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The mammalian adrenal gland (or suprarenal gland) is a small organ located on the superior aspect of the kidney. The central region of the gland, the medulla, consists of chromaffin cells, which release catecholamines into the blood during periods of stress. This is best known as the 'fight or flight' response and is regulated, in the adult animal, by neuronal signals from the cholinergic sympathetic fibres of the splanchnic nerve. Interestingly, in some mammals, such as rat and human, sympathetic innervation is immature at birth, yet the chromaffin cells can still secrete catecholamines in response to physiological stessors, e.g. hypoxia. Increased plasma catecholamines is thought to provide a vital protective role for the neonatal animal during, and following birth. This is mediated in part by promoting lung fluid absorption, surfactant secretion, heart rate stabilization, and brown fat mobilization. The observation that, in the neonate, catecholamines are secreted in the absence of functional sympathetic innervation suggests that the chromaffin cells possess other mechanisms for directly 'sensing' a fall in blood O2 tension (hypoxia). The primary goal of this thesis was to uncover the mechanisms of oxygen-sensing in developing chromaffin cells from the rat and mouse, using primary short-term cell cultures of chromaffin cells. The experimental approaches relied on patch clamp techniques to record ionic currents and membrane potential, carbon fibre electrochemistry to record catecholamine secretion from cell clusters, and fluorescent indicators to measure reactive oxygen species generation. Hypoxic chemosensitivity was found in embryonic and neonatal, but not juvenile chromaffin cells from both the rat and mouse. Exposure to hypoxia or anoxia caused a reversible suppression of whole-cell current, which was comprised of the differential modulation of three K+ currents: (1) suppression of a large-conductance Ca2+-dependent K+ current; (2) suppression of a delayed rectifier K+ current; and (3) activation of an ATP-sensitive K+ current. Hypoxia also induced membrane depolarization that was not initiated by any of these three voltage-dependent K+ currents. Additionally, hypoxia broadened action potentials in chromaffin cells that showed spontaneous activity, and this was mediated by a prolongation of the time course of membrane repolarization. All of these factors likely contribute to catecholamine secretion by enhancing the influx of Ca2+ through depolarization-activated L-type Ca2+ channels. Two sets of experiments were designed to identify the oxygen sensor in neonatal chromaffin cells. First, cells from transgenic mice, deficient in the gp91^phox component of the putative O2-sensor protein, NADPH oxidase, responded to hypoxia in the same way as wild type cell, indicating that NADPH oxidase is not primarily responsible for oxygen sensitivity in these cells. Second, inhibitors of the proximal electron transport chain (e.g. rotenone and antimycin A) mimicked and attenuated the hypoxic response, while inhibitors of the distal electron transport chain (cyanide) and uncouplers of oxidative phosphorylation (2,4-dinitrophenol) had no effect. Furthermore, reactive oxygen species production, primarily H2O2, decreased during exposure to hypoxia or inhibitors of the proximal electron transport chain, revealing a potential mitochondrial mechanism for 'sensing' of the hypoxic stimulus. Reduced oxygen availability to the electron transport chain is proposed to cause a fall in cellular reactive oxygen species (ROS), principally H2O2. This fall in ROS signals closure of Ca2+-dependent and Ca2+-independent K+ channels, which causes broadening action potentials and increases Ca2+ influx. The latter is further enhanced by the hypoxia-induced membrane depolarization, which in turn increases the probability of cell firing. The rise in intracellular Ca2+ then acts as the signal for catecholamine release from the chromaffin cells. / Thesis / Doctor of Philosophy (PhD)

Alcohol use disorder (AUD) is a relapsing–remitting condition characterized by excessive and/or continued alcohol consumption despite harmful consequences. New adjuvant tools, such as noninvasive brain stimulation techniques, might be helpful additions to conventional treatment approaches or even provide an alternative option for patients who fail to respond adequately to other treatment options. Here, we discuss the potential use of auricular transcutaneous vagus nerve stimulation (atVNS) as an ADD-ON intervention in AUD. Compared with other techniques, atVNS has the advantage of directly stimulating nuclei that synthesize GABA and catecholamines, both of which are functionally altered by alcohol intake in AUD patients. Pharmacological options targeting those neurotransmitters are widely available, but have relatively limited beneficial effects on cognition, even though restoring normal cognitive functioning, especially cognitive control, is key to maintaining abstinence. Against this background, atVNS could be a particularly useful add-on because there is substantial meta-analytic evidence based on studies in healthy individuals that atVNS can enhance cognitive control processes that are crucial to regaining control over drug intake. We discuss essential future research on using atVNS as an ADD-ON intervention in AUD to enhance clinical and cognitive outcomes by providing a translational application. Given that this novel technique can be worn like an earpiece and can be employed without medical supervision/outside the clinical settings, atVNS could be well integratable into the daily life of the patients, where the task of regaining control over drug intake is most challenging.

Toxinas que atuam em canais para Na+ operados por voltagem são as principais responsáveis pelos efeitos tóxicos do envenenamento escorpiônico e podem ser divididas em duas classes: a- e b-neurotoxinas. TsTX-V e TsTX-I da peçonha de Tityus serrulatus (TsV) são, respectivamente, exemplos destas toxinas. Neste trabalho, foram avaliados os efeitos da TsV e destas toxinas sobre a pressão arterial média (PAM) e liberação de catecolaminas em ratos conscientes e não imobilizados, previamente cateterizados, bem como a captação de GABA, dopamina (DA) e glutamato (Glu) em sinaptosomas isolados de cérebro de ratos e a concentração citoplasmática de Ca+2 ([Ca+2 ]C) em células de músculo liso vascular de aorta de ratos. As toxinas foram isoladas por cromatografia de troca iônica (TsTX-I) seguida por CLAE de fase reversa (TsTX-V). As toxinas (15 e 30 g/kg) e TsV (50 e 100 g/kg) foram injetadas intravenosamente. A PAM foi monitorada continuamente através do cateter femoral. Os níveis plasmáticos de adrenalina (ADR) e noradrenalina (NA) foram determinados por CLAE de fase reversa com detector eletroquímico, em 10 min antes e 2,5, 30 e 90 min após os tratamentos. Efeitos pressores máximos foram observados em 2,5?3,5 min. TsV induziu um intenso aumento de longa duração na PAM, bem como a TsTX-I. A TsTX-V mostrou efeitos pressores menores. TsV mostrou os maiores efeitos sobre a liberação de catecolaminas, seguido pela TsTX-I e TsTX-V com um efeito máximo em 2,5 min, seguido por uma gradual redução, permanecendo, todavia, maior que os controles. Embora ambas as classes de toxinas atuem em canais para Na+, TsTX-I mostrou efeitos mais significantes e intensos sobre a liberação de catecolaminas e pressão arterial que a TsTX-V. Parece que a toxicidade da TsTX-V não está somente relacionada à sua capacidade de liberar catecolaminas, indicando que outros neutrotransmissores podem estar envolvidos em sua toxicidade. Nem a TsV ou suas toxinas foram capazes de afetar a captação de 3H-Glu. TsTXI inibiu somente a captação de 3H-DA (IC50 = 28,41 nM). Por outro lado, TsV (0,43ng/mL) inibiu a captação de 3H-GABA e 3H-DA (~50%). TsTX-V mostrou IC50 = 9,37 nM e 22,2 nM para a captação de 3H-GABA e 3HDA, respectivamente. Esses efeitos foram abolidos pelo pré-tratamento com TTX, indicando o envolvimento de canais para Na+ neste processo. Na ausência de Ca+2 e em baixas concentrações de toxinas, a redução não é tão singnificante como na presença de Ca+2. TsTX-V não reduziu a captação de 3H-GABA em células COS-7 expressando os transportadores de GABA, GAT-1 e GAT-3, sugerindo que esta toxina reduz indiretamente o transporte. A redução da captação de 3H-GABA pelos sinaptosomas pode ser devido a rápida e intensa despolarização celular, como revelado por microscopia confocal em células de glioma C6. Assim, TsTX-V causou redução da captação de 3H-GABA e 3H-DA de uma maneira independente de Ca+2, não afetando diretamente os transportadores de GABA, mas em consequencia da despolarização, envolvendo canais para Na+ operados por voltagem. TsV e suas toxinas foram capazes de aumentar a ([Ca2+ ]C , provavelmente por interargir com canais para Na+. Quando comparado aos efeitos despolarizantes do KCl 60 mM (100 %), TsV (100 e 500 g/mL) exibiu um aumento de 49,60 ± 2,58 % e 103,66 ± 5,17 %, respectivamente, enquanto que a TsTX-I e TsTX-V (50 e 100 g/mL de cada) exibiu 43,92 ± 3,06 % e 121,8 ± 8,9 %; 52,56 ± 8,33 % e 79,5 ± 6,1 % de aumento, respectivamente. TsTX-I (100 g/mL) mostrou-se mais potente nesta preparação, visto que uma dose de 100 g/mL causou efeito muito mais intenso do que a TsTX-V na mesma concentração. É possível que as diferenças observadas sobre os efeitos induzidos pela TsTX-I e TsTX-V sejam conseqüência de alterações estruturais entre canais para Na+ presentes em vários tipos de tecidos e inervações. / Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: a- and b-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, were evaluate the effects of TsV and its toxins on mean arterial pressure (MAP) and catecholamines release in conscious unrestrained rats previously catheterized, as well as GABA, dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes and cytosolic Ca2+ concentration ([Ca2+ ]C) in vascular smooth muscle cells from rat aorta. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). The toxins (15 and 30 g/kg) and TsV (50 and 100 g/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) plasma levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5 3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity. Neither the TsV or its toxins was capable to affect the 3H-Glu uptake. TsTX-I inhibited only 3H-DA uptake (IC50 = 28.41 nM). On the other hand, TsV (0.43ng/mL) inhibited both 3H-GABA and 3H-DA uptake (~50%). TsTX-V showed IC50 = 9.37 nM and 22.2 nM for 3H-GABA and 3H-DA uptake, respectively. These effects were abolished by pre-treatment with TTX, indicating the involvement of Na+ channels in this process. In the absence of Ca2+ and at low concentrations of toxin, the reduction is not as significant as in the presence of Ca2+. TsTX-V did not reduce 3H-GABA uptake in COS-7 cells expressing GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced 3H-GABA uptake by synaptosomes could be due to fast and intense cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes reduction on 3H-GABA and 3H-DA uptake in a Ca2+-independent manner, not affecting directly GABA transporters, but, in consequence of depolarization, involving voltage-gated Na+ channels. TsV and its toxins were able to increase the ([Ca2+ ]C , probably by interact with Na+ channels. When compared to KCl 60 mM depolarizing effect (100 %), TsV (100 and 500 ?g/mL), showed an increase of 49.60 ± 2.58 % and 103.66 ± 5.17 %, respectively, whereas TsTX-I and TsTX-V (50 and 100?g/mL of each) showed 43.92 ± 3.06 % and 121.8 ± 8.9 %; 52.56 ± 8.33 % and 79.5 ± 6.1 %, respectively. TsTX-I (100 ?g/mL) showed most potent effects in this type of preparation, since induced most intense effect that TsTX-V in the same concentration. Thus, it is possible that the differences observed on the effects induced by both toxins are consequence of structural changes among Na+ channels present in several types of tissues and innervations .

Toxinas que atuam em canais para Na+ operados por voltagem são as principais responsáveis pelos efeitos tóxicos do envenenamento escorpiônico e podem ser divididas em duas classes: a- e b-neurotoxinas. TsTX-V e TsTX-I da peçonha de Tityus serrulatus (TsV) são, respectivamente, exemplos destas toxinas. Neste trabalho, foram avaliados os efeitos da TsV e destas toxinas sobre a pressão arterial média (PAM) e liberação de catecolaminas em ratos conscientes e não imobilizados, previamente cateterizados, bem como a captação de GABA, dopamina (DA) e glutamato (Glu) em sinaptosomas isolados de cérebro de ratos e a concentração citoplasmática de Ca+2 ([Ca+2 ]C) em células de músculo liso vascular de aorta de ratos. As toxinas foram isoladas por cromatografia de troca iônica (TsTX-I) seguida por CLAE de fase reversa (TsTX-V). As toxinas (15 e 30 g/kg) e TsV (50 e 100 g/kg) foram injetadas intravenosamente. A PAM foi monitorada continuamente através do cateter femoral. Os níveis plasmáticos de adrenalina (ADR) e noradrenalina (NA) foram determinados por CLAE de fase reversa com detector eletroquímico, em 10 min antes e 2,5, 30 e 90 min após os tratamentos. Efeitos pressores máximos foram observados em 2,5?3,5 min. TsV induziu um intenso aumento de longa duração na PAM, bem como a TsTX-I. A TsTX-V mostrou efeitos pressores menores. TsV mostrou os maiores efeitos sobre a liberação de catecolaminas, seguido pela TsTX-I e TsTX-V com um efeito máximo em 2,5 min, seguido por uma gradual redução, permanecendo, todavia, maior que os controles. Embora ambas as classes de toxinas atuem em canais para Na+, TsTX-I mostrou efeitos mais significantes e intensos sobre a liberação de catecolaminas e pressão arterial que a TsTX-V. Parece que a toxicidade da TsTX-V não está somente relacionada à sua capacidade de liberar catecolaminas, indicando que outros neutrotransmissores podem estar envolvidos em sua toxicidade. Nem a TsV ou suas toxinas foram capazes de afetar a captação de 3H-Glu. TsTXI inibiu somente a captação de 3H-DA (IC50 = 28,41 nM). Por outro lado, TsV (0,43ng/mL) inibiu a captação de 3H-GABA e 3H-DA (~50%). TsTX-V mostrou IC50 = 9,37 nM e 22,2 nM para a captação de 3H-GABA e 3HDA, respectivamente. Esses efeitos foram abolidos pelo pré-tratamento com TTX, indicando o envolvimento de canais para Na+ neste processo. Na ausência de Ca+2 e em baixas concentrações de toxinas, a redução não é tão singnificante como na presença de Ca+2. TsTX-V não reduziu a captação de 3H-GABA em células COS-7 expressando os transportadores de GABA, GAT-1 e GAT-3, sugerindo que esta toxina reduz indiretamente o transporte. A redução da captação de 3H-GABA pelos sinaptosomas pode ser devido a rápida e intensa despolarização celular, como revelado por microscopia confocal em células de glioma C6. Assim, TsTX-V causou redução da captação de 3H-GABA e 3H-DA de uma maneira independente de Ca+2, não afetando diretamente os transportadores de GABA, mas em consequencia da despolarização, envolvendo canais para Na+ operados por voltagem. TsV e suas toxinas foram capazes de aumentar a ([Ca2+ ]C , provavelmente por interargir com canais para Na+. Quando comparado aos efeitos despolarizantes do KCl 60 mM (100 %), TsV (100 e 500 g/mL) exibiu um aumento de 49,60 ± 2,58 % e 103,66 ± 5,17 %, respectivamente, enquanto que a TsTX-I e TsTX-V (50 e 100 g/mL de cada) exibiu 43,92 ± 3,06 % e 121,8 ± 8,9 %; 52,56 ± 8,33 % e 79,5 ± 6,1 % de aumento, respectivamente. TsTX-I (100 g/mL) mostrou-se mais potente nesta preparação, visto que uma dose de 100 g/mL causou efeito muito mais intenso do que a TsTX-V na mesma concentração. É possível que as diferenças observadas sobre os efeitos induzidos pela TsTX-I e TsTX-V sejam conseqüência de alterações estruturais entre canais para Na+ presentes em vários tipos de tecidos e inervações. / Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: a- and b-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, were evaluate the effects of TsV and its toxins on mean arterial pressure (MAP) and catecholamines release in conscious unrestrained rats previously catheterized, as well as GABA, dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes and cytosolic Ca2+ concentration ([Ca2+ ]C) in vascular smooth muscle cells from rat aorta. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). The toxins (15 and 30 g/kg) and TsV (50 and 100 g/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) plasma levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5 3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity. Neither the TsV or its toxins was capable to affect the 3H-Glu uptake. TsTX-I inhibited only 3H-DA uptake (IC50 = 28.41 nM). On the other hand, TsV (0.43ng/mL) inhibited both 3H-GABA and 3H-DA uptake (~50%). TsTX-V showed IC50 = 9.37 nM and 22.2 nM for 3H-GABA and 3H-DA uptake, respectively. These effects were abolished by pre-treatment with TTX, indicating the involvement of Na+ channels in this process. In the absence of Ca2+ and at low concentrations of toxin, the reduction is not as significant as in the presence of Ca2+. TsTX-V did not reduce 3H-GABA uptake in COS-7 cells expressing GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced 3H-GABA uptake by synaptosomes could be due to fast and intense cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes reduction on 3H-GABA and 3H-DA uptake in a Ca2+-independent manner, not affecting directly GABA transporters, but, in consequence of depolarization, involving voltage-gated Na+ channels. TsV and its toxins were able to increase the ([Ca2+ ]C , probably by interact with Na+ channels. When compared to KCl 60 mM depolarizing effect (100 %), TsV (100 and 500 ?g/mL), showed an increase of 49.60 ± 2.58 % and 103.66 ± 5.17 %, respectively, whereas TsTX-I and TsTX-V (50 and 100?g/mL of each) showed 43.92 ± 3.06 % and 121.8 ± 8.9 %; 52.56 ± 8.33 % and 79.5 ± 6.1 %, respectively. TsTX-I (100 ?g/mL) showed most potent effects in this type of preparation, since induced most intense effect that TsTX-V in the same concentration. Thus, it is possible that the differences observed on the effects induced by both toxins are consequence of structural changes among Na+ channels present in several types of tissues and innervations .

Capillary electrophoretic (CE) separations were successfully coupled to time-of-flight mass spectrometric (TOFMS) detection for the analysis of three families of biological compounds that act as mediators and/or indicators of disease, namely, catecholamines (dopamine, epinephrine, norepinephrine) and their O-methoxylated metabolites (3-methoxytyramine, norepinephrine, and normetanephrine), indolamines (serotonin, tryptophan, and 5-hydroxytryptophan), and angiotensin peptides. While electrophoretic separation techniques provided high separation efficiency, mass spectrometric detection afforded specificity unsurpassed by other types of detectors. Both catecholamines and indolamines are present in body fluids at concentrations that make it possible for them to be determined by capillary zone electrophoresis coupled to TOFMS without employing any preconcentration scheme beyond sample work up by solid phase extraction (SPE). Using this hyphenated approach, submicromolar levels of catecholamines and metanephrines in normal human urine and indolamines in human plasma were detected after the removal of the analytes from their biological matrices and after preconcentration by SPE on mixed mode cation-exchange sorbents. The CE-TOFMS and SPE methods were individualized for each group of compounds. While catecholamines and metanephrines in urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard, deuterated isotopes, considered ideal internal standards, were used for the quantitation of indolamines. Because the angiotensin peptides are present in biological fluids at much lower concentrations than the previous two families of analytes, their analysis required the application of additional preconcentration techniques. In this work, the coupling of either of two types of electrophoretic preconcentration methods - field amplified injection (FAI) and isotachophoresis (ITP) - to capillary zone electrophoresis with both UV and MS detection was evaluated. Using FAI-CE-UV, angiotensins were detected at ~1 nM concentrations. Using similar conditions but TOFMS detection, the detection limits were below 10 nM. ITP was evaluated in both single-column and two-column comprehensive arrangements. The detection limits achieved for the ITP-based techniques were approximately one order of magnitude higher than for the FAI-based preconcentration. While the potential usefulness of these techniques was demonstrated using angiotensins standards, substantial additional research would be required to allow these approaches to be applied to plasma as part of clinical assays.

An efficient integration of sensory and motor processes is crucial to goal-directed behavior. Despite this high relevance, and although cognitive theories provide clear conceptual frameworks, the neurobiological basis of these processes remains insufficiently understood. In a double-blind, randomized placebo-controlled pharmacological study, we examine the relevance of catecholamines for perception-motor integration processes. Using EEG data, we perform an in-depth analysis of the underlying neurophysiological mechanisms, focusing on sensorimotor integration processes during response inhibition. We show that the catecholaminergic system affects sensorimotor integration during response inhibition by modulating the stability of the representational content. Importantly, catecholamine levels do not affect the stability of all aspects of information processing during sensorimotor integration, but rather—as suggested by cognitive theory—of specific codes in the neurophysiological signal. Particularly fronto-parietal cortical regions are associated with the identified mechanisms. The study shows how cognitive science theory-driven pharmacology can shed light on the neurobiological basis of perception-motor integration and how catecholamines affect specific information codes relevant to cognitive control.