1,721,037 research outputs found
Monitoring and controlling GABAergic interneuron subtypes during epileptiform activity
Full text linkInhibitory synaptic transmission is of paramount importance for maintaining the delicate balance between excitation and inhibition in the brain. If this balance is perturbed in favour of excitation, epilepsy is likely to develop. Fast synaptic inhibition is mediated by type A γ-aminobutyric acid ionotropic receptors (GABAARs), which are primarily permeable to Clâ. The strength of synaptic inhibition crucially depends on the release of GABA from different populations of presynaptic interneurons and the transmembrane electrochemical gradient for Clâ in postsynaptic cells. GABAAR-mediated inhibition has been shown to oppose epileptic seizures by establishing an inhibitory restraint against spreading excitation. Of the different subtypes of GABAergic interneurons, parvalbumin-expressing (PV) interneurons that target the somatic compartment of excitatory neurons have been strongly implicated in this process. In the context of an epileptic seizure, it is thought that the inhibitory restraint is overwhelmed by runaway excitation, and the seizure front is able to spread from the pathologic epileptic focus into adjacent healthy areas, referred to as the âpenumbraâ.
In the first part of this thesis I assess the potential of using chemogenetic strategies to suppress epileptiform activity by boosting the synaptic output from three major interneuron populations in the rodent hippocampus: PV, somatostatin (SST) and vasoactive intestinal peptide (VIP) expressing interneurons. Electrophysiological recordings in an in vitro model of epilepsy reveal that the interneuron populations exhibit different effects on epileptiform events. Recruiting VIP interneurons does not change the total duration of epileptiform activity. By contrast, recruiting SST or PV interneurons produces robust suppression of epileptiform synchronisation. PV interneurons exhibit the strongest effect per cell, eliciting at least a five-fold greater reduction in epileptiform activity than the other cell types. Consistent with this, I find that in vivo chemogenetic recruitment of PV interneurons suppresses convulsive behaviours by more than 80%.
In the second part of the thesis I use a genetically-encoded reporter to investigate activity-dependent intracellular pH and Clâ concentration transients in pyramidal neurons and PV, SST and VIP interneurons. I demonstrate that pyramidal neurons and interneurons have different pH and intracellular Clâ concentration steady states, and exhibit distinct dynamics during epileptiform events. Compared to the other cell types, PV interneurons maintain a relatively stable intracellular Clâ concentration, even when challenged with epileptiform activity. This suggests that PV interneurons may be more likely to maintain a balance in their excitatory and inhibitory synaptic inputs during seizures.
In the final part of the thesis I investigate the contribution of PV interneurons to inhibitory restraint in an in vitro model of the epileptic penumbra. Although PV interneurons are recruited in response to spreading excitation, they can be overwhelmed as they enter a state referred to as âdepolarising blockâ, which is characterized by a decrease in action potential firing. To investigate the impact of this process, I use a light-activated optogenetic tool to induce brief hyperpolarisations of the PV interneuron membrane potential. This successfully reduces depolarising block in PV interneurons, enhances their action potential firing, and reduces the spread of epileptiform activity.
In conclusion, this thesis demonstrates that selective enhancement of inhibitory synaptic pathways offers potential as an anti-seizure strategy, providing valuable insights into the development of therapeutic interventions.</p
Using human induced pluripotent stem cells to reveal astrocyte-neuron interactions in health and disease
Full text linkInteractions between neurons and astrocytes underpin normal brain function. Astrocytes fulfil a variety of essential roles, including regulating synapse formation and maintenance, clearing neurotransmitters, and providing homeostatic regulation of the extracellular ionic environment. Evidence for a more active participation is also emerging, in which astrocytes respond to neuronal signals by influencing synaptic strength through gliotransmission. Most of our knowledge regarding neuron-astrocyte interactions stems from animal models, which have been important in dissecting events at a cellular and molecular level. Yet a major question for experimental neuroscience and drug development is how well findings translate from rodents to the human condition. The recent developments in induced pluripotent stem cell (iPSC) technology are providing a valuable tool with which to answer this question. Furthermore, because iPSCs can be derived from clinically diagnosed patients, this technology affords exciting new opportunities to model human disease processes.
The objective of this thesis was to establish a human co-culture system in which neuron-astrocyte interactions can be investigated. To this end, the first part of the thesis characterises the functionality of cortical human neurons and astrocytes generated using protocols that recapitulate key aspects of corticogenesis. This characterisation establishes that human iPSC-derived astrocytes satisfy many of the hallmarks of rodent astrocytes. Live cell imaging methods are used to investigate intracellular ion dynamics, which are believed to underpin how astrocytes communicate with neurons and regulate the extracellular environment. This reveals that iPSC-derived astrocytes exhibit intracellular Ca2+ signalling events both spontaneously and in response to neurotransmitters. Furthermore, through a combination of monoculture and xeno-transplantation models, iPSC-derived astrocytes are shown to respond to neuronal activity and extracellular K+ by exhibiting similar membrane depolarisation and H+ dynamics to those observed in rodent astrocytes.
The latter part of the thesis focuses on the use of iPSC co-cultures for studying astrocyte-to-neuron signalling. First, I establish that human astrocytes convey pro-maturational effects in terms of enhancing the intrinsic excitability and synaptic activity of co-cultured human neurons. Next, I develop an optogenetic paradigm for directly eliciting membrane depolarisation and Ca2+ events in astrocytes. This leads to glutamate receptor activation in nearby neurons and astrocyte stimulation over a period of days causes a potentiation of the synaptic inputs to co-cultured neurons. Finally, astrocytes are generated from individuals diagnosed with sporadic Alzheimer’s disease (sAD), whom are homozygous for the APOE4 allele - the highest genetic risk factor associated with sAD. Compared to controls, APOE4 astrocytes are found to secrete less APOE and fail to generate astrocyte-mediated increases in synaptic inputs.
Together, this work advances the use of human iPSC-derived cells for studying neuron-astrocyte interactions. iPSC-derived human astrocytes can recapitulate many of the functional properties of rodent astrocytes and can be used in different co-culture settings to examine multiple forms of astrocyte-to-neuron communication. This represents an important step towards establishing more complex human-based models, which are expected to prove useful for modelling disease processes
Investigating the role of lineage in the cortical integration of thalamic inputs
Full text linkPrimary sensory cortex receives and integrates inputs from first-order and higher-order thalamic nuclei. First-order inputs convey sensory information from the periphery and exhibit simple response properties, whereas higher-order inputs exhibit more complex response properties, provide contextual feedback, and can modulate first-order inputs. The data presented within this thesis show that the way in which cortical neurons integrate these thalamic inputs, reflects the progenitor cell from which the cortical neurons derive. Within layer 4 of mouse primary somatosensory cortex, excitatory neurons that derive from apical intermediate progenitors exhibit multi-whisker response properties and receive higher-order thalamic input, in a manner consistent with their dendritic morphology. These properties depend upon the expression levels of the transcription factor Lhx2, which when increased, abolishes the higher-order properties of apical intermediate progenitor-derived neurons, and disrupts the induction of sensory-evoked plasticity. These data reveal a lineage- dependent mechanism that establishes the integration and functional contribution of higher-order thalamic inputs within cortex
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The Oxford Mouse Polysomnography Benchmark Data Set
No full text<p>This archive contains electrophysiological recordings from freely behaving mice, with each 4-second epoch having been annotated with the corresponding vigilance state by multiple sleep experts. The recordings are either 12 or 24 hours long and consist at minimum of a frontal EEG, a parietal EEG and an EMG trace sampled at 256 Hz. Several recordings additionally contain LFP traces and/or unsorted multi-unit activity.</p>
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<p>The data comprises four groups:</p>
<p>- a pilot data set,</p>
<p>- a test data set,</p>
<p>- a sleep deprivation data set, and</p>
<p>- an optogenetic stimulation data set.</p>
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<p>The recordings in the pilot data set and the test data set have been annotated by 4-10 experienced sleep researchers from the Vyazovskiy group at the University of Oxford. They are ideally suited for benchmarking of automated methods for polysomnography. The recordings in the sleep deprivation data set and the optogenetic stimulation data set exhibit characteristics that are distinct from corresponding baseline recordings and are thus useful to test the resilience of automated methods to experimental manipulations. Sleep deprivation increases the amplitude of slow-wave activity during NREM and thus changes the spectral features used by many automated methods. The manipulation in the optogenetic stimulation data set increased the number of times the animal was (briefly) awake during sleep, resulting in increases in the transition probabilities from NREM or REM to the awake state compared to baseline recordings. Methods that leverage expectations of transition probabilities in their predictions would be expected to be sensitive to these changes.</p>
<p>This benchmark data set was created during the development of Somnotate, an automated vigilance state classifier (available at https://github.com/paulbrodersen/somnotate). The data collection is hence described in the following publications:</p>
<p>Brodersen et al. Somnotate: A probabilistic sleep stage classifier for studying vigilance state transitions. PLoS Comput Biol. 2024. DOI: 10.1371/journal.pcbi.1011793</p>
<p>Please consider citing this publication if you use Somnotate or this data set in your academic work.</p>
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Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Examining the influence of lineage relationships upon excitatory neocortical development
Full text linkThe mammalian neocortex comprises a diverse population of excitatory neurons that perform distinct roles in the neocortical circuit. These neurons are born from a heterogeneous population of progenitor cells during embryonic development and it is increasingly being recognised that individual progenitors can impart specific functional characteristics to their offspring. For example, clonally-related sister neurons in mouse neocortex are biased to form gap junctions with one another early in development, to form synaptic connections with one another as they mature, and to show similar response properties in the adult. This highlights a fundamental role for neuronal lineage in the formation of precise neocortical connectivity. However, the extent to which neuronal phenotype is determined by lineage, and the process by which this arises, is not fully understood. Nor is it known whether similar lineage relationships exist in the human neocortex. Deciphering neocortical lineage relationships has been limited by the techniques available to identify and manipulate clonally-related neurons.
In this thesis I have developed novel molecular tools for the identification and manipulation of clonally-related neurons and refined their use by in utero surgery in mice. I first developed a retrovirus encoding Cre recombinase and demonstrated that this can be successfully combined with reporter mice to capitalise on optogenetics for functional studies. In an effort to establish a reliable and unequivocal method of identifying clonally-related neurons, I then constructed a retrovirus encoding genetically-distinct RNA barcodes. I confirmed that the RNA barcode could be reliably retrieved from single neurons and used to determine clonal relationships in mouse neocortex and in an in vitro model of human neocortex derived from induced pluripotent stem cells (iPSCs). By comparing the dendritic morphology of barcoded mouse neocortical neurons, I was able to demonstrate that the dendritic arbor of clonally-related neurons is more similar than control neurons derived from different progenitors. This may contribute to specific patterns of synaptic connectivity amongst clonally-related neurons. Within the iPSC system, I demonstrate the utility of the retroviral RNA barcode and revealed that clonally-related human neocortical cells exhibit a higher probability of being gap-junction coupled. These studies advance our understanding of lineage relationships in neocortical excitatory neurons in mouse and provide the first evidence that human neocortical clones exhibit similar functional relationships to those observed in the rodent neocortex.</p
Intracellular chloride and hydrogen ion dynamics in the nervous system
Full text linkSynaptic transmission in the nervous system involves the activation of receptor proteins that permit rapid transmembrane fluxes of ions. Ionic gradients across the membrane determine the direction and driving force for the flow of ions and are therefore crucial in setting the properties of synaptic transmission. These ionic gradients are established by a variety of mechanisms, including pump and transporter proteins. However, the gradients can be affected by periods of neural activity, which in turn, are predicted to influence the properties of ongoing synaptic transmission. In this thesis I have examined the concentration gradients of two ions that play a fundamental role in synaptic transmission: chloride ions (Cl-) and protons (H+). Type A γ-Aminobutyric acid receptors (GABAARs) are primarily permeable to Cl- and mediate the majority of fast post-synaptic inhibition in the brain. The transmembrane concentration gradient for Cl- is therefore a critical parameter in governing the strength of synaptic inhibition. In the first part of the Thesis I use a combination of experimental and theoretical approaches to demonstrate that influxes of Cl- via activated GABAARs can overwhelm a neurons ability to maintain a stable Cl- concentration gradient. The consequence is that subsequent activation of GABAARs results in weaker inhibition or even excitation, which alters how the neuron integrates synaptic inputs. This process is shown to be dependent upon the level of activity of the GABAAR, the post-synaptic cells membrane potential and the cellular compartment into which the Cl- flows. These principles were extended to demonstrate that popular optogenetic strategies for silencing neural activity have different effects upon GABAAR transmission. A light-activated Cl- pump was shown to cause substantial accumulations in intracellular Cl, which meant that the strength of synaptic inhibition was significantly reduced following light offset. In the second part of the Thesis I use electrophysiological and fluorescence imaging techniques to demonstrate that the activation of GABAARs during epileptiform activity results in pronounced changes to the transmembrane Cl- gradient. Indeed, these changes convert synaptic inhibition into synaptic excitation during the course of a seizure event. As part of this work I characterise a novel, genetically-encoded reporter for measuring intracellular Cl- dynamics in different cell types and subcellular compartments. A significant advantage of this reporter is that it permits the simultaneous quantification of H+ fluxes, which are also shown to change in an activity-dependent manner and which have been a confounding factor for previous Cl- reporters. In the third and final part of the Thesis I use genetically-encoded reporters to investigate activity-dependent changes in intracellular H+ concentration. I demonstrate that markedly different pH changes occur in neurons and astrocytes during epileptiform activity. Whereas neurons become acidic, astrocytes become alkaline and the dynamics of these pH shifts exhibit a very different temporal relationship with the seizure event. In conclusion, this thesis demonstrates that the intracellular concentrations of Cl- and H+ are dynamic variables that evolve across time and space, in an activity-dependent manner. Changes in the transmembrane gradients of these two ions influence ongoing synaptic transmission. Therefore this work has significant implications for our understanding of network activity and the balance of synaptic excitation and inhibition
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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