Experiments with the ST-Integrated network were performed exclusively for comparison with the individual STRING sources. Throughout the text we refer to the original STRING network as STRINGv8.2 and to the custom integrated STRING network as ST-Integrated, whenever it matters. Additionally, we may use the term STRING to refer to either or both of these networks whenever they exhibit similar performances.Prioritization strategies were evaluated using 620 known disease-related genes automatically selected from OMIM Leave-one-out cross-validation tests were conducted . For eacPrioritizers return a real-valued function used to define a strictly ordered ranking. Let positive and negative classes of a binary classification problem denote association and non-association with disease, respectively. A binary outcome can be produced by sweeping an imposed cutoff over the ranks and plotting the corresponding receiver operating characteristic curve (ROC) of the true positive rate as a function of the false positive rate. In this case, the left-out gene ranking higher than the cutoff implies its association with the disease. AUC denotes the area under ROC and fits in r is the ratio between the number of relevant items and the total number of items retrieved at rr is thus 0 before it has been found, or MAP denotes the mean of the average precision of the rankings obtained for all left-out genes. Average precision combines precision, the probability that a randomly selected gene associated by the prioritizer is in fact related, and recall, the probability that a randomly selected gene known to be involved in the disease is associated by the prioritizer. It averages the precisions computed by truncating the ranked list after each relevant item is found, where precision at rank In this section, we investigate the impact of network features and ranking strategy on the prioritization task. We aim to validate three claims concerning the performance of Interactogeneous stategies against alternative approaches: (1) heterogeneous associations should outperform single source data, as multiple evidence increases coverage/density and reduces bias toward individual sources; (2) a full topology score resuming all paths is potentially more informative than measures based on direct links or shortest paths, deriving a whole-system perspective and avoiding excessive focus on well studied genes; (3) incorporating confidence weights should increase reliability, as it is expected to diminish the effect of false positives. Finally, we evaluate the effectiveness of the method to discover genes putatively associated with Parkinson’s disease (PD).Leave-one-out cross-validation tests were conducted. For each disease, in each evaluation run, a different gene was retained from the set of disease genes (training set). Prioritizers were applied to compute a score for every gene in the network, taking training genes as seeds. A test set was formed by adding the retained gene to a previously generated set of candidates. The genes in the test set were then ranked according to their gene-disease scores. We performed leave-one-out cross-validation in order to be able to evaluate all diseases, even those yielding only a few known genes, in most networks. Results of 5-fold cross-validation on the larger disease sets are also discussed below and made available in Full topology rankings were obtained with heat diffusion (HDiffusion) and PageRank (PRank). A measure based on shortest paths (SPaths) and three neighborhood scores, namely disease neighbor weighting (NWeight) or counting (NCount) and neighborhood overlap (EndNet), were selected for comparison. Heterogeneous association  and single source interaction (other STRING sources and NCBI PPI) networks were assessed see . Direct HDiffusion was the best method overall, consistently performing on top in all networks . PRank pHeterogeneous associations in STRING outperformed both HEFalMp and the NCBI PPI network using every method tested in this study and every of the 10 candidate sets generated from each pool of genes  and 5. CN, which was shown to be smaller for STRING and HEFalMp than for the PPI network. This could be explained by a stronger small world effect on the denser networks caused by the existence of shorter paths linking the pairs of nodes. Regarding the propagation rate, values t and t and equal N, HDiffusion pertained variations in the orders of N in the orders of In general, methods incorporating edge weights outperformed those ignoring them, in the networks where weights were available, namely STRING and HEFalMp , 2 and 3per se a specific method or class of methods, or a particular network or class of networks, in detriment of others. In this context, we aimed primarily at promoting a fair comparative evaluation procedure. For completeness, we conducted 5-fold cross-validation tests on the larger disease sets, those with 20 or more known disease genes present in the three major networks . Such results are presented in Supporting Information S1. We observed that most performance measures remained relatively stable, which allowed to maintain the relative performances of the several methods and the various networks.Leave-one-out cross-validation is not usually the preferred approach for performance evaluation, as it can lead to optimistic results. Nevertheless, its choice is widely accepted in the literature in cases where the data is scarce. In this work, the size of the seed sets was reasonably small and would prevent the application of a standard 10-fold cross-validation. Similarly, a 5-fold approach would immediately exclude 4 networks and 19 diseases due to insufficient coverage of the seed sets by the networks. On the other hand, our study focused on relative rather than absolute performances, and the leave-one-out procedure is not expected to favor Parkinson’s disease  is the second most common neurodegenerative disorder, after Alzheimer’s protein kinase, AMP-activated, gamma 2 non-catalytic subunit (PRKAG2), a gene involved in Wolf-Parkinson-White syndrome responsible for heart defects. All remaining genes have been linked to PD before. Mutations in genes synuclein, alpha (non A4 component of amyloid precursor) (SNCA) located in PARK1 and PARK4 and ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) (UCHL1) in PARK5 were identified in autosomal dominant families, while mutated parkinson protein 2, E3 ubiquitin protein ligase (parkin) (PARK2) in PARK2 and parkinson disease  7 (PARK7) in PARK7 have been implicated in autosomal recessive forms of monogenic PD Nuclear receptor subfamily 4, group A, member 2 (NR4A2), required for the development of dopaminergic neurons and associated with both autosomal dominant and sporadic PD microtubule-associated protein tau (MAPT), for which several haplotypes were indicated to influence age at onset and risk of PD NADH dehydrogenase (ubiquinone) flavoprotein 2, 24 kDa (NDUFV2), involved in electron transfer from NADH to the mitochondrial respiratory chain where the electron acceptor is believed to be ubiquinonecytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6), encoding an enzyme involved in dopamine metabolism, was included due to its multiple polymorphisms affecting susceptibility to PD The list of known PD related genes excludes recent findings and purposely lacks some of the well known disease causing factors in an attempt to assess the ability of the method to unravel novel and recover established knowledge on the disease. Additional noise was introduced with the inclusion of PRKAG2 ranked last among the seeds, confirming non-relatedness to PD. All seeds were filtered from the results. We investigate the first 20 genes, of which 18 could be associated with mechanisms of PD  gene in PARK6 known to cause autosomal recessive early-onset PD and the leucine-rich repeat kinase 2  gene in PARK8 often related to autosomal dominant late-onset variant synuclein, alpha interacting protein  known to cooperate with SNCA and PARK2 in the formation of Lewy bodies fibroblast growth factor 20 gene  possessing broad mitogenic and cell survival activities, ranked in the top 10. Variation in FGF20 has been linked to risk for PD, not confirmed by later studies ubiquitin C  and glutathione S-transferase omega 1 and 2 , hinted to influence age at onset in PD heat shock 70 kDa protein 4 . These genes are involved in mitochondrial activity, particularly linked to ubiquitination and chaperone function, whose dysfunction and impairment in PD leads to the degradation of proteins and accumulation of Lewy bodies HtrA serine peptidase 2 , located in PARK13 and found among the best 20, promotes cell death by binding apoptosis inhibitory proteins. Genetic variability in HTRA2 has been reported to cause PD, though studies showing no association have also been published solute carrier family 6 , member 3  and potassium inwardly-rectifying channel, subfamily J, member 6  tyrosine hydroxylase  monoamine oxidase B  glucosidase, beta, acid gene , top 20, significantly alter risk for sporadic and familial PD Mitochondrially encoded NADH dehydrogenase 3  is a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase complex I. Although MT-ND3 has not been specifically linked to PD, dysfunction of complex I is considered the major mitochondrial defect in PD ND2, ND5) have been filed. Presenilin 1  is known to carry mutations causing Alzheimer’s disease (AD). Impairment of presenilin-mediated signaling pathways leading to presynaptic dysfunction has been suggested as a converging event before neurodegeneration in AD and PD sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain and short cytoplasmic domain, (semaphorin) 5A  gene, involved in axonal guidance during neural development, connectivity maintenance and repair synuclein, beta , homologous to SNCA, has been found highly expressed in the substantia nigra SNCA aggregation and fibrillization SNCB might also influence age at onset In the top three, the FGF20 and KCNJ6 against TH and MAOB, for instance).Overall the ranking reflected the contribution of additional sources as, in several cases, genes with scarce literature evidence ranked higher than others with widely spread reference  were considered. These results support the reasoning that incorporating full topology with higher order neighborhoods and alternative paths (redundancies) potentiates a more comprehensive understanding of the interactome and ability to compensate for missing linkage. They further corroborate the findings in previous studies giving the lead to full topology scores against other measures based on direct neighbors, shortest paths and graph partitioning on protein-protein interaction networks Interactogeneous strategies proved successful. We used the best performing technique, combining heat diffusion ranking with the STRING network, to prioritize genes potentially involved in Parkinson’s disease. Known susceptibility genes widely referred in literature or databases were recovered. Analysis of the top ranked genes further revealed a number of genes with scarce or controversial evidence, most of which could be related to mechanisms of Parkinson’s disease: (1) impairment of mitochondrial activity and dopamine metabolism with oxidative stress leading to apoptotic death of dopaminergic neurons; (2) disruption of the ubiquitin proteasome system and consequent protein degradation with formation of Lewy bodies; (3) presynaptic dysfunction caused by perturbation of developmental signaling pathways; (4) compensatory mechanisms for cell survival and repair upon stress.Supporting Information S15-fold cross-validation results. This document contains the results obtained for all methods  on three networks  using the larger disease sets in a 5-fold cross-validation evaluation scheme.(PDF)Click here for additional data file.