423 research outputs found

    Cell-type specific cholinergic modulation in anterior cingulate and lateral prefrontal cortices of the rhesus macaque

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    The lateral prefrontal cortex (LPFC) and the anterior cingulate cortex (ACC) are two key regions of the frontal executive control network. Ascending cholinergic pathways differentially innervate these two functionally distinct cortices to modulate arousal and motivational signaling for higher-order functions. The action of acetylcholine (ACh) in sensory cortices is constrained by layer, anatomical cell type, and subcellular localization of distinct receptors, but little is known about the nature and organization of frontal-cholinergic circuitry in primates. In this dissertation, we characterized the anatomical localization of muscarinic acetylcholine receptors (mAChRs), m1 and m2–the predominant subtypes in the cortex–and their expression profiles on distinct cell types and pathways in ACC and LPFC of the rhesus monkey, using immunohistochemistry, anatomical tract-tracing, whole cell patch-clamp recordings, and single nucleus RNA sequencing. In the first series of studies (Chapter 2), we used immunohistochemistry and high-resolution confocal microscopy to reveal regional differences in m1 and m2 receptor localization on excitatory pyramidal and inhibitory neuron subpopulations and subcellular compartments in ACC (A24) versus LPFC (A46) of adult rhesus monkeys (Macaca mulatta; aged 7-11 yrs; 4 males and 2 females). The ACC exhibited a greater proportion of m2+ inhibitory neurons and a greater density of presynaptic m2+ receptors localized on inhibitory (VGAT+) terminations on pyramidal neurons compared to the LPFC. This result suggests a greater cholinergic suppression of GABAergic neurotransmission in ACC. In a second set of experiments (Chapter 3), we examined the heterogeneity of m1 and m2 laminar expression in functionally distinct ACC areas A24, A25, and A32. These differ in their connections with higher order cortical areas and limbic structures, such as the amygdala (AMY). The density of m1+ and/or m2 expressing (m1+/m2+) pyramidal neurons was significantly greater in A24 compared to A25 and to A32, while A25 exhibited a significantly greater density of m2+VGAT+ terminals. In addition, we examined the substrates for cholinergic modulation of long-range cortico-limbic processing using bidirectional neural tracers to label one specific subtype, the AMY-targeting projection neurons in these ACC areas. Compared to A24 and A32, the limbic ventral A25 had a greater density of m1+/m2+ AMY-targeting pyramidal neurons across upper layers 2-3 and deep layers 5-6, suggesting stronger cholinergic modulation of amygdalar outputs. Lastly (Chapter 4), we assessed the functional effects of cholinergic modulation on excitatory and inhibitory synaptic activity as well as the molecular signatures related to m1 and m2 receptor expression. In experiments using in vitro whole-cell patch-clamp recordings of layer 3 pyramidal neurons in ACC and LPFC, we found that application of the cholinergic agonist carbachol (CCh) significantly decreased the frequency of excitatory postsynaptic currents (EPSCs) to a greater extent in ACC A24 than in LPFC A46. Using single nucleus RNA sequencing, we found that enriched m1 and m2 transcriptional profiles in distinct cell-types and frontal areas (ACC A24 and LPFC A46) had differentially expressed genes associated with down-stream signaling cascades related to synaptic signaling and plasticity. Together, these data reveal the anatomical, functional, and transcriptomic neural substrates of diverse cholinergic modulation of local excitatory and inhibitory circuits and long-range cortico-limbic pathways in functionally-distinct ACC and LPFC frontal areas that are important for cognitive-emotional integration

    DISENTANGLING THE ROLE OF PARVALBUMIN-EXPRESSING INTERNEURONS IN STIMULUS-RESPONSE LEARNING AND COGNITIVE FLEXIBILITY

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    Habits enable animals to efficiently navigate their surroundings while tending to more cognitively demanding environmental factors. One mechanism underlying habit is known as stimulus-response (S-R) learning, which takes place in the dorsolateral striatum (DLS). However, there is limited knowledge regarding the complex striatal microcircuits involved in S-R learning and cognitive flexibility. Recently, attention has turned toward the GABAergic Parvalbumin-expressing (PV) interneurons that can modulate striatal outputs. Here, we utilized chemogenetic techniques and touchscreen cognitive assessments to analyze the influence of PV neurons on S-R learning in mice. When PV neurons were inhibited, during the acquisition of a S-R and cognitive flexibility cognitive assessment, there were no significant differences in the percent accuracy. Further exploratory analysis, however, revealed a significant difference in the male mice but not the female mice between the experimental groups for the acquisition of the S-R task. Furthermore, PV neuron inhibition did not affect performance of a previously acquired S-R task. These findings contribute to our understanding of what mechanisms are and are not necessary for the various cognitive functions in which the dorsal striatum is involved

    The role of perineuronal nets in the anterior bed nucleus of the stria terminalis in regulating emotional behaviour and neuronal transmission

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    Anxiety disorders are the most common mental health disorders suffered globally, affecting upwards of 300 million people. Despite the prevalence of anxiety disorders, the currently available therapies lack efficacy, with only 50-85% of patients experiencing at least a 50% improvement in symptoms, and can cause adverse reactions. As such, greater insight into the molecular underpinnings of anxiety disorders is essential to provide a rationale for novel effective treatments. Exposure to severe or prolonged stress promotes the development of anxiety, through aberrant changes to neuronal plasticity in the cortex, hippocampus and amygdala, three well-characterised brain regions involved in the stress response. However, the role of the bed nucleus of the stria terminalis (BNST), uniquely placed to modulate the stress response, in anxiety disorders is not fully understood. Here, I investigated the role of specialised organisations of extracellular matrix, perineuronal nets (PNNs), within the BNST in regulating emotional behaviour and neuronal transmission in relation to stress. The experimental work presented in this thesis characterises the spatiotemporal development of PNNs in the anterior BNST and identifies the anteromedial BNST as the region with densest PNN expression. Morphological studies determine that the structure of PNNs in the anteromedial BNST is the most vulnerable seven days following exposure to repeated restraint stress, a period which coincides with increased anxiety-like behaviour in the elevated plus maze and changes to plasticity of BNST neurons. However, such behavioural changes cannot be recapitulated by PNN degradation, suggesting that PNN alterations are highly specific following stress. Altogether these experiments provide evidence of a relationship between stress and PNNs in the BNST and open an avenue for future research, which may one day inform discovery of novel therapeutics for anxiety disorders.Leverhulme Trus

    Estudio de la inervación GABAérgica de neuronas de las distintas capas de la corteza cerebral

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    Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 24/03/2022Las interneuronas GABAérgicas de la corteza cerebral de los mamíferos son inhibidoras y modulan la actividad y las respuestas de las neuronas piramidales. Las interneuronas corticales se clasifican atendiendo a criterios morfológicos, electrofisiológicos, neuroquímicos, a su origen y distribución laminar y también según la región de las neuronas piramidales con las que establecen contactos sinápticos. Entre las interneuronas se encuentran las células en cesto que inervan de manera selectiva el soma neuronal, y en menor medida la región más proximal de las dendritas; y las neuronas en candelabro o axo-axónicas, que inervan selectivamente el segmento inicial del axón (SIA) de las neuronas piramidales. Para conocer si la inhibición perisomática de las neuronas piramidales es homogénea entre las distintas capas y áreas corticales, la presente Tesis Doctoral ha centrado su estudio cuantitativo en las distintas capas de la neocorteza temporal humana y la neocorteza somatosensorial primaria del ratón, incluyendo la inervación llevada a cabo por los terminales de las neuronas en cesto y en candelabro...GABAergic interneurons in the mammalian cerebral cortex are inhibitory and modulate the activity and responses of pyramidal neurons. Cortical interneurons are classified according to morphological, electrophysiological, and neurochemical criteria, their origin and laminar distribution and according to the region of the pyramidal neurons with which they establish synaptic contacts. Among the types of interneurons are basket cells, that selectively innervate the neuronal soma, and to a lesser extent, the most proximal region of the dendrites, and chandelier cells, or axo-axonal neurons, which selectively innervate the axon initial segment (AIS) of pyramidal neurons.In order to know whether the perisomatic inhibition of pyramidal neurons is homogeneous between the different layers and cortical areas, the present work has focused on the quantitative characterization of perisomatic inhibition in the different layers of the human temporal neocortex and the primary somatosensory neocortex of the mouse, including that innervation carried out by the terminals of both basket and chandelier neurons...Fac. de Ciencias BiológicasTRUEunpu

    Identification and Functional Assessment of Novel Neuromuscular Disease-Causing Genes

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    Inherited neuromuscular diseases comprise a highly heterogeneous group of disorders characterized by the impairment of the neural structures or motor unit components responsible for the generation of movement. While as single gene-associated disorder the majority of them are rare, taken together their estimated prevalence reaches 1 – 3 cases / 1000 individuals. Due to their elevated morbidity and mortality, they represent a significant health burden for the affected individuals, their families, and the healthcare systems. Moreover, their clinical and genetic heterogeneity makes their diagnosis a long and complex process, which often requires specialized diagnostic procedures and poses a challenge in about half of the cases. However, thanks to decreasing costs and increased availability of next-generation sequencing technologies, the last years had witnessed a rise in the number of novel genes associated to neuromuscular disorders. In this study, we identified three novel neuromuscular disease-causing genes: PIEZO2, whose biallelic loss-of-function mutations cause distal arthrogryposis with impaired proprioception and touch; VAMP1, whose biallelic loss-of-function mutations cause a novel presynaptic congenital myasthenic syndrome; CAPRIN1, whose specific p.Pro512Leu mutation causes a neurodegenerative disorder characterized by ataxia and muscle weakness. For PIEZO2, we identified biallelic loss-of-function mutations using exome sequencing, SNPchip-based linkage analysis, DNA microarray, and Sanger sequencing in ten affected individuals of four independent families showing arthrogryposis, hypotonia, respiratory insufficiency at birth, scoliosis, and delayed motor development. This phenotype is clearly distinct from distal arthrogryposis with ocular anomalies which characterize the autosomal dominant distal arthrogryposis 3 (DA3), distal arthrogryposis 5 (DA5), and Marden-Walker syndrome (MWKS). While these disorders are caused by heterozygous gain-of-function mutations in PIEZO2, the novel reported mutations result in the loss of PIEZO2, since they lead to nonsense-mediated mRNA decay in patient-derived fibroblast cell lines. PIEZO2 is a mechanosensitive ion channel playing a major role in light-touch sensation and proprioception. Mice ubiquitously depleted of PIEZO2 die postnatally because of respiratory distress, while individuals lacking PIEZO2 develop a neuromuscular disorder, likely due to the loss of proprioception inputs in muscles. For VAMP1, we identified biallelic loss-of-function mutations using exome or genome sequencing in two pairs of siblings from two independent families affected by a novel congenital myasthenic syndrome. Electrodiagnostic examination showed severely low compound muscle action potentials and presynaptic impairment. The two described homozygous mutations are a frameshift and a missense mutation of a highly conserved residue, therefore are likely to result in the loss of VAMP1 function. Indeed, the phenotype is resembled by VAMP1lew/lew mice, which carry a homozygous VAMP1 truncating mutation and show neurophysiological features of presynaptic impairment. For CAPRIN1, we identified the identical de novo c.1535C>T (p.Pro512Leu) missense variant using trio exome sequencing in two unrelated individuals displaying early-onset ataxia, dysarthria, cognitive decline and muscle weakness. This mutation causes the substitution of a highly conserved residue and in silico tools predict an increase in the protein aggregation propensity. Overexpression of CAPRIN1-P512L caused the formation of insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders, such as ATXN2, GEMIN5, SNRNP200, and SNCA. Upon differentiation in cortical neurons of induced pluripotent stem cell (iPSC) lines where the CAPRIN1-P512L was introduced via CRISPR/Cas9, reduced neuronal activity and altered stress granules dynamics were observed in the lines harboring the mutation. Moreover, nano-differential scanning fluorimetry revealed that CAPRIN1-P512L adopts an extended conformation, and fluorescence microscopy demonstrated that RNA greatly enhances its aggregation in vitro. Taken together, this study associates: (1) biallelic loss-of-function mutations in PIEZO2 with the autosomal recessive distal arthrogryposis with impaired proprioception and touch; (2) biallelic loss-of-function mutations in VAMP1 with an autosomal recessive presynaptic congenital myasthenic syndrome; (3) a recurrent de novo p.Pro512Leu mutation of CAPRIN1 with a neurodegenerative disorder characterized by ataxia and muscle weakness

    Behavioral and Neural Mechanisms of Serotonin Modulation of Impulsivity and Reward

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    Despite its prevalence in many psychiatric disorders, such as attention deficit hyperactivity disorder, suicidal depression, schizophrenia, and aggression and motivational disorders, impulsivity and its biological bases remain poorly understood. Subdivisions of impulsivity, including impulsive action (reduced response inhibition) and impulsive choice (reduced delay of gratification), sometimes present in an uncorrelated manner. This complexity renders pathological impulsivity difficult to treat, as different underlying causes likely result in different phenotypic presentations, despite being placed under one umbrella term. In order to study the behavior and biology of one particular facet of impulsivity, this dissertation utilizes the serotonin 1B receptor (5-HT1BR; an inhibitory G-protein coupled receptor) knockout mouse model, which presents with a specific elevation in impulsive action but not impulsive choice. In Chapter 1, I show that mice lacking the 5-HT1BR have increased impulsive action accompanied by enhanced motivation and responsiveness to palatable rewards, indicating that they may have dysregulation of subjective reward valuation. In Chapter 2, I then explore the 5- HT1BR knockout model from the perspective of behavioral inhibition, demonstrating that knockout mice have intact inhibitory learning despite having difficulty withhold responding for reward. Of particular interest to this particular presentation of impulsive action, therefore, is serotonin neuromodulation of reward circuitry in the brain. In Chapter 3, I first show behaviorally that normalizing reward value in 5-HT1BR knockout mice reduces impulsive action to the level of controls. Neurally, I then complete a series of experiments with targeted knockouts in reward-related brain regions, specifically projections to and from the nucleus accumbens shell, in addition to combined 5-HT1BR genetic heteroreceptor and viral autoreceptor knockout. Only combined Emx1+ heteroreceptor and autoreceptor knockout results in increased motivation and impulsivity similar to the whole brain knockout. On the other hand, combined VGAT+ heteroreceptor and autoreceptor knockout increases hedonic taste reactvity. This suggests that modified serotonin release in addition to multiple 5-HT1B heteroreceptor population losses synergistically modulate neural signaling to increase reward valuation and impulsive action. Together, these studies provide insight into the behavioral and biological bases of impulsive action and propose a framework for better understanding specific presentations of impulsivity

    Úloha cholinergní signalizace ve striatu v řízení chování

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    Cholinergic transmission regulates many behavioural domains, ranging from motor activity to cognition. Acetylcholine signalling is mediated by muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs, respectively). While mAChRs are slow responding metabotropic receptors, nAChRs are ion channels, mediating fast neurotransmission. There is a growing body of evidence suggesting a role of nAChRs as important modulators of behavioural functions. However, as nAChRs consist of many subtypes, depending on their composition in subunits, and as they are expressed by various neuronal populations in different brain regions, their contribution to behavioural control is very complex. To decipher their contribution, it is necessary to selectively target nAChRs expressed not only in particular regions but also by particular neurons with a defined effect on local microcircuits. The goal of the present thesis was to use different genetic strategies to induce regional- and cell-specific deletion of β2-containing nAChRs in the mouse brain, in order to characterize the functional role of these receptors. We focused our work in two brain areas, the striatum and the prefrontal cortex (PFC). In the striatum, we identified the striatal neurons that express one of the most common nicotinic subunits, the β2...Cholinergní přenos reguluje celou řadu behaviorálních domén, od motorické aktivity po kognici. Cholinergní signalizace je zprostředkována muskarinovými a nikotinovými acetylcholinovými receptory (mAChR a nAChR). Zatímco mAChR jsou pomalu reagující metabotropní receptory, nAChR jsou iontové kanály zprostředkovávající rychlou neurotransmisi. Existuje rostoucí množství důkazů potvrzujících roli nAChR jako důležitých modulátorů behaviorálních funkcí. Protože se však nAChR skládají z mnoha podtypů, v závislosti na jejich podjednotkovém složení, a protože jsou exprimovány různými populacemi neuronů v různých oblastech mozku, jejich příspěvek ke kontrole chování je velmi komplexní. K jeho dešifrování je nutné selektivně cílit na nAChR exprimované nejen v konkrétních oblastech, ale i konkrétními neurony s definovaným účinkem na lokální nervové okruhy. Cílem této práce bylo použít různé genetické strategie k vytvoření delece β2-obsahujících nAChR v konkrétních oblastech myšího mozku a typech neuronů , aby bylo možné charakterizovat funkční význam těchto receptorů. Naši práci jsme zaměřili na dvě oblasti mozku, striatum a prefrontální kůru (PFC). Ve striatu jsme identifikovali neurony exprimující jednu z nejběžnějších nikotinových podjednotek, β2 podjednotku, pomocí dvojitě fluorescenční hybridizace in situ....Katedra fyziologieDepartment of PhysiologyFaculty of SciencePřírodovědecká fakult

    High-resolution volumetric imaging constrains compartmental models to explore synaptic integration and temporal processing by cochlear nucleus globular bushy cells

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    Globular bushy cells (GBCs) of the cochlear nucleus play central roles in the temporal processing of sound. Despite investigation over many decades, fundamental questions remain about their dendrite structure, afferent innervation, and integration of synaptic inputs. Here, we use volume electron microscopy (EM) of the mouse cochlear nucleus to construct synaptic maps that precisely specify convergence ratios and synaptic weights for auditory- nerve innervation and accurate surface areas of all postsynaptic compartments. Detailed biophysically-based compartmental models can help develop hypotheses regarding how GBCs integrate inputs to yield their recorded responses to sound. We established a pipeline to export a precise reconstruction of auditory nerve axons and their endbulb terminals together with high-resolution dendrite, soma, and axon reconstructions into biophysically-detailed compartmental models that could be activated by a standard cochlear transduction model. With these constraints, the models predict auditory nerve input profiles whereby all endbulbs onto a GBC are subthreshold (coincidence detection mode), or one or two inputs are suprathreshold (mixed mode). The models also predict the relative importance of dendrite geometry, soma size, and axon initial segment length in setting action potential threshold and generating heterogeneity in sound-evoked responses, and thereby propose mechanisms by which GBCs may homeostatically adjust their excitability. Volume EM also reveals new dendritic structures and dendrites that lack innervation. This framework defines a pathway from subcellular morphology to synaptic connectivity, and facilitates investigation into the roles of specific cellular features in sound encoding. We also clarify the need for new experimental measurements to provide missing cellular parameters, and predict responses to sound for further in vivo studies, thereby serving as a template for investigation of other neuron classes
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