Receptors on Microglia.

Microglial cells are the most receptive cells in the central nervous system (CNS), expressing several classes of receptors reflecting their immune heritage and newly acquired neural specialisation. Microglia possess, depending on the particular context, receptors to neurotransmitters and neuromodulators as well as immunocompetent receptors. This rich complement allows microglial cells to monitor the functional status of the nervous system, contribute actively to the regulation of neural activity and plasticity and homeostasis, and guard against pathogens as well as other challenges to the CNS's integrity and function.

Interactions between the Nicotinic and Endocannabinoid Receptors at the Plasma Membrane.

Compartmentalization, together with transbilayer and lateral asymmetries, provide the structural foundation for functional specializations at the cell surface, including the active role of the lipid microenvironment in the modulation of membrane-bound proteins. The chemical synapse, the site where neurotransmitter-coded signals are decoded by neurotransmitter receptors, adds another layer of complexity to the plasma membrane architectural intricacy, mainly due to the need to accommodate a sizeable number of molecules in a minute subcellular compartment with dimensions barely reaching the micrometer. In this review, we discuss how nature has developed suitable adjustments to accommodate different types of membrane-bound receptors and scaffolding proteins via membrane microdomains, and how this "effort-sharing" mechanism has evolved to optimize crosstalk, separation, or coupling, where/when appropriate. We focus on a fast ligand-gated neurotransmitter receptor, the nicotinic acetylcholine receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as a paradigmatic example.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

Nanoscale Sub-Compartmentalization of the Dendritic Spine Compartment.

Compartmentalization of the membrane is essential for cells to perform highly specific tasks and spatially constrained biochemical functions in topographically defined areas. These membrane lateral heterogeneities range from nanoscopic dimensions, often involving only a few molecular constituents, to micron-sized mesoscopic domains resulting from the coalescence of nanodomains. Short-lived domains lasting for a few milliseconds coexist with more stable platforms lasting from minutes to days. This panoply of lateral domains subserves the great variety of demands of cell physiology, particularly high for those implicated in signaling. The dendritic spine, a subcellular structure of neurons at the receiving (postsynaptic) end of central nervous system excitatory synapses, exploits this compartmentalization principle. In its most frequent adult morphology, the mushroom-shaped spine harbors neurotransmitter receptors, enzymes, and scaffolding proteins tightly packed in a volume of a few femtoliters. In addition to constituting a mesoscopic lateral heterogeneity of the dendritic arborization, the dendritic spine postsynaptic membrane is further compartmentalized into spatially delimited nanodomains that execute separate functions in the synapse. This review discusses the functional relevance of compartmentalization and nanodomain organization in synaptic transmission and plasticity and exemplifies the importance of this parcelization in various neurotransmitter signaling systems operating at dendritic spines, using two fast ligand-gated ionotropic receptors, the nicotinic acetylcholine receptor and the glutamatergic receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as paradigmatic examples.

A review on alcohol: from the central action mechanism to chemical dependency / Uma revisão sobre o álcool: do mecanismo de ação central à dependência química

SummaryIntroduction:alcohol is a psychotropic depressant of the central nervous system (CNS) that promotes simultaneous changes in several neuronal pathways, exerting a profound neurological impact that leads to various behavioral and biological alterations.Objectives:to describe the effects of alcohol on the CNS, identifying the signaling pathways that are modified and the biological effects resulting from its consumption.Methods:a literature review was conducted and articles published in different languages over the last 15 years were retrieved.Results:the studies reviewed describe the direct effect of alcohol on several neurotransmitter receptors (gamma-aminobutyric acid [GABA], glutamate, endocannabinoids AEA and 2-AG, among others), the indirect effect of alcohol on the limbic and opioid systems, and the effect on calcium and potassium channels and on proteins regulated by GABA in the hippocampus.Discussion and conclusion:the multiple actions of alcohol on the CNS result in a general effect of psychomotor depression, difficulties in information storage and logical reasoning and motor incoordination, in addition to stimulating the reward system, a fact that may explain the development of addiction. Knowledge on the neuronal signaling pathways that are altered by alcohol allows the identification of effectors which could reduce its central action, thus, offering new therapeutic perspectives for the rehabilitation of alcohol addicts.

ResumoIntrodução:o álcool é uma substância psicotrópica depressora do sistema nervoso central (SNC), que promove alteração simultânea de inúmeras vias neuronais, gerando profundo impacto neurológico e traduzindo-se em diversas alterações biológicas e comportamentais.Objetivos:descrever as ações do álcool sobre o SNC, identificando as vias de sinalização modificadas e os efeitos biológicos gerados pelo seu consumo.Métodos:revisão bibliográfica, priorizando trabalhos multilinguísticos publicados nos últimos 15 anos.Resultados:são descritas ação direta do álcool em inúmeros receptores de neurotransmissores (ácido gama-aminobutírico – GABA, glutamato, endocanabinoides AEA e 2-AG, entre outros), ação indireta do álcool no sistema límbico e opioide, ação sobre canais de cálcio, potássio e proteínas reguladas por GABA no hipocampo, além de ações centrais mediadas pela deficiência de vitamina B1.Conclusão:a ação multifocal do álcool sobre o SNC resulta em efeito geral de depressão psicomotora, dificuldades no armazenamento de informações e no raciocínio lógico, incoordenação motora, além da estimulação do sistema de recompensa, o que pode explicar o desenvolvimento da dependência química. O conhecimento das vias de sinalização neuronais alteradas pelo álcool permite reconhecer a descrição de efetores que possam reduzir sua ação central e, assim, vislumbrar novas perspectivas terapêuticas para a reabilitação de adictos a essa substância.

Identification, localization, and quantification of neuronal cell membrane receptors with plasmonic probes: role of protein kinase D1 in their distribution.

Detecting, imaging, and being able to localize the distribution of several cell membrane receptors on a single neuron are very important topics in neuroscience research. In the present work, the distribution of metabotropic glutamate receptor 1a (mGluR1a) density on neuron cells on subcellular length scales is determined by evaluating the role played by protein kinase D1 (PKD1) in the trafficking of membrane proteins, comparing the distribution of mGluR1a in experiments performed in endogenous PKD1 expression with those in the presence of kinase-inactive protein kinase D1 (PKD1-kd). The localization, distribution, and density of cell surface mGluR1a were evaluated using 90 nm diameter Au nanoparticle (NP) probes specifically functionalized with a high-affinity and multivalent labeling function, which allows not only imaging NPs where this receptor is present but also quantifying by optical means the NP density. This is so because the NP generates a density (ρ)-dependent SERS response that facilitated a spatial mapping of the mGluR1a density distribution on subcellular length scales (dendrites and axons) in an optical microscope. The measured ρ values were found to be significantly higher on dendrites than on axons for endogenous PKD1, while an increase of ρ on axons was observed when PKD1 is altered. The spatial distribution of the NP immunolabels through scanning electron microscopy (SEM) confirmed the results obtained by fluorescence bright-field analysis and dark-field spectroscopy and provided additional structural details. In addition, it is shown using electrodynamic simulations that SERS spectroscopy could be a very sensitive tool for the spatial mapping of cell membrane receptors on subcellular length scales, as SERS signals are almost linearly dependent on NP density and therefore give indirect information on the distribution of cell membrane proteins. This result is important since the calibration of the ρ-dependent near-field enhancement of the Au immunolabels through correlation of SERS and SEM paves the way toward quantitative immunolabeling studies of cell membrane proteins involved in neuron polarity. From the molecular biology point of view, this study shows that in cultured hippocampal pyramidal cells mGluR1a is predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive protein kinase D1 (PKD1-kd) dramatically and selectively alters the intracellular trafficking and membrane delivery of mGluR1a-containing vesicles.

The MAP1B case: an old MAP that is new again.

The functions of microtubule-associated protein 1B (MAP1B) have historically been linked to the development of the nervous system, based on its very early expression in neurons and glial cells. Moreover, mice in which MAP1B is genetically inactivated have been used extensively to show its role in axonal elongation, neuronal migration, and axonal guidance. In the last few years, it has become apparent that MAP1B has other cellular and molecular functions that are not related to its microtubule-stabilizing properties in the embryonic and adult brain. In this review, we present a systematic review of the canonical and novel functions of MAP1B and propose that, in addition to regulating the polymerization of microtubule and actin microfilaments, MAP1B also acts as a signaling protein involved in normal physiology and pathological conditions in the nervous system.

Purificação de células troco de lipoaspirado humano por aptâmeros de DNA, seguida da caracterização dos fenótipos obtidos da diferenciação neuronal / Human adipose mesechymal stem cell separation by DNA aptamers followed by the characterization of the obtained phenotypes from neuronal differentiation

Células tronco mesenquimais de tecido adiposo, são uma promissora ferramenta para aplicações clínicas em terapias celular e regenerativa, em vista da facilidade de sua extração e da maior quantidade de células por unidade de massa de tecido quando comparado a outras fontes clássicas de células mesenquimais como medula óssea. O protocolo clássico de extração e purificação dessas células, depende de sua adesão em plástico e xeno-materiais demandando muito tempo para ser utilizado por médicos para auxiliar pacientes em procedimentos de emergência. Estas células são capazes se diferenciar em diversos tipos celulares, o que as torna boas candidatas para terapia celular, embora sua capacidade de transdiferenciação para fenótipos neuronais seja ainda discutida. Neste trabalho demonstramos um novo processo para isolar essas células na base de epitopos específicos expressos (assinatura molecular de superfície) utilizando aptâmeros como ligantes de alta afinidade para estes sitios. Aptâmeros, moléculas de DNA simples fita identificadas a partir de uma biblioteca combinatória de sequencias de DNA simples-fita foram identificados por ciclos reiterativos de seleção in vitro (SELEX) utilizando células tronco do lipoaspirado como alvo. Dois aptâmeros isolados, denominados APT9 e APT11, foram capazes de identificar subpopulações (15,8 e 23,7% respectivamente) dentre as células tronco mesenquimais (classicamente CD29+/CD90+/CD45-) e separá-las usando nano-partículas magnéticas acopladas aos aptâmeros. Além disso, seguindo uma indução para diferenciação neuronal, as células tronco mesenquimais passam a apresentar morfologia neuronal e apresentam expressão e atividade de diversos receptores de neurotransmissores, avaliados por PCR real-time e imageamento de variações da concentração de cálcio intracelular ápos stimulação com vários agonistas de receptores metatrópicos e ionotrópicos. Ao longo da diferenciação, os níveis transcricionais de mRNA de receptores de cininas (B1 e B2), nicotínicos (alfa 7), muscarínicos (M1, M3 e M4), glutamatérgicos (AMPA2 e mGluR2), purinérgicos (P2Y1 e P2Y4) e GABAergicos (GABA-A, subunidade 3) e da óxido nítrico sintase neural aumentaram quando comparados aos níveis das células não diferenciadas, enquanto que os níveis de expressão de outros receptores incluindo purinérgicos P2X1, P3X4, P2X7 e P2Y6 e muscarínico M5 diminuíram. Os níveis de atividade das classes dos receptores estudados, por imageamento de variações da concentração de cálcio intrac, aumentaram para a maioria dos agonistas analisados durante a diferenciação neuronal com exceção para respostas induzidas por glutamato e NMDA. Células diferenciadas expressavam altos níveis de antígenos específicos de neurônios como ß3-tubulina, NF-H, NeuN e MAP-2 indicando uma diferenciação em fenótipo neuronal bem sucedida. Desta maneira, esta tese, ao identificar aptâmeros, prove uma inovadora solução para médicos usarem as células tronco mesenquimais dentro de uma sala de cirurgia, através de um método que é capaz de purificar essas células em um tempo clínico viável, com pureza e sem contato com contaminantes. Além disso, nós mostramos aqui que com um protocolo como o proposto para diferenciação neuronal, nós poderíamos induzir essas células para se diferenciar em neurônios, através da ativação de fatores de transcrição específicos, levando às células tronco mesenquimais a serem possivelmente utilizadas em terapias celulares de reparo neuronal

Adipose mesenchymal stem cells are promising tools for clinical applications in cellular and regeneration therapies, in view of easiness of extraction and higher amount of isolated stem cells per mass of tissue when compared to other classical mesenchymal stem cell sources including bone marrow. The classical protocol to extract and purify these cells, depending on plastic adherence and xeno-materials, is too time consuming to be used by physicians to help patients at emergency procedures. These cells are able to differentiate into various cell types, making them good candidates for cell therapy, however their capability for transdifferentiation into neural phenotypes is yet discussed. Here we show a novel process to isolate these cells using their surface molecular signature and aptamers, ssDNA molecules identified through the SELEX technique, denominated APT9 and APT11 that are able to identify subpopulations (15,8 and 23,7% respectively) within the mesenchymal stem cells (classically CD29+/CD90+/CD45-) and separate them using magnetic nano-particles attached to the aptamers. Moreover, following induction to neural differentiation, mesenchymal cells presents neuronal morphology and present expression and activity of several neurotransmitter receptors, as evaluated by real-time PCR and calcium imaging. During this process, mRNA transcription levels of bradykinin (B1 and B2), cholinergic (alpha 7), muscarinic (M1, M3 and M4), glutamatergic (AMPA2 and mGlu2), purinergic (P2Y1 and P2Y4) and GABAergic (GABA-A, subunit 3) receptors and neuronal nitric oxide synthase were augmented when compared to levels of undifferentiated cells, while the expression levels of other receptors including purinergic P2X1, P2X4, P2X7 and P2Y6 and muscarinic M5 receptors were down-regulated. Activity levels of the studied receptor classes, as studied by calcium imaging, increased for most of the agonists analyzed during the neuronal differentiation with the exception for glutamate- and NMDA-induced receptor responses. Differentiated cells expressed high levels of neuron-specific antigens such as ß3-tubulin, NF-H, NeuN and MAP-2, indicating a successful differentiation into neuronal phenotypes. This thesis, by identifying aptamers, provides a novel solution for physicians to use mesenchymal stem cells inside a surgery room, by using a method that are able to purify the cells in a clinical viable time, with purity and no contact with contaminats. Furthermore, we show here that with a protocol as provided for neuronal differentiation, we could induce these cells to differentiate into neurons, by activating specific transcription factors,making mesenchymal stem cells to possibly be used in neuronal repair cell therapies

Aspectos neurobiológicos implicados en el miedo animal / Neurobiological aspects involved in animal fear

El miedo es una emoción que sirve para la expresión de comportamientos defensivos en situaciones de peligro. Posee un sustrato biológico, con base en el funcionamiento coordinado de los diferentes sistemas orgánicos. Particularmente, el sistema nervioso en su actividad intrínseca genera la vivencia y la acción motriz derivada. En efecto, se ha hallado la intervención de varias estructuras neuroanatómicas como la amígdala e hipotálamo, así como un gran conjunto de moléculas distintas como neurotransmisores y sus receptores. La interacción anatomofuncional causa la emoción. Al igual que se cuenta con la capacidad de producir el miedo, también se puede regular su generación. Para este mecanismo se encuentran determinadas estructuras neuroanatómicas como la corteza prefrontal y orbitofrontal, y sustancias como el GABA y los opiáceos, que inhiben o reducen la actividad en las zonas activas que actúan en el miedo. El equilibrio entre la activación y la inhibición posibilita la ocurrencia del miedo en las circunstancias requeridas y no de una manera descontextualizada o generalizada. En esta revisión se presenta una descripción de diferentes aspectos relevantes en la generación y regulación de la emoción.

Fear is an emotion that is useful for expressing defensive behaviors in dangerous situations. It has a biological support based on the coordinating functionality of different organic systems. Particularity, the nervous system in its intrinsic activity generates the experience and the derived motor action. In fact, researchers have discovered the participation of several neuroanatomical structures such as the amygdala and hypothalamus, as well as a wide range of molecules such as neurotransmitters and their receptors. The anatomical and physiological interactions cause emotion. Since the ability to produce fear exists, the nervous system may regulate it, too. Certain anatomical structures are found for this mechanism such as the prefrontal and orbitofrontal cortex and molecules like GABA and opiates, which inhibited or reduced the activity in the active zones that act upon fear. The balance between activation and inhibition enables the event of fear in the circumstances required and not in an out-of-context or generalized manner. This review presents a description of different relevant aspects in thegeneration and regulation of the emotion.