DNA methylation and demethylation in honeybee long-term memory formation

Trabalho final de mestrado integrado em Medicina (Cardiologia), apresentado à Faculdade de Medicina da Universidade de Coimbra.A estenose aórtica paradoxal define-se por uma área valvular aórtica ≤ 1 cm2, um volume sistólico indexado < 35 mL/m2 e um gradiente médio transvalvular < 40 mmHg, apesar de uma fração de ejeção do ventrículo esquerdo preservada (> 50%). Caracteriza-se por uma marcada remodelagem concêntrica do ventrículo esquerdo, com predomínio de disfunção diastólica, e está associada a uma rigidez vascular sistémica aumentada. A prevalência desta doença varia entre os 3-35% dos doentes com estenose aórtica grave e afeta sobretudo doentes idosos, do género feminino e com multimorbilidade. A estenose aórtica paradoxal é um desafio diagnóstico, particularmente pelas inconsistências associadas à equação de continuidade. Têm surgido novos métodos de avaliação na literatura para auxiliar no diagnóstico do estado de baixo fluxo sistólico, bem como para estudar a remodelagem ventricular esquerda. Até há pouco tempo existia algum consenso na literatura sobre o facto da estenose aórtica paradoxal representar um estadio mais avançado da doença valvular aórtica. Como tal, na presença de sintomas, a substituição valvular aórtica parecia ser o tratamento mais indicado. No entanto, outros autores mostraram resultados diferentes e assim, aumentaram a discussão sobre a abordagem e gestão deste fenótipo da estenose aórtica degenerativa. A presente tese tem por objetivo elaborar uma revisão sistemática da literatura sobre a estenose aórtica paradoxal, e abordar aspetos relacionados com a sua demografia, semiologia, avaliação diagnóstica, implicações terapêuticas e prognóstico.Paradoxical aortic stenosis is defined by an aortic valve area ≤ 1cm2, an indexed systolic volume < 35 mL/m2 and a transvalvular gradient < 40 mmHg, despite a preserved left ventricular ejection fraction (> 50%). It’s characterized by an extensive concentric cardiac remodelling of the left ventricle, mainly impairing diastolic function, and has been associated with an increased systemic valvular stiffness. This disease prevalence varies from 3 up to 35% of the patients with severe aortic stenosis, specially affecting the elderly, women and those with multi-morbilities. Paradoxical aortic stenosis is a diagnostic challenge due to the inconsistencies associated with the continuity equation. In order to assess the low flow state and to study the left ventricle remodelling, new methods have emerged. Until now there has been some agreement between authors regarding paradoxical aortic stenosis being a more advanced form of aortic valve disease. Hence, in the presence of symptoms, aortic valve replacement presents as an appropriate therapeutic approach. On the contrary, other authors have shown different results, and so fired up the debate concerning the management of this degenerative aortic stenosis phenotype. The aim of the present paper is to systematically review the literature on paradoxical aortic stenosis and to cast light on its demography, semiology, diagnostic workup, therapeutic approach and prognosis

DNA Methylation Mediates the Discriminatory Power of Associative Long-Term Memory in Honeybees

Memory is created by several interlinked processes in the brain, some of which require long-term gene regulation. Epigenetic mechanisms are likely candidates for regulating memory-related genes. Among these, DNA methylation is known to be a long lasting genomic mark and may be involved in the establishment of long-term memory. Here we demonstrate that DNA methyltransferases, which induce and maintain DNA methylation, are involved in a particular aspect of associative long-term memory formation in honeybees, but are not required for short-term memory formation. While long-term memory strength itself was not affected by blocking DNA methyltransferases, odor specificity of the memory (memory discriminatory power) was. Conversely, perceptual discriminatory power was normal. These results suggest that different genetic pathways are involved in mediating the strength and discriminatory power of associative odor memories and provide, to our knowledge, the first indication that DNA methyltransferases are involved in stimulus-specific associative long-term memory formation

DNA methylation mediates neural processing after odor learning in the honeybee

DNA methyltransferases (Dnmts) - epigenetic writers catalyzing the transfer of methyl-groups to cytosine (DNA methylation) - regulate different aspects of memory formation in many animal species. In honeybees, Dnmt activity is required to adjust the specificity of olfactory reward memories and bees' relearning capability. The physiological relevance of Dnmt-mediated DNA methylation in neural networks, however, remains unknown. Here, we investigated how Dnmt activity impacts neuroplasticity in the bees' primary olfactory center, the antennal lobe (AL) an equivalent of the vertebrate olfactory bulb. The AL is crucial for odor discrimination, an indispensable process in forming specific odor memories. Using pharmacological inhibition, we demonstrate that Dnmt activity influences neural network properties during memory formation in vivo. We show that Dnmt activity promotes fast odor pattern separation in trained bees. Furthermore, Dnmt activity during memory formation increases both the number of responding glomeruli and the response magnitude to a novel odor. These data suggest that Dnmt activity is necessary for a form of homoeostatic network control which might involve inhibitory interneurons in the AL network

The Speed of Smell: Odor-Object Segregation within Milliseconds

Segregating objects from background, and determining which of many concurrent stimuli belong to the same object, remains one of the most challenging unsolved problems both in neuroscience and in technical applications. While this phenomenon has been investigated in depth in vision and audition it has hardly been investigated in olfaction. We found that for honeybees a 6-ms temporal difference in stimulus coherence is sufficient for odor-object segregation, showing that the temporal resolution of the olfactory system is much faster than previously thought

Dnmts and Tet target memory-associated genes after appetitive olfactory training in honey bees

DNA methylation and demethylation are epigenetic mechanisms involved in memory formation. In honey bees DNA methyltransferase (Dnmt) function is necessary for long-term memory to be stimulus specific (i.e. to reduce generalization). So far, however, it remains elusive which genes are targeted and what the time-course of DNA methylation is during memory formation. Here, we analyse how DNA methylation affects memory retention, gene expression, and differential methylation in stimulus-specific olfactory long-term memory formation. Out of 30 memory-associated genes investigated here, 9 were upregulated following Dnmt inhibition in trained bees. These included Dnmt3 suggesting a negative feedback loop for DNA methylation. Within these genes also the DNA methylation pattern changed during the first 24 hours after training. Interestingly, this was accompanied by sequential activation of the DNA methylation machinery (i.e. Dnmts and Tet). In sum, memory formation involves a temporally complex epigenetic regulation of memory-associated genes that facilitates stimulus specific long-term memory in the honey bee

Corrigendum : Dnmts and Tet target memory-associated genes after appetitive olfactory training in honey bees

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DNA Methylation Adjusts the Specificity of Memories Depending on the Learning Context and Promotes Relearning in Honeybees

The activity of the epigenetic writers DNA methyltransferases (Dnmts) after olfactory reward conditioning is important for both stimulus-specific long-term memory (LTM) formation and extinction. It, however, remains unknown which components of memory formation Dnmts regulate (e.g., associative vs. non-associative) and in what context (e.g., varying training conditions). Here, we address these aspects in order to clarify the role of Dnmt-mediated DNA methylation in memory formation. We used a pharmacological Dnmt inhibitor and classical appetitive conditioning in the honeybee Apis mellifera, a well characterized model for classical conditioning. We quantified the effect of DNA methylation on naïve odor and sugar responses, and on responses following olfactory reward conditioning. We show that (1) Dnmts do not influence naïve odor or sugar responses, (2) Dnmts do not affect the learning of new stimuli, but (3) Dnmts influence odor-coding, i.e., 'correct' (stimulus-specific) LTM formation. Particularly, Dnmts reduce memory specificity when experience is low (one-trial training), and increase memory specificity when experience is high (multiple-trial training), generating an ecologically more useful response to learning. (4) In reversal learning conditions, Dnmts are involved in regulating both excitatory (re-acquisition) and inhibitory (forgetting) processes.publishe