Computational Modeling of Genetic Contributions to Excitability and Neural Coding in Layer V Pyramidal Cells: Applications to Schizophrenia Pathology

Pyramidal cells in layer V of the neocortex are one of the most widely studied neuron types in the mammalian brain. Due to their role as integrators of feedforward and cortical feedback inputs, they are well-positioned to contribute to the symptoms and pathology in mental disorders—such as schizophrenia—that are characterized by a mismatch between the internal perception and external inputs. In this modeling study, we analyze the input/output properties of layer V pyramidal cells and their sensitivity to modeled genetic variants in schizophrenia-associated genes. We show that the excitability of layer V pyramidal cells and the way they integrate inputs in space and time are altered by many types of variants in ion-channel and Ca2+ transporter-encoding genes that have been identified as risk genes by recent genome-wide association studies. We also show that the variability in the output patterns of spiking and Ca2+ transients in layer V pyramidal cells is altered by these model variants. Importantly, we show that many of the predicted effects are robust to noise and qualitatively similar across different computational models of layer V pyramidal cells. Our modeling framework reveals several aspects of single-neuron excitability that can be linked to known schizophrenia-related phenotypes and existing hypotheses on disease mechanisms. In particular, our models predict that single-cell steady-state firing rate is positively correlated with the coding capacity of the neuron and negatively correlated with the amplitude of a prepulse-mediated adaptation and sensitivity to coincidence of stimuli in the apical dendrite and the perisomatic region of a layer V pyramidal cell. These results help to uncover the voltage-gated ion-channel and Ca2+ transporter-associated genetic underpinnings of schizophrenia phenotypes and biomarkers

The neural mechanisms of mindfulness-based pain relief: a functional magnetic resonance imaging-based review and primer.

The advent of neuroimaging methodologies, such as functional magnetic resonance imaging (fMRI), has significantly advanced our understanding of the neurophysiological processes supporting a wide spectrum of mind-body approaches to treat pain. A promising self-regulatory practice, mindfulness meditation, reliably alleviates experimentally induced and clinical pain. Yet, the neural mechanisms supporting mindfulness-based pain relief remain poorly characterized. The present review delineates evidence from a spectrum of fMRI studies showing that the neural mechanisms supporting mindfulness-induced pain attenuation differ across varying levels of meditative experience. After brief mindfulness-based mental training (ie, less than 10 hours of practice), mindfulness-based pain relief is associated with higher order (orbitofrontal cortex and rostral anterior cingulate cortex) regulation of low-level nociceptive neural targets (thalamus and primary somatosensory cortex), suggesting an engagement of unique, reappraisal mechanisms. By contrast, mindfulness-based pain relief after extensive training (greater than 1000 hours of practice) is associated with deactivation of prefrontal and greater activation of somatosensory cortical regions, demonstrating an ability to reduce appraisals of arising sensory events. We also describe recent findings showing that higher levels of dispositional mindfulness, in meditation-naïve individuals, are associated with lower pain and greater deactivation of the posterior cingulate cortex, a neural mechanism implicated in self-referential processes. A brief fMRI primer is presented describing appropriate steps and considerations to conduct studies combining mindfulness, pain, and fMRI. We postulate that the identification of the active analgesic neural substrates involved in mindfulness can be used to inform the development and optimization of behavioral therapies to specifically target pain, an important consideration for the ongoing opioid and chronic pain epidemic

Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms

Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3′-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability

A novel brain partition highlights the modular skeleton shared by structure and function

Elucidating the intricate relationship between brain structure and function, both in healthy and pathological conditions, is a key challenge for modern neuroscience. Recent progress in neuroimaging has helped advance our understanding of this important issue, with diffusion images providing information about structural connectivity (SC) and functional magnetic resonance imaging shedding light on resting state functional connectivity (rsFC). Here, we adopt a systems approach, relying on modular hierarchical clustering, to study together SC and rsFC datasets gathered independently from healthy human subjects. Our novel approach allows us to find a common skeleton shared by structure and function from which a new, optimal, brain partition can be extracted. We describe the emerging common structure-function modules (SFMs) in detail and compare them with commonly employed anatomical or functional parcellations. Our results underline the strong correspondence between brain structure and resting-state dynamics as well as the emerging coherent organization of the human brain.Work supported by Ikerbasque: The Basque Foundation for Science, Euskampus at UPV/EHU, Gobierno Vasco (Saiotek SAIO13-PE13BF001) and Junta de Andalucía (P09-FQM-4682) to JMC; Ikerbasque Visiting Professor to SS; Junta de Andalucía (P09-FQM-4682) and Spanish Ministry of Economy and Competitiveness (FIS2013-43201-P) to MAM; the European Union’s Seventh Framework Programme (ICT-FET FP7/2007-2013, FET Young Explorers scheme) under grant agreement n. 284772 BRAIN BOW (www.brainbowproject.eu) and by the Joint Italy—Israel Laboratory on Neuroscience to PB. For results validation (figure S8), data were provided by the Human Connectome Project, WU-Minn Consortium (Principal Investigators: David Van Essen and Kamil Ugurbil; 1U54MH091657) funded by the 16 NIH Institutes and Centers that support the NIH Blueprint for Neuroscience Research; and by the McDonnell Center for Systems Neuroscience at Washington University

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

Study and characterisation of human HEK293 cell line as a platform for recombinant protein production

El present treball es centra en l'estudi de la producció de proteïnes recombinants en línies cel·∙lulars de mamífer. Concretament, s'ha realitzat l'estudi de tres estratègies de bioprocés, totes elles basades en el cultiu de cèl·∙lules HEK293. Com a proteïna model per a l'expressió de proteïnes heteròlogues s'ha triat la proteïna CapPCV2, la qual conforma la càpsida viral del Circovirus porcí serotip 2 (PCV2). Aquest virus és l'agent causal de PCVDS (porcine circovirus diseases o malaties derivades de circovirus porcí). Aquest terme engloba un conjunt de malalties i síndromes que tenen un elevat impacte econòmic en la indústria porcina. El projecte s'ha enfocat des de la perspectiva de desenvolupament i optimització del bioprocés i, en conseqüència, l'increment de la producció volumètrica ha estat la força impulsora de tot el treball. En primer lloc es presenten els estudis per a la selecció del medi de cultiu i suplements nutricionals. El creixement cel·∙lular depèn en gran mesura de les característiques nutricionals i fisicoquímiques del medi en que se les cultiva. Per tant, trobar el medi adequat és un dels factors clau per a l'expansió del cultiu cel·∙lular. L'estudi inicial de medis de cultiu va permetre augmentar sis vegades la densitat de cèl·∙lules viables en comparació al medi original en que es cultivaven. D'altra banda, s'han explorat diferents estratègies de cultiu, i com a resultat s'ha implementat una estratègia de fed-­-batch que ha permès arribar a densitats cel·∙lulars de 26.8x106 cell/mL. En el segon i tercer capítol de resultats, s'avaluen tres estratègies diferents per a la producció de la proteïna recombinant CapPCV2 (r-­-CapPCV2). La primera estratègia ha estat la infecció de cèl·∙lules HEK293 amb un vector adenoviral que codifica el gen de la CapPCV2 (vector generat dins del treball d'aquesta tesis doctoral). Els paràmetres d'infecció s'han estudiat en profunditat per tal de trobar els paràmetres d'infecció (medi de cultiu, MOI (multiplicitat d'infecció), TOI (temps d'infecció) i TOH (temps de recollida)) per a la millora de la producció de la proteïna i el vector adenoviral. La segona i tercera estratègia estan basades en la generació de línies cel·∙lulars estables. Concretament, s'ha generat una línia cel·∙lular productora de r-­-CapPCV2 a partir de la integració a l'atzar del vector plasmídic en el genoma de la cèl·∙lula. D'altra banda, s'han generat línies cel·∙lulars amb la integració dirigida del gen en llocs prèviament caracteritzats com d'altra transcripció genètica. La integració dirigida s'ha efectuat mitjançant la tecnologia RMCE (recombinant mediated cassette exchange, o bescanvi de casset mitjançada per recombinació). Després de la comparació de les productivitats específiques i volumètriques aconseguides amb cada estratègia, el millor productor va ser seleccionat. Nogensmenys, r-­-CapPCV2 es produeix en quantitats molt baixes i per tant no ha sigut possible dissenyar un procés de producció rentable i altres alternatives de producció s'haurien d'estudiar en un futur. Finalment, l'estudi d'un comportament metabòlic particular observat en les cèl·lules en cultiu s'ha adreçat des d'una perspectiva fisiològica i metabòlica. A certes condicions extracel·∙lulars, s'ha observat que les cèl·∙lules HEK293 poden consumir de manera simultània glucosa i lactat durant el seu creixement exponencial. Després d'un ampli estudi d'aquestes condicions, s'ha determinat que el canvi de la producció d'àcid làctic (que és el principal problema dels cultius d'alta densitat de cèl·∙lules de mamífer) cap al consum d'aquest metabòlit pot ser generat des de el començament del cultiu quan el pH és de 6.6 i la concentració de lactat és de 4-­-8mM. En aquestes condicions, ni el creixement cel·∙lular ni la producció de proteïna es veuen afectades negativament. A la llum d'aquests resultats, es genera la hipòtesi de que les cèl·∙lules HEK293 poden co-­-transportar el lactat extracel·lular i els protons com un mecanisme de detoxificació del pH. D'altra banda, l'aplicació de l'anàlisi de balanç de fluxos (FBA) ha revelat que quan la glucosa i el lactat es consumeixen simultàniament s'aconsegueix un metabolisme "equilibrat", és a dir els fluxos de la glicòlisi i el cicle TCA esdevenen similars, evitant l'acumulació de piruvat en el citosol, la seva transformació a làctic i finalment la secreció d'aquest metabòlit. Aquest comportament és totalment oposat al que s'observa de forma general en els cultius de cèl·lules de mamífer en creixement exponencial, on els elevats fluxos de la glicòlisi troben una limitació en els fluxos d'entrada a la mitocòndria (és a dir, del cicle TCA) i conseqüentment el lactat és produït i secretat al medi. La construcció d'un model metabòlic i l'aplicació de FBA permetrà fer prediccions in silico de comportaments metabòlics causats per la sobreexpressió o el silenciament de gens diana. Aquesta estratègia obre la possibilitat de generar línies cel·∙lulars que presentin un metabolisme optimitzat per tal d'estudiar estratègies de cultiu més eficients per a l'increment de la densitat cel·∙lular i productivitat de proteïna recombinant.The thesis is focused on the study of recombinant protein production in mammalian cell lines. In particular, the study of three different approaches of different bioprocesses based on HEK293 cells has been addressed. As a model protein for recombinant expression, CapPCV2 has been selected. This protein makes up the viral capsid of Porcine circovirus serotype 2 (PCV2), which is the causative agent of PCVDs (porcine circovirus diseases), a group of diseases with major impact in pig's industry worldwide. This project has been addressed from the perspective of bioprocess development and optimization and therefore, the increment of volumetric production of cells, virus and proteins have been the driving force of the research. Firstly, cell culture media and nutritional supplementation studies are presented. Cell growth relies in high extent to the nutritional and physicochemical characteristics of the media in which cells are cultured and therefore, finding the proper cell media is one of the key factors for cell culture expansion. The initial media study resulted in a 6-­-fold increment of the maximal viable cell achieved in the original media. Besides, different cell culture strategies have been explored, which resulted in a fed-­-batch strategy that allowed reaching maximal viable cell densities of 26.8x106 cell/mL, which represents 13-­-fold increment on maximal viable cell density originally reached. In the second and third chapter of results, three different approaches for the expression of recombinant CapPCV2 (r-­-CapPCV2) are evaluated and discussed. As a first approach, a viral recombinant adenovirus encoding for the gene CapPCV2 has been generated and used as viral vector for the production of the recombinant protein in HEK293 cells. Besides, a deep study of the main parameters that affect the infection performance has been carried out and discussed in order to find the best media, MOI (multiplicity of infection), TOI (time of infection) and TOH (time of harvest) for adenovirus and recombinant protein production. This study was performed with an adenovirus expressing the reporter gene GFP and thereafter, the best infection parameters encountered were applied for the production of r-­-CapPCV2 (media: SFMTransFx-­-293 supplemented with 4mM glutaMAX, 5% FBS and 10%CB5; MOI:1; TOI:1x106 cell/mL) and TOH:48hpi). The second and third strategies are both based on the generation of stable producer cell lines, but one strategy relies on illegitimate (or random) integration of the gene in the HEK293 genome ,whereas the other strategy is a site-­-directed integration of the gene in previously characterized hot-­-spots (i.e. high-­-active transcribed regions from genome). The site-­-directed integration was performed using RMCE technology (Recombinant mediated cassette exchange). After the comparison of the specific and volumetric productivities achieved with each approach, the best producer has been selected. Nevertheless, r-­-CapPCV2 was poorly produced so it was unfeasible to develop/design a cost-­-effective industrial bioprocess and other alternatives must be studied in the future. Finally, the study of an unexpected metabolic behaviour observed in HEK293 cells cultured in our lab has been addressed from a physiologic and metabolic perspective. HEK293 cells could concomitantly consume glucose and lactate in exponentially growing cultures at particular environmental conditions. After a deep study of these conditions, it was found out that the switch from lactate secretion (which is the main drawback of mammalian high cell density cultures) to lactate consumption can be triggered from the beginning of cell culture at pH0=6.6 together with the addition of 4-­-12mM of lactate to media. Remarkably, under these conditions nor cell growth neither protein production were negatively affected. Form these results, we hypothesize that HEK293 can co-­-transport lactate and H+ to the cytosol as a pH-­-detoxification mechanism. Moreover, the application of flux balance analysis permitted to find out that when lactate and glucose are consumed together a "more balanced" metabolism is achieved, meaning that glycolytic and TCA fluxes became similar, avoiding pyruvate accumulation at the cytosol and consequently, lactate formation. This is totally opposed to the extensively observed metabolism of exponentially growing mammalian cell lines, where the high flux through the glycolytic pathway encounters a limitation on the fluxes entering the mitochondria (hence, the TCA cycle) and consequently lactate is produced and secreted to media. The construction of a HEK293 metabolic model and the application of FBA will allow making in silico predictions of metabolic beahaviours after the upregulation or downregulation of target genes. This strategy may open the possibility of generate engineered HEK293 cell lines with an optimised metabolism in order to study more efficient cell culture strategies towards the achievement of higher cell densities and product titres

Intrinsic functional connectivity differentiates minimally conscious from unresponsive patients

Despite advances in resting state functional magnetic resonance imaging investigations, clinicians remain with the challenge of how to implement this paradigm on an individualized basis. Here, we assessed the clinical relevance of resting state functional magnetic resonance imaging acquisitions in patients with disorders of consciousness by means of a systems-level approach. Three clinical centres collected data from 73 patients in minimally conscious state, vegetative state/unresponsive wakefulness syndrome and coma. The main analysis was performed on the data set coming from one centre (Liège) including 51 patients (26 minimally conscious state, 19 vegetative state/unresponsive wakefulness syndrome, six coma; 15 females; mean age 49 ± 18 years, range 11-87; 16 traumatic, 32 non-traumatic of which 13 anoxic, three mixed; 35 patients assessed \u3e1 month post-insult) for whom the clinical diagnosis with the Coma Recovery Scale-Revised was congruent with positron emission tomography scanning. Group-level functional connectivity was investigated for the default mode, frontoparietal, salience, auditory, sensorimotor and visual networks using a multiple-seed correlation approach. Between-group inferential statistics and machine learning were used to identify each network\u27s capacity to discriminate between patients in minimally conscious state and vegetative state/unresponsive wakefulness syndrome. Data collected from 22 patients scanned in two other centres (Salzburg: 10 minimally conscious state, five vegetative state/unresponsive wakefulness syndrome; New York: five minimally conscious state, one vegetative state/unresponsive wakefulness syndrome, one emerged from minimally conscious state) were used to validate the classification with the selected features. Coma Recovery Scale-Revised total scores correlated with key regions of each network reflecting their involvement in consciousness-related processes. All networks had a high discriminative capacity (\u3e80%) for separating patients in a minimally conscious state and vegetative state/unresponsive wakefulness syndrome. Among them, the auditory network was ranked the most highly. The regions of the auditory network which were more functionally connected in patients in minimally conscious state compared to vegetative state/unresponsive wakefulness syndrome encompassed bilateral auditory and visual cortices. Connectivity values in these three regions discriminated congruently 20 of 22 independently assessed patients. Our findings point to the significance of preserved abilities for multisensory integration and top-down processing in minimal consciousness seemingly supported by auditory-visual crossmodal connectivity, and promote the clinical utility of the resting paradigm for single-patient diagnostics

Functional connectivity and neuronal variability of resting state activity in bipolar disorder-reduction and decoupling in anterior cortical midline structures

Introduction: The cortical midline structures seem to be involved in the modulation of different resting state networks, such as the default mode network (DMN) and salience network (SN). Alterations in these systems, in particular in the perigenual anterior cingulate cortex (PACC), seem to play a central role in bipolar disorder (BD). However, the exact role of the PACC, and its functional connections to other midline regions (within and outside DMN) still remains unclear in BD. Methods: We investigated functional connectivity (FC), standard deviation (SD, as a measure of neuronal variability) and their correlation in bipolar patients (n?=?40) versus healthy controls (n?=?40), in the PACC and in its connections in different frequency bands (standard: 0.01?0.10 Hz; Slow-5: 0.01?0.027 Hz; Slow-4: 0.027?0.073 Hz). Finally, we studied the correlations between FC alterations and clinical-neuropsychological parameters and we explored whether subgroups of patients in different phases of the illness present different patterns of FC abnormalities. Results: We found in BD decreased FC (especially in Slow-5) from the PACC to other regions located predominantly in the posterior DMN (such as the posterior cingulate cortex (PCC) and inferior temporal gyrus) and in the SN (such as the supragenual anterior cingulate cortex and ventrolateral prefrontal cortex). Second, we found in BD a decoupling between PACC-based FC and variability in the various target regions (without alteration in variability itself). Finally, in our subgroups explorative analysis, we found a decrease in FC between the PACC and supragenual ACC (in depressive phase) and between the PACC and PCC (in manic phase). Conclusions: These findings suggest that in BD the communication, that is, information transfer, between the different cortical midline regions within the cingulate gyrus does not seem to work properly. This may result in dysbalance between different resting state networks like the DMN and SN. A deficit in the anterior DMN-SN connectivity could lead to an abnormal shifting toward the DMN, while a deficit in the anterior DMN-posterior DMN connectivity could lead to an abnormal shifting toward the SN, resulting in excessive focusing on internal contents and reduced transition from idea to action or in excessive focusing on external contents and increased transition from idea to action, respectively, which could represent central dimensions of depression and mania. If confirmed, they could represent diagnostic markers in BD