http://hdl.handle.net/10230/6237 2013-05-21T21:40:13Z http://hdl.handle.net/10230/20563 Sequencing primate genomes: what have we learned? Marquès i Bonet, Tomàs, 1975-; Ryder, Oliver A.; Eichler, Evan E. We summarize the progress in whole-genome sequencing and analyses of primate genomes. These emerging genome datasets have broadened our understanding of primate genome evolution revealing unexpected and complex patterns of evolutionary change. This includes the characterization of genome structural variation, episodic changes in the repeat landscape, differences in gene expression, new models regarding speciation, and the ephemeral nature of the recombination landscape. The functional characterization of genomic differences important in primate speciation and adaptation remains a significant challenge. Limited access to biological materials, the lack of detailed phenotypic data and the endangered status of many critical primate species have significantly attenuated research into the genetic basis of primate evolution. Next-generation sequencing technologies promise to greatly expand the number of available primate genome sequences; however, such draft genome sequences will likely miss critical genetic differences within complex genomic regions unless dedicated efforts are put forward to understand the full spectrum of genetic variation. 2009-01-01T00:00:00Z http://hdl.handle.net/10230/20558 A burst of segmental duplications in the genome of the African great ape ancestor Marquès i Bonet, Tomàs, 1975-; Kidd, Jeffrey M.; Ventura, Mario; Graves, Tina A.; Cheng, Ze; Hillier, LaDeana W.; Jiang, Zhaoshi; Baker, Carl; Malfavon-Borja, Ray; Fulton, Lucinda A.; Alkan, Can; Aksay, Gozde; Girirajan, Santhosh; Siswara, Priscillia; Chen, Lin; Cardone, Maria Francesca; Navarro i Cuartiellas, Arcadi, 1969-; Mardis, Elaine R.; Wilson, Richard K.; Eichler, Evan E. It is generally accepted that the extent of phenotypic change between human and great apes is dissonant with the rate of molecular change. Between these two groups, proteins are virtually identical, cytogenetically there are few rearrangements that distinguish ape-human chromosomes, and rates of single-base-pair change and retrotransposon activity have slowed particularly within hominid lineages when compared to rodents or monkeys. Studies of gene family evolution indicate that gene loss and gain are enriched within the primate lineage. Here, we perform a systematic analysis of duplication content of four primate genomes (macaque, orang-utan, chimpanzee and human) in an effort to understand the pattern and rates of genomic duplication during hominid evolution. We find that the ancestral branch leading to human and African great apes shows the most significant increase in duplication activity both in terms of base pairs and in terms of events. This duplication acceleration within the ancestral species is significant when compared to lineage-specific rate estimates even after accounting for copy-number polymorphism and homoplasy. We discover striking examples of recurrent and independent gene-containing duplications within the gorilla and chimpanzee that are absent in the human lineage. Our results suggest that the evolutionary properties of copy-number mutation differ significantly from other forms of genetic mutation and, in contrast to the hominid slowdown of single-base-pair mutations, there has been a genomic burst of duplication activity at this period during human evolution. 2009-01-01T00:00:00Z http://hdl.handle.net/10230/20557 The origins and impact of primate segmental duplications Marquès i Bonet, Tomàs, 1975-; Girirajan, Santosh; Eichler, Evan E. Duplicated sequences are substrates for the emergence of new genes and are an important source of genetic instability associated with rare and common diseases. Analyses of primate genomes have shown an increase in the proportion of interspersed segmental duplications (SDs) within the genomes of humans and great apes. This contrasts with other mammalian genomes that seem to have their recently duplicated sequences organized in a tandem configuration. In this review, we focus on the mechanistic origin and impact of this difference with respect to evolution, genetic diversity and primate phenotype. Although many genomes will be sequenced in the future, resolution of this aspect of genomic architecture still requires high quality sequences and detailed analyses. 2009-01-01T00:00:00Z http://hdl.handle.net/10230/20531 Sequencing human-gibbon breakpoints of synteny reveals mosaic new insertions at rearrangement sites Girirajan, Santosh; Chen, Lin; Graves, Tina; Marquès i Bonet, Tomàs, 1975-; Ventura, Mario; Fronick, Catrina; Fulton, Lucinda; Rocchi, Mariano; Fulton, Robert S.; Wilson, Richard K.; Mardis, Elaine R.; Eichler, Evan E. The gibbon genome exhibits extensive karyotypic diversity with an increased rate of chromosomal rearrangements during evolution. In an effort to understand the mechanistic origin and implications of these rearrangement events, we sequenced 24 synteny breakpoint regions in the white-cheeked gibbon (Nomascus leucogenys, NLE) in the form of high-quality BAC insert sequences (4.2 Mbp). While there is a significant deficit of breakpoints in genes, we identified seven human gene structures involved in signaling pathways (DEPDC4, GNG10), phospholipid metabolism (ENPP5, PLSCR2), beta-oxidation (ECH1), cellular structure and transport (HEATR4), and transcription (ZNF461), that have been disrupted in the NLE gibbon lineage. Notably, only three of these genes show the expected evolutionary signatures of pseudogenization. Sequence analysis of the breakpoints suggested both nonclassical nonhomologous end-joining (NHEJ) and replication-based mechanisms of rearrangement. A substantial number (11/24) of human-NLE gibbon breakpoints showed new insertions of gibbon-specific repeats and mosaic structures formed from disparate sequences including segmental duplications, LINE, SINE, and LTR elements. Analysis of these sites provides a model for a replication-dependent repair mechanism for double-strand breaks (DSBs) at rearrangement sites and insights into the structure and formation of primate segmental duplications at sites of genomic rearrangements during evolution. 2009-01-01T00:00:00Z http://hdl.handle.net/10230/20530 Death and resurrection of the human IRGM gene Bekpen, Cemalettin; Marquès i Bonet, Tomàs, 1975-; Alkan, Can; Antonacci, Francesca; Leogrande, Maria Bruna; Ventura, Mario; Kidd, Jeffrey M.; Siswara, Priscillia; Howard, Jonathan C.; Eichler, Evan E. Immunity-related GTPases (IRG) play an important role in defense against intracellular pathogens. One member of this gene family in humans, IRGM, has been recently implicated as a risk factor for Crohn's disease. We analyzed the detailed structure of this gene family among primates and showed that most of the IRG gene cluster was deleted early in primate evolution, after the divergence of the anthropoids from prosimians ( about 50 million years ago). Comparative sequence analysis of New World and Old World monkey species shows that the single-copy IRGM gene became pseudogenized as a result of an Alu retrotransposition event in the anthropoid common ancestor that disrupted the open reading frame (ORF). We find that the ORF was reestablished as a part of a polymorphic stop codon in the common ancestor of humans and great apes. Expression analysis suggests that this change occurred in conjunction with the insertion of an endogenous retrovirus, which altered the transcription initiation, splicing, and expression profile of IRGM. These data argue that the gene became pseudogenized and was then resurrected through a series of complex structural events and suggest remarkable functional plasticity where alleles experience diverse evolutionary pressures over time. Such dynamism in structure and evolution may be critical for a gene family locked in an arms race with an ever-changing repertoire of intracellular parasites. 2009-01-01T00:00:00Z http://hdl.handle.net/10230/20529 Gorilla genome structural variation reveals evolutionary parallelisms with chimpanzee Ventura, Mario; Catacchio, Claudia R.; Alkan, Can; Marquès i Bonet, Tomàs, 1975-; Saijadian, Saba; Graves, Tina A.; Hormozdiari, Fereydoun; Navarro i Cuartiellas, Arcadi, 1969-; Malig, Maika; Baker, Carl; Lee, Choli; Turner, Emily H.; Chen, Lin; Kidd, Jeffrey M.; Archidiacono, Nicoletta; Shendure, Jay; Wilson, Richard K.; Eichler, Evan E. Structural variation has played an important role in the evolutionary restructuring of human and great ape genomes. Recent analyses have suggested that the genomes of chimpanzee and human have been particularly enriched for this form of genetic variation. Here, we set out to assess the extent of structural variation in the gorilla lineage by generating 10-fold genomic sequence coverage from a western lowland gorilla and integrating these data into a physical and cytogenetic framework of structural variation. We discovered and validated over 7665 structural changes within the gorilla lineage, including sequence resolution of inversions, deletions, duplications, and mobile element insertions. A comparison with human and other ape genomes shows that the gorilla genome has been subjected to the highest rate of segmental duplication. We show that both the gorilla and chimpanzee genomes have experienced independent yet convergent patterns of structural mutation that have not occurred in humans, including the formation of subtelomeric heterochromatic caps, the hyperexpansion of segmental duplications, and bursts of retroviral integrations. Our analysis suggests that the chimpanzee and gorilla genomes are structurally more derived than either orangutan or human genomes. 2011-01-01T00:00:00Z http://hdl.handle.net/10230/20473 Pathway databases and tools for their exploitation: benefits, current limitations and challenges Bauer-Mehren, Anna; Furlong, Laura I.; Sanz, Ferran In past years, comprehensive representations of cell signalling pathways have been developed by manual curation from literature, which requires huge effort and would benefit from information stored in databases and from automatic retrieval and integration methods. Once a reconstruction of the network of interactions is achieved, analysis of its structural features and its dynamic behaviour can take place. Mathematical modelling techniques are used to simulate the complex behaviour of cell signalling networks, which ultimately sheds light on the mechanisms leading to complex diseases or helps in the identification of drug targets. A variety of databases containing information on cell signalling pathways have been developed in conjunction with methodologies to access and analyse the data. In principle, the scenario is prepared to make the most of this information for the analysis of the dynamics of signalling pathways. However, are the knowledge repositories of signalling pathways ready to realize the systems biology promise? In this article we aim to initiate this discussion and to provide some insights on this issue. 2009-01-01T00:00:00Z http://hdl.handle.net/10230/20472 Gene-disease network analysis reveals functional modules in mendelian, complex and environmental diseases Bauer-Mehren, Anna; Bundschus, Markus; Rautschka, Michael; Mayer, Miguel Ángel; Sanz, Ferran; Furlong, Laura I. Abstract BACKGROUND: Scientists have been trying to understand the molecular mechanisms of diseases to design preventive and therapeutic strategies for a long time. For some diseases, it has become evident that it is not enough to obtain a catalogue of the disease-related genes but to uncover how disruptions of molecular networks in the cell give rise to disease phenotypes. Moreover, with the unprecedented wealth of information available, even obtaining such catalogue is extremely difficult. PRINCIPAL FINDINGS: We developed a comprehensive gene-disease association database by integrating associations from several sources that cover different biomedical aspects of diseases. In particular, we focus on the current knowledge of human genetic diseases including mendelian, complex and environmental diseases. To assess the concept of modularity of human diseases, we performed a systematic study of the emergent properties of human gene-disease networks by means of network topology and functional annotation analysis. The results indicate a highly shared genetic origin of human diseases and show that for most diseases, including mendelian, complex and environmental diseases, functional modules exist. Moreover, a core set of biological pathways is found to be associated with most human diseases. We obtained similar results when studying clusters of diseases, suggesting that related diseases might arise due to dysfunction of common biological processes in the cell. CONCLUSIONS: For the first time, we include mendelian, complex and environmental diseases in an integrated gene-disease association database and show that the concept of modularity applies for all of them. We furthermore provide a functional analysis of disease-related modules providing important new biological insights, which might not be discovered when considering each of the gene-disease association repositories independently. Hence, we present a suitable framework for the study of how genetic and environmental factors, such as drugs, contribute to diseases. AVAILABILITY: The gene-disease networks used in this study and part of the analysis are available at http://ibi.imim.es/DisGeNET/DisGeNETweb.html#Download 2011-01-01T00:00:00Z http://hdl.handle.net/10230/20467 Automatic filtering and substantiation of drug safety signals Bauer-Mehren, Anna; van Mullingen, Erik M.; Avillach, Paul; Carrascosa, Maria del Carmen; García Serna, Ricard; Piñero, Janet; Singh, Barat; Lopes, Pedro; Oliveira, José Luis; Diallo, Gayo; Ahlberg Helgee, Ernest; Boyer, Scott; Mestres, Jordi; Sanz, Ferran; Kors, Jan A.; Furlong, Laura I. Drug safety issues pose serious health threats to the population and constitute a major cause of mortality worldwide. Due to the prominent implications to both public health and the pharmaceutical industry, it is of great importance to unravel the molecular mechanisms by which an adverse drug reaction can be potentially elicited. These mechanisms can be investigated by placing the pharmaco-epidemiologically detected adverse drug reaction in an information-rich context and by exploiting all currently available biomedical knowledge to substantiate it. We present a computational framework for the biological annotation of potential adverse drug reactions. First, the proposed framework investigates previous evidences on the drug-event association in the context of biomedical literature (signal filtering). Then, it seeks to provide a biological explanation (signal substantiation) by exploring mechanistic connections that might explain why a drug produces a specific adverse reaction. The mechanistic connections include the activity of the drug, related compounds and drug metabolites on protein targets, the association of protein targets to clinical events, and the annotation of proteins (both protein targets and proteins associated with clinical events) to biological pathways. Hence, the workflows for signal filtering and substantiation integrate modules for literature and database mining, in silico drug-target profiling, and analyses based on gene-disease networks and biological pathways. Application examples of these workflows carried out on selected cases of drug safety signals are discussed. The methodology and workflows presented offer a novel approach to explore the molecular mechanisms underlying adverse drug reactions 2012-01-01T00:00:00Z http://hdl.handle.net/10230/20451 The EU-ADR Web Platform: delivering advanced pharmacovigilance tools Oliveira, José Luis; Lopes, Pedro; Nunes, Tiago; Campos, David; Boyer, Scott; Ahlberg Helgee, Ernst; van Mulligen, Erik M.; Kors, Jan A.; Singh, Barat; Furlong, Laura I.; Sanz, Ferran; Bauer-Mehren, Anna; Carrascosa, María del Carmen; Mestres, Jordi; Avillach, Paul; Diallo, Gayo; Díaz Acedo, Carlos; van der Lei, Johan PURPOSE: Pharmacovigilance methods have advanced greatly during the last decades, making post-market drug assessment an essential drug evaluation component. These methods mainly rely on the use of spontaneous reporting systems and health information databases to collect expertise from huge amounts of real-world reports. The EU-ADR Web Platform was built to further facilitate accessing, monitoring and exploring these data, enabling an in-depth analysis of adverse drug reactions risks. METHODS: The EU-ADR Web Platform exploits the wealth of data collected within a large-scale European initiative, the EU-ADR project. Millions of electronic health records, provided by national health agencies, are mined for specific drug events, which are correlated with literature, protein and pathway data, resulting in a rich drug-event dataset. Next, advanced distributed computing methods are tailored to coordinate the execution of data-mining and statistical analysis tasks. This permits obtaining a ranked drug-event list, removing spurious entries and highlighting relationships with high risk potential. RESULTS: The EU-ADR Web Platform is an open workspace for the integrated analysis of pharmacovigilance datasets. Using this software, researchers can access a variety of tools provided by distinct partners in a single centralized environment. Besides performing standalone drug-event assessments, they can also control the pipeline for an improved batch analysis of custom datasets. Drug-event pairs can be substantiated and statistically analysed within the platform's innovative working environment. CONCLUSIONS: A pioneering workspace that helps in explaining the biological path of adverse drug reactions was developed within the EU-ADR project consortium. This tool, targeted at the pharmacovigilance community, is available online at https://bioinformatics.ua.pt/euadr/. Copyright © 2012 John Wiley & Sons, Ltd. 2012-01-01T00:00:00Z http://hdl.handle.net/10230/16981 La difusa frontera entre biología y computación (o hacia la mecanización de la ciencia) Guigó Serra, Roderic 2007-01-01T00:00:00Z http://hdl.handle.net/10230/16729 Motivational effects of cannabinoids are mediated by μ-opioid and k-opioid receptor Ghozland, Sandy; Matthes, Hans W. D.; Simonin, Frederic; Filliol, Dominique; Kieffer, Brigitte L.; Maldonado, Rafael Repeated THC administration produces motivational and somatic adaptive changes leading to dependence in rodents. To investigate the molecular basis for cannabinoid dependence and its possible relationship with the endogenous opioid system, we explored Δ9-tetrahydrocannabinol (THC) activity in mice lacking μ-, δ- or κ-opioid receptor genes. Acute THCinduced hypothermia, antinociception, and ypolocomotion remained unaffected in these mice, whereas THC tolerance and withdrawal were minimally modified in mutant animals. In contrast, profound phenotypic changes are observed in several place conditioning protocols that reveal both THC rewarding and aversive properties. Absence of μ receptors abolishes THC place preference. Deletion of κ receptors ablates THC place aversion and furthermore unmasks THC place preference. Thus, an opposing activity of μ- and κ-opioid receptors in modulating reward pathways forms the basis for the dual euphoric–dysphoric activity of THC. 2002-01-01T00:00:00Z http://hdl.handle.net/10230/16675 Stable complexes involving acetylcholinesterase and amyloid-ß-peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils Álvarez, Alejandra; Alarcón, Rodrigo; Opazo, Carlos; Campos, Eliseo O.; Muñoz López, Francisco José; Calderón, Frances H.; Dajas, Federico; Gentry, Mary K.; Doctor, Bhupendra P.; Mello, Fernando G. de; Inestrosa, Nibaldo C. Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain. 1998-01-01T00:00:00Z http://hdl.handle.net/10230/16674 Activity of the δ-Opioid Receptor Is Partially Reduced, Whereas Activity of the κ-Receptor Is Maintained in Mice Lacking the μ-Receptor Matthes, Hans W. D.; Smadja, C.; Valverde, Olga; Vonesch, J. L.; Foutz, A. S.; Boudinot, E.; Denavit-Saubié, M.; Severini, Cintia; Negri, Lucia; Roques, B. P.; Maldonado, Rafael; Kieffer, Brigitte L. Previous pharmacological studies have indicated the possible existence of functional interactions between μ-, δ- and κ-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of δ- and κ-opioid receptors in mice lacking the μ-opioid receptor (MOR). Measurements of agonist-induced [35S]GTPγS binding and adenylyl cyclase inhibition showed that functional coupling of δ- and κ-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[d-penicillamine2,d-penicillamine5] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR −/− mice. δ-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR −/− mice. Analgesic and respiratory responses produced by the selective κ-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of δ- and κ-receptor signaling properties in mice lacking μ-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the κ-agonist further suggest that the κ-receptor mainly acts independently from the μ-receptor in vivo. Reduced δ-analgesia and the absence of δ-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between μ-receptors and central δ-receptors in specific neuronal pathways. 1998-01-01T00:00:00Z http://hdl.handle.net/10230/16673 K(+) channel expression distinguishes subpopulations of parvalbumin- and somatostatin-containing neocortical interneurons Chow, Alan; Erisir, A.; Farb, C.; Nadal, M. S.; Ozaita Mintegui, Andrés; Lau, David; Welker, E.; Rudy, Bernardo Kv3.1 and Kv3.2 K+ channel proteins form similar voltage-gated K+ channels with unusual properties, including fast activation at voltages positive to −10 mV and very fast deactivation rates. These properties are thought to facilitate sustained high-frequency firing. Kv3.1 subunits are specifically found in fast-spiking, parvalbumin (PV)-containing cortical interneurons, and recent studies have provided support for a crucial role in the generation of the fast-spiking phenotype. Kv3.2 mRNAs are also found in a small subset of neocortical neurons, although the distribution of these neurons is different. We raised antibodies directed against Kv3.2 proteins and used dual-labeling methods to identify the neocortical neurons expressing Kv3.2 proteins and to determine their subcellular localization. Kv3.2 proteins are prominently expressed in patches in somatic and proximal dendritic membrane as well as in axons and presynaptic terminals of GABAergic interneurons. Kv3.2 subunits are found in all PV-containing neurons in deep cortical layers where they probably form heteromultimeric channels with Kv3.1 subunits. In contrast, in superficial layer PV-positive neurons Kv3.2 immunoreactivity is low, but Kv3.1 is still prominently expressed. Because Kv3.1 and Kv3.2 channels are differentially modulated by protein kinases, these results raise the possibility that the fast-spiking properties of superficial- and deep-layer PV neurons are differentially regulated by neuromodulators. Interestingly, Kv3.2 but not Kv3.1 proteins are also prominent in a subset of seemingly non-fast-spiking, somatostatin- and calbindin-containing interneurons, suggesting that the Kv3.1–Kv3.2 current type can have functions other than facilitating high-frequency firing. 1999-01-01T00:00:00Z http://hdl.handle.net/10230/16672 Involvement of the extracellular signal-regulated kinase cascade for cocaine-rewarding properties Valjent, Emmanuel; Corvol, Jean-Christophe; Pagès, Christiane; Besson, Marie-J.; Maldonado, Rafael; Caboche, Jocelyne A central feature of drugs of abuse is to induce gene expression in discrete brain structures that are critically involved in behavioral responses related to addictive processes. Although extracellular signal-regulated kinase (ERK) has been implicated in several neurobiological processes, including neuronal plasticity, its role in drug addiction remains poorly understood. This study was designed to analyze the activation of ERK by cocaine, its involvement in cocaine-induced early and long-term behavioral effects, as well as in gene expression. We show, by immunocytochemistry, that acute cocaine administration activates ERK throughout the striatum, rapidly but transiently. This activation was blocked when SCH 23390 [a specific dopamine (DA)-D1 antagonist] but not raclopride (a DA-D2 antagonist) was injected before cocaine. Glutamate receptors of NMDA subtypes also participated in ERK activation, as shown after injection of the NMDA receptor antagonist MK 801. The systemic injection of SL327, a selective inhibitor of the ERK kinase MEK, before cocaine, abolished the cocaine-induced ERK activation and decreased cocaine-induced hyperlocomotion, indicating a role of this pathway in events underlying early behavioral responses. Moreover, the rewarding effects of cocaine were abolished by SL327 in the place-conditioning paradigm. Because SL327 antagonized cocaine-induced c-fos expression and Elk-1 hyperphosphorylation, we suggest that the ERK intracellular signaling cascade is also involved in the prime burst of gene expression underlying long-term behavioral changes induced by cocaine. Altogether, these results reveal a new mechanism to explain behavioral responses of cocaine related to its addictive properties. 2000-01-01T00:00:00Z http://hdl.handle.net/10230/16671 Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins Lau, David; Vega-Saenz de Miera, Eleazar; Contreras, Diego; Ozaita Mintegui, Andrés; Harvey, Michael; Chow, Alan; Noebels, Jeffrey L.; Paylor, Richard; Morgan, James I.; Leonard, Christopher S.; Rudy, Bernardo Voltage-gated K+ channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 −/− mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing. 2000-01-01T00:00:00Z http://hdl.handle.net/10230/16670 Cannabinoid withdrawal syndrome is reduced in pre-proenkephalin knock-out mice Valverde, Olga; Maldonado, Rafael; Valjent, Emmanuel; Zimmer, Anne M.; Zimmer, Andreas The functional interactions between the endogenous cannabinoid and opioid systems were evaluated in pre-proenkephalin-deficient mice. Antinociception induced in the tail-immersion test by acute Delta9-tetrahydrocannabinol was reduced in mutant mice, whereas no difference between genotypes was observed in the effects induced on body temperature, locomotion, or ring catalepsy. During a chronic treatment with Delta9-tetrahydrocannabinol, the development of tolerance to the analgesic responses induced by this compound was slower in mice lacking enkephalin. In addition, cannabinoid withdrawal syndrome, precipitated in Delta9-tetrahydrocannabinol-dependent mice by the injection of SR141716A, was significantly attenuated in mutant mice. These results indicate that the endogenous enkephalinergic system is involved in the antinociceptive responses of Delta9-tetrahydrocannabinol and participates in the expression of cannabinoid abstinence. 2000-01-01T00:00:00Z http://hdl.handle.net/10230/16669 Bradykinin, but not muscarinic, inhibition of M-current in rat sympathetic ganglion neurons involves phospholipase C-β4 Haley, Jane E.; Abogadie, Fe C.; Fernández-Fernández, José Manuel; Dayrell, Mariza; Vallis, Yvonne; Buckley, Noel J.; Brown, David A. Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (I-K(M)), which can be inhibited by activation of M-1 muscarinic receptors (M-1 mAChR) and bradykinin (BK) B-2 receptors. Inhibition by the M1 mAChR agonist oxotremorine methiodide (Oxo-M) is mediated, at least in part, by the pertussis toxin-insensitive G-protein G alpha (q) (Caulfield et al., 1994; Haley et al., 1998a), whereas BK inhibition involves G alpha (q) and/or G alpha (11) (Jones et al., 1995). G alpha (q) and G alpha (11) can stimulate phospholipase C-beta (PLC-beta), raising the possibility that PLC is involved in I-K(M) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-beta1, - beta2, - beta3, and - beta4 in rat SCG. We have tested the role of two PLC isoforms (PLC-beta1 and PLC-beta4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3'-untranslated regions of PLC-beta1 and PLC-beta4, were injected into the nucleus of dissociated SCG neurons. Injected cells showed reduced antibody staining for the relevant PLC-beta isoform when compared to uninjected cells 48 hr later. BK inhibition of I-K(M) was significantly reduced 48 hr after injection of the PLC-beta4, but not the PLC-beta1, antisense-encoding plasmid. Neither PLC-beta antisense altered M-1 mAChR inhibition by Oxo-M. These data support the conclusion of Cruzblanca et al. (1998) that BK, but not M-1 mAChR, inhibition of I-K(M) involves PLC and extends this finding by indicating that PLC-beta4 is involved. 2000-01-01T00:00:00Z http://hdl.handle.net/10230/16668 Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient Mice Otto, Christiane; Kovalchuk, Yury; Wolfer, David Paul; Gass, Peter; Martin, Miguel; Zuschratter, Werner; Gröne, Hermann Josef; Kellendonk, Christoph; Tronche, François; Maldonado, Rafael; Lipp, Hans-Peter; Konnerth, Arthur; Schütz, Günter The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, harboring either a complete or a forebrain-specific inactivation of PAC1. Mutants from both strains show a deficit in contextual fear conditioning, a hippocampus-dependent associative learning paradigm. In sharp contrast, amygdala-dependent cued fear conditioning remains intact. Interestingly, no deficits in other hippocampus-dependent tasks modeling declarative learning such as the Morris water maze or the social transmission of food preference are observed. At the cellular level, the deficit in hippocampus-dependent associative learning is accompanied by an impairment of mossy fiber long-term potentiation (LTP). Because the hippocampal expression of PAC1 is restricted to mossy fiber terminals, we conclude that presynaptic PAC1-mediated signaling at the mossy fiber synapse is involved in both LTP and hippocampus-dependent associative learning. 2001-01-01T00:00:00Z