<p>The sequencing of genomes has caused a growing demand for functional analysis of gene products. This research field named proteomics is derived from the term proteome, which by analogy to genome is defined as all proteins expressed by a cell or a tissue. Proteomics is however methodologically restricted to the analysis of proteins with higher molecular weights. The development of a technology which includes peptides with low molecular weight and small proteins is needed, since peptides play a central role in many biological processes. </p><p>To study endogenous peptides and hormones, the peptidome, an improved method comprising rapid deactivation in combination with nano-flow liquid chromatography (LC) and mass spectrometry (MS) was developed. The method has been used to investigate endogenous peptides in brains of mouse and rat. Several novel peptides have been discovered together with known neuropeptides. </p><p>To elucidate the <i>post mortem</i> time influence on peptides and proteins, a time course study was performed using peptidomics and proteomics technologies. Already after three minutes a substantial amount of protein fragments emerged in the peptidomics study and some endogenous peptides were drastically reduced with increasing <i>post mortem</i> time. Of about 1500 proteins investigated, 53 were found to be significantly changed at 10 minutes <i>post mortem</i> as compared to control. Moreover, using western blot the level of MAPK phosphorylation was shown to decrease by 95% in the 10 minutes <i>post mortem </i>sample. </p><p>A database, SwePep (a repository of endogenous peptides, hormones and small proteins), was constructed to facilitate identification using MS. The database also contains additional information concerning the peptides such as physical properties. A method for analysis of LC-MS data, including scanning for, and further profiling of, biologically significant peptides was developed. We show that peptides present in different amounts in groups of samples can be automatically detected.</p><p>The peptidome approach was used to investigate levels of peptides in two animal models of Parkinson’s disease. PEP-19, was found to be significantly decreased in the striatum of MPTP lesioned parkinsonian mice. The localization and expression was further investigated by imaging MALDI MS and by <i>in situ</i> hybridization. The brain peptidome of reserpine treated mice was investigated and displayed a number of significantly altered peptides. This thesis demonstrates that the peptidomics approach allows for the study of complex biochemical processes.</p>

The computational ability of principal neurons and interneurons in the brain and their ability to work together in concert are thought to underlie higher order cognitive processes such as learning, memory, and attention. Dendrites play a very important role in neuronal information processing because they receive and integrate incoming input and can undergo experience-dependent changes that will alter the future output of the neuron. Here, I have used whole-cell patch clamp recordings and fluorescent Ca2+-imaging to examine the modulation of dendritic excitability in principal neurons of the rat and human hippocampus and neocortex. First, I determined that dendrites of dentate granule cells of the hippocampus are tuned to high frequencies of both afferent input and backpropagating action potentials. Under these conditions they are also capable of generating regenerative dendritic activity that can propagate to the soma, which is prone to modulation. In particular, Neuropeptide Y (NPY) Y1 receptors can decrease frequency-dependent dendritic Ca2+ influx. Dopamine D1 receptors (D1Rs) have an opposite effect; they potentiate frequency-dependent dendritic excitability. These two neuromodulators also have an opposing effect on plasticity, with dopamine acting to induce, and NPY acting to inhibit long-term potentiation (LTP). Parallel observations of D1-induced LTP and an NPY-mediated decrease in dendritic excitability in rodents were complemented by findings in human dentate granule cells. Second, I examined the role of NPY receptors on dendrites of layer 5 pyramidal neurons. In these neurons I found that NPY acts post-synaptically on distal dendrites via the Y1 receptor to inhibit frequency-dependent Ca2+-currents, similar to the findings in dentate granule cells. NPY also decreased regenerative Ca2+ currents caused by the appropriate pairing of pre- and post-synaptic input. Together, these observations demonstrate that the role of NPY in the hippocampus and neocortex is not solely as an anti-epileptic agent. NPY release, likely to occur during high frequency oscillatory activity, can act locally to limit dendritic excitability, which can have a profound effect on plasticity. In the dentate gyrus, NPY can inhibit a D1R induced increased dendritic excitability and resultant changes in synaptic strength. These findings will further the understanding of dendritic information processing in the hippocampus and neocortex.

G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution. (c) 2006 Elsevier Ltd. All rights reserved.

The three evolutionarily related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are ligands to at least five G-protein coupled receptors in mammals, which are denoted by numbers. NPY has many physiological effects including stimulation of appetite and regulation of circadian rhythm and blood pressure. This work describes the ancient origin of the NPY receptor genes as deduced from molecular cloning of six receptors in four distantly related vertebrate species. Three of the receptors have been functionally expressed in vitro to determine ligand binding properties. The first Y2 receptor from any non-mammalian species was cloned from the chicken. The receptor was found to exhibit substantial structural and pharmacological differences to mammalian Y2, but showed similar anatomical distribution. A receptor was cloned in a primitive vertebrate, an agnathan fish, the river lamprey Lampetra fluviatilis. Phylogenetic analyses indicated that it represents an orthologue to the ancestor of Y4 and the teleost subtypes Yb and Yc. Three NPY receptors were cloned from a shark, the spiny dogfish Squalus acanthias. These were found to correspond to the three mammalian subtypes Y1, Y4 and y6, and was thereby the first complete Y1 subfamily in any species outside the mammalian lineage. This suggests that all three receptor subtypes arose in the common ancestor of sharks and mammals 420-450 million years ago. The sixth described receptor was cloned from the zebrafish, Danio rerio, and was shown to have equal identity to all three mammalian Y1 subfamily receptors. Phylogenetic analyses including the shark and lamprey sequences suggested that Yb may represent a fourth Y1 subfamily gene. It has previously been found that the genes for Y1, Y4 and y6 are located on separate chromosomes. Taken together, these results show that the NPY receptor family expanded by chromosomal duplications early in vertebrate evolution, prior to the origin of gnathostomes. This work will be important for the determination of the time points for the origin of the many functions of NPY as well as for the understanding of the processes that shaped the vertebrate genome.

The sequencing of genomes has caused a growing demand for functional analysis of gene products. This research field named proteomics is derived from the term proteome, which by analogy to genome is defined as all proteins expressed by a cell or a tissue. Proteomics is however methodologically restricted to the analysis of proteins with higher molecular weights. The development of a technology which includes peptides with low molecular weight and small proteins is needed, since peptides play a central role in many biological processes. To study endogenous peptides and hormones, the peptidome, an improved method comprising rapid deactivation in combination with nano-flow liquid chromatography (LC) and mass spectrometry (MS) was developed. The method has been used to investigate endogenous peptides in brains of mouse and rat. Several novel peptides have been discovered together with known neuropeptides. To elucidate the post mortem time influence on peptides and proteins, a time course study was performed using peptidomics and proteomics technologies. Already after three minutes a substantial amount of protein fragments emerged in the peptidomics study and some endogenous peptides were drastically reduced with increasing post mortem time. Of about 1500 proteins investigated, 53 were found to be significantly changed at 10 minutes post mortem as compared to control. Moreover, using western blot the level of MAPK phosphorylation was shown to decrease by 95% in the 10 minutes post mortem sample. A database, SwePep (a repository of endogenous peptides, hormones and small proteins), was constructed to facilitate identification using MS. The database also contains additional information concerning the peptides such as physical properties. A method for analysis of LC-MS data, including scanning for, and further profiling of, biologically significant peptides was developed. We show that peptides present in different amounts in groups of samples can be automatically detected. The peptidome approach was used to investigate levels of peptides in two animal models of Parkinson’s disease. PEP-19, was found to be significantly decreased in the striatum of MPTP lesioned parkinsonian mice. The localization and expression was further investigated by imaging MALDI MS and by in situ hybridization. The brain peptidome of reserpine treated mice was investigated and displayed a number of significantly altered peptides. This thesis demonstrates that the peptidomics approach allows for the study of complex biochemical processes.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 261-288).

The lateral septum is heavily implicated in anxiety regulation, with lesions or pharmacological inhibition of this region suppressing rats' defensive responses in various rat models of anxiety. My first objective was to explore the functional relationship between the lateral septum and its major afferent structure, the ventral hippocampus. Although these structures are extensively connected, it was not clear if they work in concert to regulate anxiety-like behaviours. This idea was tested using a pharmacological disconnection technique, whereby communication between these two structures was disabled by infusing the GABAA agonist muscimol into one side of the lateral septum and the contralateral side of the ventral hippocampus. Increases in open-arm exploration were evident when muscimol was co-infused into one side of the lateral septum and the contralateral ventral hippocampus. By contrast, open arm exploration was not altered when muscimol was co-infused into one side of the lateral septum and the ipsilateral ventral hippocampus. These results support the contention that the ventral hippocampus and the lateral septum regulate rats' open arm exploration in a serial fashion, and that this involves ipsilateral projections from the former to the latter site. My second objective was to further characterize the neuropharmacological aspects of lateral septal regulation of behavioural defence. The lateral septum contains high levels of NPY Y1 and Y2 receptor binding sites in the brain, yet little is known about their contribution in anxiety regulation at this site. Therefore, the second aim of my thesis was to characterize the contribution of NPY and its Y1 and Y2 receptor subtypes in the lateral septal regulation of anxiety in the elevated plus maze, novelty-induced suppression of feeding, and shock-probe burying tests. I determined that distinct NPY receptors differentially contribute to NPY-mediated anxiolysis in a test specific manner, with the Y1 receptor mediating NPY-induced anxiolysis in the novelty-induced suppression of feeding test, and the Y2 receptor mediating NPY13-36-induced anxiolysis in the plus-maze test. Taken together, the results from these studies reinforce the view that the regulation of anxiety involves a variety of different, yet overlapping neural processes. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2012-09-25 18:02:11.172

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