Modulation des neurones dopaminergiques du mésencéphale par la neurotensine

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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.

Modulation des neurones GABAergiques du mésencéphale ventral

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Modulation des neurones dopaminergiques du mésencéphale par la neurotensine

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

INVESTIGATING THE MECHANISM OF AUTOIMMUNE DISEASE ASSOCIATED-LQTS

The human ether-a-go-go-related gene (hERG) protein passes the rapidly activating delayed rectifier potassium channel (IKr), and malfunction of hERG protein/IKr is the primary cause of acquired long QT syndrome (LQTS). Autoimmune diseases are significantly correlated with prolonged QT intervals, for which autoantibodies have been implicated. The anti-Ro52 autoantibody is the most frequently evaluated, and importantly has been correlated with prolonged QT intervals. Pathological anti-Ro52-hERG interactions have been discussed as a mechanism for autoimmune disease-related LQTS. However, the mechanism is unclear, and it does not explain LQTS in autoimmune diseases which do not commonly express anti-Ro52. In this thesis, I investigated the effects of anti-Ro52 on hERG/IKr function. Through Western blot analysis, whole-cell patch-clamp, and immunofluorescence, I show that anti-Ro52 chronically (12 h) reduced hERG protein expression and hERG current by over 50%, but did not acutely block the channel. My work revealed a novel mechanism in which the Fc portion of anti-Ro52 interacts with the extracellular S5-pore linker of the channel to induce internalization through a tyrosine phosphorylation dependent pathway. This phenomenon extends beyond anti-Ro52 IgG, as other IgG, regardless of their antigen binding specificity, have the potential to reduce hERG expression/current. Rather, the ability of IgG to reduce hERG expression and current is dependent on the IgG subclass, as we show mouse IgG2A was the only mouse IgG subclass which reduced hERG expression. These results provide a novel explanation for autoimmune disease associated LQTS. It also has implications in the development of safe monoclonal antibody drugs.

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.

Impaired GABA Plasticity at ovBNST Synapses Predicts Compulsive Drinking in Schedule-Induced Polydipsia

Schedule-Induced Polydipsia (SIP) is an animal model of adjunctive drinking induced when a hungry rat receives food on a fixed interval of time. This model has been implemented as a model of compulsive behaviour and may represent a powerful tool to understand the neural mechanisms of compulsion. The bed nucleus of the stria terminalis (BNST) is thought to translate challenges to energy homeostasis into consummatory behaviours, and is therefore likely to contribute to drinking behaviours displayed by food restricted rats in the SIP paradigm. Furthermore, the BNST seems implicated in various compulsive behaviors, including compulsive water drinking in rats. Therefore, the goal of this project was to determine whether compulsive drinking in the SIP paradigm was associated with alterations in transmission at oval BNST (ovBNST) synapses. Rats undergoing the SIP procedure had restricted food access (1-hours/day) for a total of 29 days. After 7 days of food restriction and for the next 21 consecutive days, the rats had daily 2-hour access to operant conditioning chambers where they were presented with a 45-mg food pellet every minute. Water consumed during these 2-hour sessions was measured and the rats that drank 15 ml or more water for a minimum of 3 consecutive days were considered High Drinkers (HD; n=17) or otherwise, Low Drinkers (LD; n=13). Brain slices whole-cell patch clamp recordings conducted 18-hours after the last SIP training showed that chronic food restriction changed low frequency stimulation (LFS) - induced long-term potentiation of ovBNST inhibitory synaptic transmission (iLTP) into LFS - induced long-term depression (iLTD) in a majority of neurons, regardless of drinking behaviours. However, ad libitum access to food between the last day of SIP training and brain slice recordings (18-hour refeed) rescued LFS-induced iLTP in LD but not in HD, suggesting that impaired bi-directional plasticity of ovBNST synapses may contribute to compulsive drinking in the SIP paradigm.

Biodentine Reduces Tumor Necrosis Factor Alpha-induced TRPA1 Expression in Odontoblastlike Cells

INTRODUCTION: The transient receptor potential (TRP) ion channels have emerged as important cellular sensors in both neuronal and non-neuronal cells, with TRPA1 playing a central role in nociception and neurogenic inflammation. The functionality of TRP channels has been shown to be modulated by inflammatory cytokines. The aim of this study was to investigate the effect of inflammation on odontoblast TRPA1 expression and to determine the effect of Biodentine (Septodent, Paris, France) on inflammatory-induced TRPA1 expression.

METHODS: Immunohistochemistry was used to study TRPA1 expression in pulp tissue from healthy and carious human teeth. Pulp cells were differentiated to odontoblastlike cells in the presence of 2 mmol/L beta-glycerophosphate, and these cells were used in quantitative polymerase chain reaction, Western blotting, calcium imaging, and patch clamp studies.

RESULTS: Immunofluorescent staining revealed TRPA1 expression in odontoblast cell bodies and odontoblast processes, which was more intense in carious versus healthy teeth. TRPA1 gene expression was induced in cultured odontoblastlike cells by tumor necrosis factor alpha, and this expression was significantly reduced in the presence of Biodentine. The functionality of the TRPA1 channel was shown by calcium microfluorimetry and patch clamp recording, and our results showed a significant reduction in tumor necrosis factor alpha-induced TRPA1 responses after Biodentine treatment.

CONCLUSIONS: In conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses.

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.

Effect of insulin resistance on monounsaturated fatty acid levels : a multi-cohort non-targeted metabolomics and mendelian randomization study

Insulin resistance (IR) and impaired insulin secretion contribute to type 2 diabetes and cardiovascular disease. Both are associated with changes in the circulating metabolome, but causal directions have been difficult to disentangle. We combined untargeted plasma metabolomics by liquid chromatography/mass spectrometry in three non-diabetic cohorts with Mendelian Randomization (MR) analysis to obtain new insights into early metabolic alterations in IR and impaired insulin secretion. In up to 910 elderly men we found associations of 52 metabolites with hyperinsulinemic-euglycemic clamp-measured IR and/or β-cell responsiveness (disposition index) during an oral glucose tolerance test. These implicated bile acid, glycerophospholipid and caffeine metabolism for IR and fatty acid biosynthesis for impaired insulin secretion. In MR analysis in two separate cohorts (n = 2,613) followed by replication in three independent studies profiled on different metabolomics platforms (n = 7,824 / 8,961 / 8,330), we discovered and replicated causal effects of IR on lower levels of palmitoleic acid and oleic acid. A trend for a causal effect of IR on higher levels of tyrosine reached significance only in meta-analysis. In one of the largest studies combining "gold standard" measures for insulin responsiveness with non-targeted metabolomics, we found distinct metabolic profiles related to IR or impaired insulin secretion. We speculate that the causal effects on monounsaturated fatty acid levels could explain parts of the raised cardiovascular disease risk in IR that is independent of diabetes development.

Projeto, implementação e análise de um conversor CC-CC de alto ganho e alto rendimento para aplicações fotovoltaicas

In recent years the photovoltaic generation has had greater insertion in the energy mix of the most developed countries, growing at annual rates of over 30%. The pressure for the reduction of pollutant emissions, diversification of the energy mix and the drop in prices are the main factors driving this growth. Grid tied systems plays an important role in alleviating the energy crisis and diversification of energy sources. Among the grid tied systems, building integrated photovoltaic systems suffers from partial shading of the photovoltaic modules and consequently the energy yield is reduced. In such cases, classical forms of modules connection do not produce good results and new techniques have been developed to increase the amount of energy produced by a set of modules. In the parallel connection technique of photovoltaic modules, a high voltage gain DC-DC converter is required, which is relatively complex to build with high efficiency. The current-fed isolated converters explored in this work have some desirable characteristics for this type of application, such as: low input current ripple and input voltage ripple, high voltage gain, galvanic isolation, feature high power capacity and it achieve soft switching in a wide operating range. This study presents contributions to the study of a high gain and high efficiency DC-DC converter for use in a parallel system of photovoltaic generation, being possible the use in a microinverter or with central inverter. The main contributions of this work are: analysis of the active clamping circuit operation proposing that the clamp capacitor connection must be done on the negative node of the power supply to reduce the input current ripple and thus reduce the filter requirements; use of a voltage doubler in the output rectifier to reduce the number of components and to extend the gain of the converter; detailed study of the converter components in order to raise the efficiency; obtaining the AC equivalent model and control system design. As a result, a DC-DC converter with high gain, high efficiency and without electrolytic capacitors in the power stage was developed. In the final part of this work the DC-DC converter operation connected to an inverter is presented. Besides, the DC bus controller is designed and are implemented two maximum power point tracking algorithms. Experimental results of full system operation connected to an emulator and subsequently to a real photovoltaic module are also given.

Effect of remote ischaemic preconditioning in cardiac dysfunction and end-organ injury following cardiac surgery with cardiopulmonary bypass in children: A translational approach investigating clinical outcome and myocardial molecular biology

Congenital heart disease (CHD) is the most common birth defect, causing an important rate of morbidity and mortality. Treatment of CHD requires surgical correction in a significant percentage of cases which exposes patients to cardiac and end organ injury. Cardiac surgical procedures often require the utilisation of cardiopulmonary bypass (CPB), a system that replaces heart and lungs function by diverting circulation into an external circuit. The use of CPB can initiate potent inflammatory responses, in addition a proportion of procedures require a period of aortic cross clamp during which the heart is rendered ischaemic and is exposed to injury. High O2 concentrations are used during cardiac procedures and when circulation is re-established to the heart which had adjusted metabolically to ischaemia, further injury is caused in a process known as ischaemic reperfusion injury (IRI). Several strategies are in place in order to protect the heart during surgery, however injury is still caused, having detrimental effects in patients at short and long term. Remote ischaemic preconditioning (RIPC) is a technique proposed as a potential cardioprotective measure. It consists of exposing a remote tissue bed to brief episodes of ischaemia prior to surgery in order to activate protective pathways that would act during CPB, ischaemia and reperfusion. This study aimed to assess RIPC in paediatric patients requiring CHD surgical correction with a translational approach, integrating clinical outcome, marker analysis, cardiac function parameters and molecular mechanisms within the cardiac tissue. A prospective, single blinded, randomized, controlled trial was conducted applying a RIPC protocol to randomised patients through episodes of limb ischaemia on the day before surgery which was repeated right before the surgery started, after anaesthesia induction. Blood samples were obtained before surgery and at three post-operative time points from venous lines, additional pre and post-bypass blood samples were obtained from the right atrium. Myocardial tissue was resected during the ischaemic period of surgery. Echocardiographic images were obtained before the surgery started after anaesthetic induction and the day after surgery, images were stored for later off line analysis. PICU surveillance data was collected including ventilation parameters, inotrope use, standard laboratory analysis and six hourly blood gas analysis. Pre and post-operative quantitation of markers in blood specimens included cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), inflammatory mediators including interleukins IL-6, IL-8, IL-10, tumour necrosis factor (TNF-α), and the adhesion molecules ICAM-1 and VCAM-1; the renal marker Cystatin C and the cardiovascular markers asymmetric dymethylarginine (ADMA) and symmetric dymethylarginine (SDMA). Nitric oxide (NO) metabolites and cyclic guanosine monophosphate (cGMP) were measured before and after bypass. Myocardial tissue was processed at baseline and after incubation at hyperoxic concentration during four hours in order to mimic surgical conditions. Expression of genes involved in IRI and RIPC pathways was analysed including heat shock proteins (HSPs), toll like receptors (TLRs), transcription factors nuclear factor κ-B (NF- κ-B) and hypoxia inducible factor 1 (HIF-1). The participation of hydrogen sulfide enzymatic genes, apelin and its receptor were explored. There was no significant difference according to group allocation in any of the echocardiographic parameters. There was a tendency for higher cTnI values and inotropic score in control patients post-operatively, however this was not statistically significant. BNP presented no significant difference according to group allocation. Inflammatory parameters tended to be higher in the control group, however only TNF- α was significantly higher. There was no difference in levels of Cystatin C, NO metabolites, cGMP, ADMA or SDMA. RIPC patients required shorter PICU stay, all other clinical and laboratory analysis presented no difference related to the intervention. Gene expression analysis revealed interesting patterns before and after incubation. HSP-60 presented a lower expression at baseline in tissue corresponding to RIPC patients, no other differences were found. This study provided with valuable descriptive information on previously known and newly explored parameters in the study population. Demographic characteristics and the presence of cyanosis before surgery influenced patterns of activity in several parameters, numerous indicators were linked to the degree of injury suffered by the myocardium. RIPC did not reduce markers of cardiac injury or improved echocardiographic parameters and it did not have an effect on end organ function; some effects were seen in inflammatory responses and gene expression analysis. Nevertheless, an important clinical outcome indicator, PICU length of stay was reduced suggesting benefit from the intervention. Larger studies with more statistical power could determine if the tendency of lower injury and inflammatory markers linked to RIPC is real. The present results mostly support findings of larger multicentre trials which have reported no cardiac benefit from RIPC in paediatric cardiac surgery.

Regulation of electrical transmission by protein kinase C in Aplysia bag cell neurons

Electrical synapses are composed of gap junctions, made from paired hemi-channels that allow for the transfer of current from one neuron to another. Gap junctions mediate electrical transmission in neurons, where they synchronize spiking and promote rapid transmission, thereby influencing the coordination, pattern, and frequency of firing. In the marine snail, Aplysia calfornica, two clusters of neuroendocrine bag cell neurons use electrical synapses to synchronize a 30-min burst of action potentials, known as the afterdischarge, which releases egg-laying hormone and induces reproduction. In culture, paired bag cell neurons present a junctional conductance that is non-rectifying and largely voltage-independent. During the afterdischarge, PKC is activated, which is known to increase voltage-gated Ca2+ current; yet, little is understood as to how this pathway impacts electrical transmission. The transfer of presynaptic spike-like waveforms (generated in voltage-clamp) to the postsynaptic cell (measured in current-clamp) was monitored with or without PKC activation. It was found that pretreatment with the PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced junctional conductance between bag cell neurons. Furthermore, in control, presynaptic action potential waveforms mainly evoked postsynaptic electrotonic potentials at both -60 and -40 mV. However, with PKC activation the presynaptic stimulus consistently elicited postsynaptic action potentials from resting potentials of -40 mV, and would occasionally result in firing from repetitive input at -60 mV. Moreover, to assess whether this enhanced electrical transmission genuinely reflects a greater junctional conductance or a change in postsynaptic responsiveness, a fast-phase junctional-like current was applied to single bag cell neurons. Neurons in PMA always fired action potentials in response to current injection as opposed to control, which were less likely to spike. This outcome did not change when the junctional-like current was artificially enhanced in control conditions. Also, in response to fast- and slow-phase electrotonic potential (ETP) waveforms, Ca2+ current was markedly larger in single PMA-treated neurons. These findings suggest that PKC activation may contribute to afterdischarge fidelity by recruiting postsynaptic Ca2+ current to promote synchronous network firing. Finally, Aplysia gap junction genes (innexins) were transfected into mouse N2A cells and characterized. This revealed a biophysical and pharmacological profile similar to native gap junctions.