Single prazosin infusion in prelimbic cortex Fosters extinction of amphetamine-induced conditioned place preference

Exposure to drug-associated cues to induce extinction is a useful strategy to contrast cue-induced drug seeking. Norepinephrine (NE) transmission in medial prefrontal cortex has a role in the acquisition and extinction of conditioned place preference induced by amphetamine. We have reported recently that NE in prelimbic cortex delays extinction of amphetamine-induced conditioned place preference (CPP). A potential involvement of α1-adrenergic receptors in the extinction of appetitive conditioned response has been also suggested, although their role in prelimbic cortex has not been yet fully investigated. Here, we investigated the effects of the α1-adrenergic receptor antagonist prazosin infusion in the prelimbic cortex of C57BL/6J mice on expression and extinction of amphetamine-induced CPP. Acute prelimbic prazosin did not affect expression of amphetamine-induced CPP on the day of infusion, while in subsequent days it produced a clear-cut advance of extinction of preference for the compartment previously paired with amphetamine (Conditioned stimulus, CS). Moreover, prazosin-treated mice that had extinguished CS preference showed increased mRNA expression of brain-derived neurotrophic factor (BDNF) and post-synaptic density 95 (PSD-95) in the nucleus accumbens shell or core, respectively, thus suggesting that prelimbic α1-adrenergic receptor blockade triggers neural adaptations in subcortical areas that could contribute to the extinction of cue-induced drug-seeking behavior. These results show that the pharmacological blockade of α1-adrenergic receptors in prelimbic cortex by a single infusion is able to induce extinction of amphetamine-induced CPP long before control (vehicle) animals, an effect depending on contingent exposure to retrieval, since if infused far from or after reactivation it did not affect preference. Moreover, they suggest strongly that the behavioral effects depend on post-treatment neuroplasticity changes in corticolimbic network, triggered by a possible “priming” effect of prazosin, and point to a potential therapeutic power of the antagonist for maladaptive memories

Of Single Nucleotides and Single Cells: Charting the Genotype-Phenotype Map at High Resolution Using \u3ci\u3eDrosophila melanogaster\u3c/i\u3e

Understanding the mechanisms by which genetic variation brings about phenotypic variation is essential for understanding variation in complex traits. Drosophila melanogaster is a powerful model organism for such studies. Flies are easy to raise in the laboratory under controlled genetic and environmental conditions and many genetic tools are widely available. To chart the genotype-phenotype map, we need to study how individual genetic variants contribute to phenotypic variation, as well as how environmental perturbations influence gene expression. Regarding the former, I generated single nucleotide substitutions in Obp56h in a common genetic background. Obp56h, a member of the Odorant binding protein multigene family, is a small gene in a favorable genomic location for CRISPR-Cas9 mediated deletion. After deletion, I reinserted the gene at the endogenous locus with individual allelic variants chosen from those segregating in a wild-derived inbred population to produce five lines varying at single nucleotides in a common genetic background. Different alleles, both within and near the gene (potentially regulatory) and both common and rare, have different, large effects on organismal fitness traits as well as on genome-wide coregulated ensembles of transcripts. These effects are at the level of mean and microenvironmental variance in both fitness traits and the transcriptome. However, these alleles have only small effects on fitness traits in the wild-derived inbred population indicating that the effects of individual alleles can be context-specific and are perhaps suppressed in natural populations via epistatic interactions. Next, I studied how acute cocaine consumption and developmental alcohol exposure affect the transcriptome at single-cell resolution. The Drosophila brain is small, allowing for comprehensive whole-brain studies. Further, previous studies have characterized effects of acute cocaine consumption and developmental alcohol exposure on flies, which resemble those in humans. Single-cell RNA sequencing revealed that the transcriptomes of cells in the fly brain are affected in a cell-type and sex-dependent manner after the flies consumed fixed amounts of cocaine or are exposed to developmental alcohol exposure. These effects are sexually dimorphic, with males showing a greater degree of differential expression and are particularly prominent in glial and mushroom body cells. Developmental alcohol exposure leads to a similar, but different, sexually dimorphic and cell-type dependent pattern of differential expression as cocaine consumption. Some mechanisms are shared between the experimental paradigms indicating common processes. The strategies used in the studies described in this dissertation can be generally applied to explore genotype-phenotype relationships at high resolution

Interpretation of psychiatric genome-wide association studies with multispecies heterogeneous functional genomic data integration.

Genome-wide association studies and other discovery genetics methods provide a means to identify previously unknown biological mechanisms underlying behavioral disorders that may point to new therapeutic avenues, augment diagnostic tools, and yield a deeper understanding of the biology of psychiatric conditions. Recent advances in psychiatric genetics have been made possible through large-scale collaborative efforts. These studies have begun to unearth many novel genetic variants associated with psychiatric disorders and behavioral traits in human populations. Significant challenges remain in characterizing the resulting disease-associated genetic variants and prioritizing functional follow-up to make them useful for mechanistic understanding and development of therapeutics. Model organism research has generated extensive genomic data that can provide insight into the neurobiological mechanisms of variant action, but a cohesive effort must be made to establish which aspects of the biological modulation of behavioral traits are evolutionarily conserved across species. Scalable computing, new data integration strategies, and advanced analysis methods outlined in this review provide a framework to efficiently harness model organism data in support of clinically relevant psychiatric phenotypes

Understanding the Implications of Anandamide, an Endocannabinoid in an Early Land Plant, Physcomitrella patens

Endocannabinoid signaling is well studied in mammals and known to be involved in numerous pathological and physiological processes. Fatty acid amide hydrolase (FAAH) terminates endocannabinoid signaling in mammals. In Physcomitrella patens, we identified nine orthologs of FAAH (PpFAAH1 to PpFAAH9) with the characteristic catalytic triad and amidase signature sequence. Kinetics of PpFAAH1 showed specificity towards anandamide (AEA) at 37°C and pH 8.0. Further biophysical and bioinformatic analyses revealed that, structurally, PpFAAH1 to PpFAAH4 were closely associated to the plant FAAH whereas PpFAAH6 to PpFAAH9 were more closely associated to the animal FAAH. A substrate entry gate or ‘dynamic paddle’ in FAAH is fully formed in vertebrates but absent or not fully developed in non-vertebrates and plants. In planta analysis revealed that PpFAAH responded differently with saturated and unsaturated N-acylethanolamines (NAEs). In vivo amidohydrolase activity showed specificity associated with developmental stages. Additionally, overexpression of PpFAAH1 indicated the need for NAEs in developmental transition. To understand and identify key molecules related to endocannabinoid signaling in P. patens, we used high-throughput RNA sequencing. We analyzed temporal expression of mRNA and long non-coding RNA (lncRNA) in response not only to exogenous anandamide but also its precursor arachidonic acid and abscisic acid (ABA, a stress hormone). From the 40 RNA-seq libraries generated, we identified 4244 novel lncRNAs. The highest number of differentially expressed genes (DEGs) for both mRNA and lncRNA were detected on short-term exposure (1 h) to AEA. Furthermore, gene ontology enrichment analysis showed that 17 genes related to activation of the G protein-coupled receptor signaling pathway were highly expressed along with a number of genes associated with organelle relocation and localization. We identified key signaling components of AEA that showed significant difference when compared with ABA. This study provides a fundamental understanding of novel endocannabinoid signaling in early land plants and a future direction to elucidate its functional role

Valence in the Nucleus Accumbens - identifying neural populations in appetitive and aversive responses

Dissertação de mestrado em Ciências da SaúdeO núcleo accumbens (NAc) é reconhecido como um componente essencial do circuito de recompensa, estando associado ao processamento de eventos recompensadores e aversivos e contribuindo para comportamentos motivados. O NAc é uma “interface límbico-motora”, e estudos mostram o seu envolvimento em codificar valência - o valor intrínseco de uma certa experiência e consequentes respostas emocionais e motivacionais. O NAc é constituído maioritariamente por neurónios espinhosos médios GABAérgicos (MSNs), divididos naqueles que expressam o recetor de dopamina D1 (D1-MSNs) e nos que expressam o recetor de dopamina D2 (D2-MSNs). Estas populações foram tradicionalmente segregadas anatomicamente (via direta vs indireta) e funcionalmente (recompensa e valência positiva vs aversão e valência negativa). No entanto, muitos estudos recentes desafiaram esta segregação simplista. Porém, é ainda incerto quais as populações neuronais que codificam valência no NAc, e esta é uma questão crucial na compreensão de distúrbios com défices emocionais. Nesta dissertação, avaliámos os níveis de ativação neuronal associados com estímulos de valência negativa (choque na pata) ou positiva (cocaína) no NAc, usando a amígdala basolateral (BLA) e central (CeA) como regiões controlo, usadas por codificarem valência. Também caracterizámos um vetor viral controlado por c-fos para estabelecer uma estratégia de marcação de ativação neuronal para trabalho futuro. Além disso, efetuámos ativação optogenética de neurónios responsivos a estímulos na BLA, usando o mesmo vetor, para induzir comportamentos de valência e validar a nossa metodologia. Os nossos dados mostram que o NAc core (NAcc) e a BLA contêm populações neuronais que respondem ao choque, medido por uma maior densidade de células c-fos+ em comparação os controlos. Porém, a cocaína não induziu alterações significativas de ativação neuronal. Quanto ao vetor (conduzindo expressão de channelrhodopsin-eYFP), determinámos que 16h pós-exposição a estímulo seria o período mais adequado para observar marcação viral. Ao usar este vetor e expor murganhos a estímulos positivos ou negativos, dados de densitometria de fluorescência mostraram apenas uma tendência para maior ativação neuronal na BLA após o choque na pata, sem efeitos no NAc, em comparação com animais controlo; sem efeitos devido à cocaína. Por último, ativação optogenética de neurónios responsivos a choque na pata da BLA induziu uma tendência de evasão num teste de preferência de lugar em tempo real (RTPP), contudo essa mesma ativação não induziu preferência de lugar condicionada (CPP). Embora os nossos dados mostrem ativação neuronal no NAcc e BLA em resposta a choque, a falta de diferenças na exposição a cocaína, junto com dados de densitometria e optogenética, indicam a necessidade de desenvolver novas ferramentas para marcar neurónios que codifiquem valência no NAc.The nucleus accumbens (NAc) is recognized as an essential component of the reward circuit, being associated with processing of both rewarding and aversive events, and contributing for motivated behaviours. The NAc is a “limbic-motor interface”, and evidence shows its involvement in valence encoding – the intrinsic value of a given experience and consequent emotional and motivational responses. The NAc is mainly constituted by GABAergic medium spiny neurons (MSNs), divided into those expressing dopamine receptor D1 (D1-MSNs) and those expressing dopamine receptor D2 (D2-MSNs). These populations have been traditionally segregated anatomically (direct vs indirect pathway) and functionally (reward and positive valence vs aversion and negative valence). However, many recent studies have challenged this simplistic segregation. Yet, it is still uncertain which neuronal populations encode valence in the NAc, and this is a crucial question in the understanding of disorders with emotional deficits. In this thesis work we evaluated the neuronal activation levels associated with negative- (footshock) or positive-valence stimuli (cocaine) in the NAc, using the basolateral (BLA) and central (CeA) amygdala as control regions, used because they encode valence. We also characterized a c-fos-driven viral vector in order to establish a neuronal activation labelling strategy for future work. Furthermore, we performed optogenetic activation of stimulus-responsive neurons in the BLA, using the same vector, to induce valence-specific behavioural responses and validate our methodology. Our data shows that the NAc core (NAcc) and the BLA contain neuronal populations that respond to shock, measured by a higher c-fos+ cell density in comparison to controls. However, cocaine induced no significant changes in neuronal activation. Regarding the vector (driving channelrhodopsin-eYFP expression), we found that 16h-post stimulus exposure would be the more adequate timeframe to observe neuronal labelling. When using this vector and exposing mice to positive or negative stimuli, densitometry fluorescence data showed only a tendency for higher neuronal activation in the BLA after footshock, with no effects in the NAc, in comparison with control animals; with no effects due to cocaine. Lastly, optogenetic activation of footshock-responsive neurons in the BLA induced a tendency for avoidance in a real time place preference test (RTPP), yet this same activation did not induce conditioned place preference (CPP). While our data show neuronal activation in the NAcc and BLA in response to shock, the lack of differences with cocaine exposure together with densitometry and optogenetics data, indicates the need to develop new tools to label valence encoding neurons in the NAc.O apoio financeiro foi prestado através de bolsas da Fundação Bial através dos projetos 30/2016 e 175/20; do Fundo Europeu de Desenvolvimento Regional (FEDER), no âmbito do projeto PTDC/MED-NEU/29071/2017 (REWSTRESS), através do COMPETE 2020; da Fundação “La Caixa” (ID100010434; LCF/PR/HR20/52400020); de fundos europeus da European Research Council (ERC) – ERC Consolidator “VALENCE”, No 101003187; da Plataforma de Microscopia Científica do ICVS, membro da PPBI - Portuguese Platform of Bioimaging (PPBI -POCI-01-0145-FEDER-022122); e por Fundos Nacionais, por meio da Fundação para a Ciência e Tecnologia (FCT) - projetos UIDB/50026/2020 e UIDP/50026/2020

Effects of diet on resource utilization by a model human gut microbiota containing Bacteroides cellulosilyticus WH2, a symbiont with an extensive glycobiome

The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities