Fiber Scaffolds of Poly (glycerol-dodecanedioate) and its Derivative via Electrospinning for Neural Tissue Engineering

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

Bayesian Inference in Large-scale Problems

Many modern applications fall into the category of "large-scale" statistical problems, in which both the number of observations n and the number of features or parameters p may be large. Many existing methods focus on point estimation, despite the continued relevance of uncertainty quantification in the sciences, where the number of parameters to estimate often exceeds the sample size, despite huge increases in the value of n typically seen in many fields. Thus, the tendency in some areas of industry to dispense with traditional statistical analysis on the basis that "n=all" is of little relevance outside of certain narrow applications. The main result of the Big Data revolution in most fields has instead been to make computation much harder without reducing the importance of uncertainty quantification. Bayesian methods excel at uncertainty quantification, but often scale poorly relative to alternatives. This conflict between the statistical advantages of Bayesian procedures and their substantial computational disadvantages is perhaps the greatest challenge facing modern Bayesian statistics, and is the primary motivation for the work presented here.

Two general strategies for scaling Bayesian inference are considered. The first is the development of methods that lend themselves to faster computation, and the second is design and characterization of computational algorithms that scale better in n or p. In the first instance, the focus is on joint inference outside of the standard problem of multivariate continuous data that has been a major focus of previous theoretical work in this area. In the second area, we pursue strategies for improving the speed of Markov chain Monte Carlo algorithms, and characterizing their performance in large-scale settings. Throughout, the focus is on rigorous theoretical evaluation combined with empirical demonstrations of performance and concordance with the theory.

One topic we consider is modeling the joint distribution of multivariate categorical data, often summarized in a contingency table. Contingency table analysis routinely relies on log-linear models, with latent structure analysis providing a common alternative. Latent structure models lead to a reduced rank tensor factorization of the probability mass function for multivariate categorical data, while log-linear models achieve dimensionality reduction through sparsity. Little is known about the relationship between these notions of dimensionality reduction in the two paradigms. In Chapter 2, we derive several results relating the support of a log-linear model to nonnegative ranks of the associated probability tensor. Motivated by these findings, we propose a new collapsed Tucker class of tensor decompositions, which bridge existing PARAFAC and Tucker decompositions, providing a more flexible framework for parsimoniously characterizing multivariate categorical data. Taking a Bayesian approach to inference, we illustrate empirical advantages of the new decompositions.

Latent class models for the joint distribution of multivariate categorical, such as the PARAFAC decomposition, data play an important role in the analysis of population structure. In this context, the number of latent classes is interpreted as the number of genetically distinct subpopulations of an organism, an important factor in the analysis of evolutionary processes and conservation status. Existing methods focus on point estimates of the number of subpopulations, and lack robust uncertainty quantification. Moreover, whether the number of latent classes in these models is even an identified parameter is an open question. In Chapter 3, we show that when the model is properly specified, the correct number of subpopulations can be recovered almost surely. We then propose an alternative method for estimating the number of latent subpopulations that provides good quantification of uncertainty, and provide a simple procedure for verifying that the proposed method is consistent for the number of subpopulations. The performance of the model in estimating the number of subpopulations and other common population structure inference problems is assessed in simulations and a real data application.

In contingency table analysis, sparse data is frequently encountered for even modest numbers of variables, resulting in non-existence of maximum likelihood estimates. A common solution is to obtain regularized estimates of the parameters of a log-linear model. Bayesian methods provide a coherent approach to regularization, but are often computationally intensive. Conjugate priors ease computational demands, but the conjugate Diaconis--Ylvisaker priors for the parameters of log-linear models do not give rise to closed form credible regions, complicating posterior inference. In Chapter 4 we derive the optimal Gaussian approximation to the posterior for log-linear models with Diaconis--Ylvisaker priors, and provide convergence rate and finite-sample bounds for the Kullback-Leibler divergence between the exact posterior and the optimal Gaussian approximation. We demonstrate empirically in simulations and a real data application that the approximation is highly accurate, even in relatively small samples. The proposed approximation provides a computationally scalable and principled approach to regularized estimation and approximate Bayesian inference for log-linear models.

Another challenging and somewhat non-standard joint modeling problem is inference on tail dependence in stochastic processes. In applications where extreme dependence is of interest, data are almost always time-indexed. Existing methods for inference and modeling in this setting often cluster extreme events or choose window sizes with the goal of preserving temporal information. In Chapter 5, we propose an alternative paradigm for inference on tail dependence in stochastic processes with arbitrary temporal dependence structure in the extremes, based on the idea that the information on strength of tail dependence and the temporal structure in this dependence are both encoded in waiting times between exceedances of high thresholds. We construct a class of time-indexed stochastic processes with tail dependence obtained by endowing the support points in de Haan's spectral representation of max-stable processes with velocities and lifetimes. We extend Smith's model to these max-stable velocity processes and obtain the distribution of waiting times between extreme events at multiple locations. Motivated by this result, a new definition of tail dependence is proposed that is a function of the distribution of waiting times between threshold exceedances, and an inferential framework is constructed for estimating the strength of extremal dependence and quantifying uncertainty in this paradigm. The method is applied to climatological, financial, and electrophysiology data.

The remainder of this thesis focuses on posterior computation by Markov chain Monte Carlo. The Markov Chain Monte Carlo method is the dominant paradigm for posterior computation in Bayesian analysis. It has long been common to control computation time by making approximations to the Markov transition kernel. Comparatively little attention has been paid to convergence and estimation error in these approximating Markov Chains. In Chapter 6, we propose a framework for assessing when to use approximations in MCMC algorithms, and how much error in the transition kernel should be tolerated to obtain optimal estimation performance with respect to a specified loss function and computational budget. The results require only ergodicity of the exact kernel and control of the kernel approximation accuracy. The theoretical framework is applied to approximations based on random subsets of data, low-rank approximations of Gaussian processes, and a novel approximating Markov chain for discrete mixture models.

Data augmentation Gibbs samplers are arguably the most popular class of algorithm for approximately sampling from the posterior distribution for the parameters of generalized linear models. The truncated Normal and Polya-Gamma data augmentation samplers are standard examples for probit and logit links, respectively. Motivated by an important problem in quantitative advertising, in Chapter 7 we consider the application of these algorithms to modeling rare events. We show that when the sample size is large but the observed number of successes is small, these data augmentation samplers mix very slowly, with a spectral gap that converges to zero at a rate at least proportional to the reciprocal of the square root of the sample size up to a log factor. In simulation studies, moderate sample sizes result in high autocorrelations and small effective sample sizes. Similar empirical results are observed for related data augmentation samplers for multinomial logit and probit models. When applied to a real quantitative advertising dataset, the data augmentation samplers mix very poorly. Conversely, Hamiltonian Monte Carlo and a type of independence chain Metropolis algorithm show good mixing on the same dataset.

Descending Control of Limb Movements in Drosophila melanogaster

Because the interactions between feedforward influences are inextricably linked during many motor outputs (including but not limited to walking), the contribution of descending inputs to the generation of movements is difficult to study. Here we take advantage of the relatively small number of descending neurons (DNs) in the Drosophila melanogaster model system. We first characterize the number and distribution of the DN populations, then present a novel load free preparation, which enables the study of descending control on limb movements in a context where sensory feedback can be is reduced while leaving the nervous system, musculature, and cuticle of the animal relatively intact. Lastly we use in-vivo whole cell patch clamp electrophysiology to characterize the role of individual DNs in response to specific sensory stimuli and in relationship to movement. We find that there are approximately 1100 DNs in Drosophila that are distributed across six clusters. Input from these DNs is not necessary for coordinated motor activity, which can be generated by the thoracic ganglion, but is necessary for the specific combinations of joint movements typically observed in walking. Lastly, we identify a particular cluster of DNs that are tuned to sensory stimuli and innervate the leg neuromeres. We propose that a multi-layered interaction between these DNs, other DNs, and motor circuits in the thoracic ganglia enable the diverse but well-coordinated range of motor outputs an animal might exhibit.

β-Arrestin-mediated Regulation of the Human Ether-a-go-go-Related Gene (hERG) Potassium Channel

The human ether-a-go-go-related gene (hERG) encodes the voltage-gated K+ channel, hERG (Kv11.1). This channel passes the rapidly-activating delayed rectifier K+ current (IKr), which is important for cardiac repolarization. A reduction in IKr due to loss-of-function mutations or drug interactions causes long QT syndrome (LQTS), which can lead to cardiac arrhythmias and sudden cardiac death. The density of hERG channels in the plasma membrane is a key determinant of normal physiological function, and is balanced by trafficking to and from the cell surface. Many LQTS-associated hERG mutations result in a trafficking deficiency of otherwise functional channels. Thus, elucidating mechanisms of hERG regulation at the plasma membrane is useful for the prevention and treatment of LQTS. We previously demonstrated that M3 muscarinic receptor activation increases mature hERG expression through a Gq protein-dependent protein kinase C (PKC) pathway. In addition to conventional Gq protein-coupling, M3 receptors recruit β-arrestins upon agonist binding. Traditionally known for their role in receptor desensitization and internalization, β-arrestins also act as adaptor proteins to facilitate G protein-independent signaling. In the present work, I investigated the exclusive effect of β-arrestin signaling on hERG expression by utilizing an arrestin-biased M3 designer receptor (M3D-arr) exclusively activated by clozapine-N-oxide (CNO). By expressing M3D-arr in hERG-HEK cells and treating with CNO under various conditions, I found that M3D-arr activation increased mature hERG expression and current. Within this paradigm, M3D-arr recruited β-arrestin to the plasma membrane, and promoted the PI3K-dependent activation of Akt. I further found that the activated Akt acted through phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) and Rab11 to facilitate endosomal recycling of hERG channels to the plasma membrane.

Subfornical organ neurons integrate cardiovascular and metabolic signals

The subfornical organ (SFO) is a critical circumventricular organ involved in the control of cardiovascular and metabolic homeostasis. Despite the abundant literature clearly demonstrating the ability of SFO neurons to sense and respond to a plethora of circulating signals that influence various physiological systems, investigation of how simultaneously sensed signals interact and are integrated in the SFO is lacking. In this study, we use patch clamp techniques to investigate how the traditionally classified ‘cardiovascular’ hormone angiotensin II (ANG), ‘metabolic’ hormone cholecystokinin (CCK) and ‘metabolic’ signal glucose interact and are integrated in the SFO. Sequential bath-application of CCK (10nM) and ANG (10nM) onto dissociated SFO neurons revealed that: 63% of responsive SFO neurons depolarized to both CCK & ANG; 25% depolarized to ANG only; and 12% hyperpolarized to CCK only. We next investigated the effects of glucose by incubating and recording neurons in either hypo-, normo- or hyperglycemic conditions for a minimum of 24 hours and comparing the proportions of responses to ANG (n=55) or CCK (n=83) application in each condition. A hyperglycemic environment was associated with a larger proportion of depolarizing responses to ANG (X2, p<0.05), and a smaller proportion of depolarizing responses along with a larger proportion of hyperpolarizing responses to CCK (X2, p<0.01). These data demonstrate that SFO neurons excited by CCK are also excited by ANG, suggesting that CCK may influence fluid intake or blood pressure via the SFO, complementary to the well-understood actions of ANG at this site. Additionally, the demonstration that glucose environment affects the responsiveness of neurons to both these hormones highlights the ability of SFO neurons to integrate multiple metabolic and cardiovascular signals to affect transmission of information from the circulation to the brain, which has important implications for this structure’s critical role regulation of autonomic function.

Electrophysiological study of the effects of arginine vasopressin in the rat juxtacapsular nucleus of the bed nucleus of the stria terminalis

Arginine vasopressin (AVP), a nine amino acid neuropeptide (CYFQNCPRG- NH2) fulfills a dual function: (i) in the periphery, AVP acts as a peptide hormone and (ii) in the CNS, AVP is a neuromodulatory peptide. AVP produces its effects through 3 AVP receptors (AVPRs). AVPR1a and AVPR1b are expressed in the CNS and periphery, whilst AVPR2 is not found centrally but instead solely expressed in the kidneys. Recent evidence revealed a high density of AVP-binding sites in the juxtacapsular nucleus of the bed nucleus of the stria terminalis (jxBNST). While in other regions of the brain, AVP acts at AVPRs to regulate an array of biological processes, including male-typical social behaviours, social memory, stress adaptation, fear, anxiety, and fluid homeostasis, its role in the jxBNST remains elusive. Furthermore, the neurophysiological properties of AVP in the jxBNST are unknown so this study aimed to examine how AVP modulates synaptic transmission in the rat jxBNST. The BNST being one of the most notable sexually dimorphic brain regions and AVPR expression being influenced by gonadal steroids, we investigated the putative influence of sex on the modulatory effects of AVP in the jxBNST. Finally, due to AVP being released at a substantially higher concentration following periods of water deprivation, we examined changes in AVPs modulatory role following water deprivation. Male and female Long Evans rats were euthanized and brain slice whole-cell voltage-clamp electrophysiology was done in the jxBNST to measure the effects of AVP on synaptic transmission of GABA synapses. Exogenous application of AVP produced three responses; either postsynaptic long-term potentiation (LTP) of GABAA-inhibitory postsynaptic currents (IPSC), postsynaptic long-term depression (LTD) of GABAA-IPSC, or no change in GABAA-IPSC amplitudes. Interestingly, the proportion of neurons responding in each of these ways did not differ between sexes and within females was not estrous cycle-dependent. Finally, although not statistically significant, 24-hour water deprivation abolished GABAA-LTD, an effect that was not a consequence of social isolation. Taken together, our data show that AVP modulates GABAA synaptic transmission in the jxBNST in fluid homeostasis- but not sex-dependent manner.

Effects of menthol on rat tail artery mediated by TRPM8 and voltage-gated calcium channels

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold sensing role in sensory neurons, it is expressed and functional in several non-neuronal tissues, including vasculature. We previously demonstrated that menthol causes vasoconstriction and vasodilatation in isolated arteries, depending on vascular tone. Here we investigated calcium's role in responses mediated by TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology and ratiometric Ca2+ recording. Isometric contraction studies examined actions of TRPM8 ligands in the presence/absence of L-type calcium channel blocker. Menthol (300 μM), a concentration typically used to induce TRPM8 currents, strongly inhibited L-type voltage-dependent Ca2+ current (L-ICa) in myocytes, especially it's sustained component, most relevant for depolarisation-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol. Menthol-induced increases in PE-induced vasoconstrictions were mediated predominantly by Ca2+-release from sarcoplasmic reticulum, since they were significantly inhibited by cyclopiazonic acid. Pre-incubation of vascular rings with a TRPM8 antagonist strongly inhibited menthol-induced increases in PE-induced vasoconstrictions, thus confirming specific role of TRPM8. Finally, two other common TRPM8 agonists, WS-12 and icilin, inhibited L-ICa. Thus, TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels, and largely obscure TRPM8-mediated vasoconstriction.

TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles

Purpose: Activation of the transient receptor potential channels, TRPC6, TRPM4, and TRPP1 (PKD2), has been shown to contribute to the myogenic constriction of cerebral arteries. In the present study we sought to determine the potential role of various mechanosensitive TRP channels to myogenic signaling in arterioles of the rat retina.

Methods: Rat retinal arterioles were isolated for RT-PCR, Fura-2 Ca2+ microfluorimetry, patch-clamp electrophysiology, and pressure myography studies. In some experiments, confocal immunolabeling of wholemount preparations was used to examine the localization of specific mechanosensitive TRP channels in retinal vascular smooth muscle cells (VSMCs).

Results: Reverse transcription-polymerase chain reaction analysis demonstrated mRNA expression for TRPC1, M7, V1, V2, V4, and P1, but not TRPC6 or M4, in isolated retinal arterioles. Immunolabeling revealed plasma membrane, cytosolic and nuclear expression of TRPC1, M7, V1, V2, V4, and P1 in retinal VSMCs. Hypoosmotic stretch-induced Ca2+ influx in retinal VSMCs was reversed by the TRPV2 inhibitor tranilast and the nonselective TRPP1/V2 antagonist amiloride. Inhibitors of TRPC1, M7, V1, and V4 had no effect. Hypoosmotic stretch-activated cation currents were similar in Na+ and Cs+ containing solutions suggesting no contribution by TRPP1 channels. Direct plasma membrane stretch triggered cation current activity that was blocked by tranilast and specific TRPV2 pore-blocking antibodies and mimicked by the TRPV2 activator, Δ9-tetrahydrocannabinol. Preincubation of retinal arterioles with TRPV2 blocking antibodies prevented the development of myogenic tone.

Conclusions: Our results suggest that retinal VSMCs express a range of mechanosensitive TRP channels, but only TRPV2 appears to contribute to myogenic signaling in this vascular bed.

Marqueurs électrophysiologiques des dysfonctions attentionnelles et de leur récupération suite à un traumatisme craniocérébral léger

L’objectif principal de cette thèse était d’obtenir, via l’électrophysiologie cognitive, des indices de fonctionnement post-traumatisme craniocérébral léger (TCCL) pour différents niveaux de traitement de l’information, soit l’attention sélective, les processus décisionnels visuoattentionnels et les processus associés à l’exécution d’une réponse volontaire. L’hypothèse centrale était que les mécanismes de production des lésions de même que la pathophysiologie caractérisant le TCCL engendrent des dysfonctions visuoattentionnelles, du moins pendant la période aiguë suivant le TCCL (i.e. entre 1 et 3 mois post-accident), telles que mesurées à l’aide d’un nouveau paradigme électrophysiologique conçu à cet effet. Cette thèse présente deux articles qui décrivent le travail effectué afin de rencontrer ces objectifs et ainsi vérifier les hypothèses émises. Le premier article présente la démarche réalisée afin de créer une nouvelle tâche d’attention visuospatiale permettant d’obtenir les indices électrophysiologiques (amplitude, latence) et comportementaux (temps de réaction) liés aux processus de traitement visuel et attentionnel précoce (P1, N1, N2-nogo, P2, Ptc) à l’attention visuelle sélective (N2pc, SPCN) et aux processus décisionnels (P3b, P3a) chez un groupe de participants sains (i.e. sans atteinte neurologique). Le deuxième article présente l’étude des effets persistants d’un TCCL sur les fonctions visuoattentionelles via l’obtention des indices électrophysiologiques ciblés (amplitude, latence) et de données comportementales (temps de réaction à la tâche et résultats aux tests neuropsychologiques) chez deux cohortes d’individus TCCL symptomatiques, l’une en phase subaigüe (3 premiers mois post-accident), l’autre en phase chronique (6 mois à 1 an post-accident), en comparaison à un groupe de participants témoins sains. Les résultats des articles présentés dans cette thèse montrent qu’il a été possible de créer une tâche simple qui permet d’étudier de façon rapide et peu coûteuse les différents niveaux de traitement de l’information impliqués dans le déploiement de l’attention visuospatiale. Par la suite, l’utilisation de cette tâche auprès d’individus atteints d’un TCCL testés en phase sub-aiguë ou en phase chronique a permis d’objectiver des profils d’atteintes et de récupération différentiels pour chacune des composantes étudiées. En effet, alors que les composantes associées au traitement précoce de l’information visuelle (P1, N1, N2) étaient intactes, certaines composantes attentionnelles (P2) et cognitivo-attentionnelles (P3a, P3b) étaient altérées, suggérant une dysfonction au niveau des dynamiques spatio-temporelles de l’attention, de l’orientation de l’attention et de la mémoire de travail, à court et/ou à long terme après le TCCL, ceci en présence de déficits neuropsychologiques en phase subaiguë surtout et d’une symptomatologie post-TCCL persistante. Cette thèse souligne l’importance de développer des outils diagnostics sensibles et exhaustifs permettant d’objectiver les divers processus et sous-processus cognitifs susceptible d’être atteints après un TCCL.

Global slowing of network oscillations in mouse neocortex by diazepam

SCHEFFZUK, C. , KUKUSHKA, V. , VYSSOTSKI, A. L. , DRAGUHN, A. , TORT, A. B. L. , BRANKACK, J. . Global slowing of network oscillations in mouse neocortex by diazepam. Neuropharmacology , v. 65, p. 123-133, 2013.

Ocular manifestations of microcephaly with or without chorioretinopathy, lymphedema or intellectual disability (MCLID) syndrome associated with mutations in KIF11

Purpose Microcephaly with or without chorioretinopathy, lymphedema or intellectual disability (MCLID) is an autosomal dominant condition. Mutations in KIF11 have been found to be causative in approximately 75% of cases. This study describes the ocular phenotype in patients with confirmed KIF11 mutations. Methods Standard ophthalmic examination and investigation including visual acuity, refraction and fundus examination was carried out in all patients. Fundus autofluorescence imaging (FAF) was performed in three patients, and four patients underwent spectral domain optical coherence tomography (OCT). Flash electroretinography (ERG) was performed in seven patients, and five underwent additional pattern electroretinography (PERG). Results The patients ranged in age from 2 to 10 years. Most presented with visual acuity loss. Fundus examination revealed lacunae of chorioretinal atrophy. Pigmentary macular changes and optic disc pallor were present in three of seven patients. Fundus autofluorescence demonstrated hypoautofluorescence at the macula in two of three patients. The lacunae of chorioretinal atrophy were hypoautofluorescent. The OCT showed atrophic maculae in three of four patients. Follow-up in one patient showed no deterioration of the vision over a 9-year period. The lesions appear not to be progressive on the follow-up imaging. Electrophysiology showed generalized rod and cone dysfunction and severe macular dysfunction. Inner retinal dysfunction was evident in three of seven patients. Conclusions Patients with KIF11 mutations show a specific ocular phenotype with variable expressivity and intrafamilial variability. Macular atrophy and dysfunction have not been consistently documented before. The fundus lesions appear non-progressive. The findings assist in providing an accurate diagnosis and thus improving the management and follow-up of patients with this syndrome.

Contact Force in Atrial Fibrillation: Role of Atrial Rhythm and Ventricular Contractions: Co-Force Atrial Fibrillation Study

In an experimental model, variable and intermittent contact force (CF) resulted in a significant decrease in lesion volume. In humans, variability of CF during pulmonary vein isolation has not been characterized. Methods and Results-In 20 consecutive patients undergoing CF-guided circumferential pulmonary vein isolation, 914 radiofrequency applications (530 in sinus rhythm and 384 in atrial fibrillation) were analyzed. The variability of the 60% CF range (CF60%) was 17 ± 9.6 g. Hundred seventy-one (19%) applications were delivered with constant, 717 (78%) with variable, and 26 (3%) with intermittent CF. The mean CF and force-time integral were significantly higher during applications with variable than with intermittent or constant CF. There was no significant difference in CF variability, CF60% variability, and force-time integral between applications delivered in sinus rhythm and atrial fibrillation. The main reasons for CF variability were systolo-diastolic heart movement (29%) and respiration (27%). In 10 additional patients, during adenosine-induced atrioventricular block, the minimum CF significantly increased at 19 sites (5.3 ± 4.4 versus 13.4 ± 5.9 g; P < 0.001) and at 16 sites intermittent or variable CF became constant. At only 1 site systolo-diastolic movement remained the main reason for variable CF. Conclusions-CF during pulmonary vein isolation remains highly variable despite efforts to optimize contact. CF and CF parameters were similar during sinus rhythm and atrial fibrillation. The main reasons for CF variability are systolodiastolic heart movement and respiration. The systolo-diastolic peaks and nadirs of CF are because of ventricular contractions at the large majority of pulmonary vein isolation sites.

Optimization of a multielectrode array (MEA)-based approach to study the impact of Aβ on the SH-SY5Y cell line

The human brain stores, integrates, and transmits information recurring to millions of neurons, interconnected by countless synapses. Though neurons communicate through chemical signaling, information is coded and conducted in the form of electrical signals. Neuroelectrophysiology focus on the study of this type of signaling. Both intra and extracellular approaches are used in research, but none holds as much potential in high-throughput screening and drug discovery, as extracellular recordings using multielectrode arrays (MEAs). MEAs measure neuronal activity, both in vitro and in vivo. Their key advantage is the capability to record electrical activity at multiple sites simultaneously. Alzheimer’s disease (AD) is the most common neurodegenerative disease and one of the leading causes of death worldwide. It is characterized by neurofibrillar tangles and aggregates of amyloid-β (Aβ) peptides, which lead to the loss of synapses and ultimately neuronal death. Currently, there is no cure and the drugs available can only delay its progression. In vitro MEA assays enable rapid screening of neuroprotective and neuroharming compounds. Therefore, MEA recordings are of great use in both AD basic and clinical research. The main aim of this thesis was to optimize the formation of SH-SY5Y neuronal networks on MEAs. These can be extremely useful for facilities that do not have access to primary neuronal cultures, but can also save resources and facilitate obtaining faster high-throughput results to those that do. Adhesion-mediating compounds proved to impact cell morphology, viability and exhibition of spontaneous electrical activity. Moreover, SH-SY5Y cells were successfully differentiated and demonstrated acute effects on neuronal function after Aβ addition. This effect on electrical signaling was dependent on Aβ oligomers concentration. The results here presented allow us to conclude that the SH-SY5Y cell line can be successfully differentiated in properly coated MEAs and be used for assessing acute Aβ effects on neuronal signaling.

Global slowing of network oscillations in mouse neocortex by diazepam

SCHEFFZUK, C. , KUKUSHKA, V. , VYSSOTSKI, A. L. , DRAGUHN, A. , TORT, A. B. L. , BRANKACK, J. . Global slowing of network oscillations in mouse neocortex by diazepam. Neuropharmacology , v. 65, p. 123-133, 2013.

Low doses of a neonicotinoid insecticide modify pheromone response thresholds of central but not peripheral olfactory neurons in a pest insect

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