28 research outputs found
Metoprolol Impairs Mesenteric and Posterior Cerebral Artery Function in Mice
Background/Rationale: In addition to their established cardioprotective role, ÎČ-adrenergic antagonists also increase the risk of stroke and mortality. We propose that a vascular mechanism could contribute to cerebral tissue ischemia in ÎČ-blocked patients.
Methods: Cardiac output (CO), mean arterial pressure (MAP) and microvascular brain oxygen tension (PBrmvO2) were measured in anesthesized mice treated with metoprolol (3mgâąkg-1, i.v.). Dose-response curves (DRCs) for adrenergic-agonists were generated in mesenteric resistance arteries (MRAs; isoproterenol, clenbuterol) and posterior cerebral arteries (PCAs; phenylephrine, isoproterenol) before and after metoprolol treatment.
Results: Metoprolol reduced CO, maintained MAP and increased systemic vascular resistance (SVR) resulting in a decreased PBrmvO2 in mice. Metoprolol attenuated ÎČ-adrenergic mediated vasodilation in both MRAs and PCAs.
Conclusions: Metoprolol reduced brain perfusion in mice. A decrease in CO contributed however, metoprolol also inhibited ÎČ-adrenergic vasodilation of mesenteric and cerebral arteries. This provides evidence in support of a vascular mechanism for cerebral ischemia in ÎČ-blocked patients.MAS
Vers une cartographie cellule entiÚre de molécule unique dynamique
Imaging of single molecules inside living cells confers insight to biological function at its most granular level. Single molecules experience a nanoscopic environment that is complicated, and in general, poorly understood. The modality of choice for probing this environment is live-cell localisation microscopy, where trajectories of single molecules can be captured. For many years, the great stumbling block in comprehension of physical processes at this scale was the lack of information accessible; statistical significance and robust assertions are hardly possible from a few dozen trajectories. It is the onset of high-density single-particle tracking that has dramatically reframed the possibilities of such studies. Importantly, the consequential amounts of data it provides invites the use of powerful statistical tools that assign probabilistic descriptions to experimental observations. In this thesis, Bayesian inference tools have been developed to elucidate the behaviour of single molecules via the mapping of motion parameters. As a readout, maps describe heterogeneities at local and whole-cell scales. Importantly, they grant quantitative details into basic cellular processes. This thesis uses the mapping approach to study receptor-scaffold interactions inside neurons and non-neuronal cells. A promising system in which interactions are patterned is also examined. It is shown that interactions of different types of chimeric glycine receptors to the gephyrin scaffold protein may be described and distinguished in situ. Finally, the prospects of whole-cell mapping in three-dimensions are evaluated based on a discussion of state-of-the-art volumetric microscopy techniques.La comprĂ©hension fondamentale de fonctions biologiques est accordĂ©e par lâimagerie de molĂ©cules uniques dans les cellules vivantes. Cet environnement nanoscopique est compliquĂ© et largement mal compris. La microscopie de localisation permet cet environnement dâĂȘtre sondĂ© avec le suivi de molĂ©cules uniques. Depuis longtemps, lâobstacle principal qui empĂȘcher la comprĂ©hension de processus physiques Ă cette Ă©chelle Ă©tait la pĂ©nurie dâinformation accessible; de fortes hypothĂšses ne peuvent pas ĂȘtre Ă©tablies Ă partir de quelques dizaines de trajectoires de molĂ©cules uniques. Lâintroduction des techniques de suivi de molĂ©cules Ă haute densitĂ© a recadrĂ© les possibilitĂ©s. Dans cette thĂšse, les outils dâinfĂ©rence Bayesienne ont Ă©tĂ© dĂ©veloppĂ© pour Ă©lucider le comportement de molĂ©cules uniques Ă partir la cartographie de leurs paramĂštres physiques de mouvement. Ces cartes dĂ©crivent, entre autre, les hĂ©tĂ©rogĂ©nĂ©itĂ©s aux Ă©chelles locales mais aussi Ă lâĂ©chelle de la cellule entiĂšre. Notamment elles dĂ©voilent les dĂ©tails quantitatifs sur les processus cellulaires de base. En utilisant cette approche cartographique, les interactions entre rĂ©cepteurs et protĂ©ines dâĂ©chafaudage chez les neurones et les cellules non-neuronales sont Ă©tudiĂ©s. Une Ă©tude sur les interactions imprimĂ©es dans la cellule est Ă©galement effectuĂ©e grace Ă un systĂšme prometteur dâimpression de protĂ©ine. Les diffĂ©rents types dâinteractions entre constructions chimĂ©riques du rĂ©cepteur de glycine et de protĂ©ines dâĂ©chafaudage de gĂ©phyrine sont dĂ©crits et distinguĂ© in situ. Enfin, les perspectives vis-Ă -vis lâobtention de cartes tridimensionnelles de dynamiques de molĂ©cules uniques sont Ă©galement commentĂ©es
Vers une cartographie cellule entiÚre de molécule unique dynamique
La comprĂ©hension fondamentale de fonctions biologiques est accordĂ©e par lâimagerie de molĂ©cules uniques dans les cellules vivantes. Cet environnement nanoscopique est compliquĂ© et largement mal compris. La microscopie de localisation permet cet environnement dâĂȘtre sondĂ© avec le suivi de molĂ©cules uniques. Depuis longtemps, lâobstacle principal qui empĂȘcher la comprĂ©hension de processus physiques Ă cette Ă©chelle Ă©tait la pĂ©nurie dâinformation accessible; de fortes hypothĂšses ne peuvent pas ĂȘtre Ă©tablies Ă partir de quelques dizaines de trajectoires de molĂ©cules uniques. Lâintroduction des techniques de suivi de molĂ©cules Ă haute densitĂ© a recadrĂ© les possibilitĂ©s. Dans cette thĂšse, les outils dâinfĂ©rence Bayesienne ont Ă©tĂ© dĂ©veloppĂ© pour Ă©lucider le comportement de molĂ©cules uniques Ă partir la cartographie de leurs paramĂštres physiques de mouvement. Ces cartes dĂ©crivent, entre autre, les hĂ©tĂ©rogĂ©nĂ©itĂ©s aux Ă©chelles locales mais aussi Ă lâĂ©chelle de la cellule entiĂšre. Notamment elles dĂ©voilent les dĂ©tails quantitatifs sur les processus cellulaires de base. En utilisant cette approche cartographique, les interactions entre rĂ©cepteurs et protĂ©ines dâĂ©chafaudage chez les neurones et les cellules non-neuronales sont Ă©tudiĂ©s. Une Ă©tude sur les interactions imprimĂ©es dans la cellule est Ă©galement effectuĂ©e grace Ă un systĂšme prometteur dâimpression de protĂ©ine. Les diffĂ©rents types dâinteractions entre constructions chimĂ©riques du rĂ©cepteur de glycine et de protĂ©ines dâĂ©chafaudage de gĂ©phyrine sont dĂ©crits et distinguĂ© in situ. Enfin, les perspectives vis-Ă -vis lâobtention de cartes tridimensionnelles de dynamiques de molĂ©cules uniques sont Ă©galement commentĂ©es.Imaging of single molecules inside living cells confers insight to biological function at its most granular level. Single molecules experience a nanoscopic environment that is complicated, and in general, poorly understood. The modality of choice for probing this environment is live-cell localisation microscopy, where trajectories of single molecules can be captured. For many years, the great stumbling block in comprehension of physical processes at this scale was the lack of information accessible; statistical significance and robust assertions are hardly possible from a few dozen trajectories. It is the onset of high-density single-particle tracking that has dramatically reframed the possibilities of such studies. Importantly, the consequential amounts of data it provides invites the use of powerful statistical tools that assign probabilistic descriptions to experimental observations. In this thesis, Bayesian inference tools have been developed to elucidate the behaviour of single molecules via the mapping of motion parameters. As a readout, maps describe heterogeneities at local and whole-cell scales. Importantly, they grant quantitative details into basic cellular processes. This thesis uses the mapping approach to study receptor-scaffold interactions inside neurons and non-neuronal cells. A promising system in which interactions are patterned is also examined. It is shown that interactions of different types of chimeric glycine receptors to the gephyrin scaffold protein may be described and distinguished in situ. Finally, the prospects of whole-cell mapping in three-dimensions are evaluated based on a discussion of state-of-the-art volumetric microscopy techniques
Mechanisms of anaesthetic depression of neocortical arousal
The most widely accepted hypotheses suggest that general anaesthetics interrupt conscious processes in the brain by decreasing synaptic excitation or by potentiating synaptic inhibition, especially in the neocortex. The putative transmitters in the neurological systems that generate neocortical arousal include acetylcholine, glutamate and Îł-aminobutyrate (GABA). The primary objective here was to determine the neuronal mechanisms by which anaesthetics may obtund this arousal.
The majority of the investigations were carried out on pyramidal neurons in layers IV and V of guinea pig neocortex (in vitro slices), using intracellular
recording and pharmacological, including microiontophoretic, techniques. Bath applications of structurally dissimilar anaesthetics, isoflurane - a halogenated ether, and Althesin - a steroidal preparation, in concentrations of 0.5-2.5 minimum alveolar concentration (MAC) and 10-1300 ÎŒM, respectively, produced a small hyperpolarization (3-5 mV) which was associated with an increase in input conductance (10-30%). The lower concentrations (0.5-1.5 MAC and 10-200 ÎŒM) of these agents which are most relevant to the production of unconsciousness did not significantly affect the passive membrane properties. However, they produced striking decreases in spontaneous activities and the repetitive spike firing evoked by orthodromic (electrical) stimulation or intracellular current injections.
Because the observed changes in membrane properties could not explain the reduction in neuronal excitability, the effects of anaesthetics were investigated extensively on excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). The application of isoflurane or Althesin induced a dose-dependent, reversible depression in the amplitude of EPSPs,
with ECâ
âs of 1 MAC and ~50 ÎŒM, respectively. The IPSPs also were reduced in a dose-dependent manner. In order to eliminate possible shunting of the EPSPs by the GABA-activated Cl-conductance that produces the IPSP in the observed EPSP-IPSP sequence, a GABA[symbol omitted]-antagonist, bicuculline, was additionally
applied. Despite this IPSP-blockade, the anaesthetics strongly depressed the EPSPs as well as epileptiform activities evoked by subpial electrical stimulation.
In cognizance of the possibility that a postsynaptic attenuation of responsiveness to transmitter substances may be involved in the EPSP depression, the neuronal sensitivities to acetylcholine, glutamate. and GABA were determined. Anaesthetic administration markedly reduced the depolarizations and associated conductance changes evoked by dendritic applications of acetylcholine, glutamate and N-methyl-D-aspartate (NMDA). The hyperpolarizing responses to somatic applications of GABA were not affected significantly whereas the depolarizing effects observed with its dendritic application were slightly depressed. Same degree of selectivity also was evident from the lower ECâ
âs for the isoflurane- and Althesin-induced depressions of responses to acetylcholine compared with glutamate. Under in vitro conditions of hypomagnesia the responses to acetylcholine were totally blocked and the order of depression in the responses to GABA and glutamate was reversed; this may be of importance in the mechanism for the known increase in anaesthetic requirements in clinical syndromes associated with hypomagnesaemia.
Because the genesis of synaptic transients is affected by CaÂČâș influx
or disposition, the interactions of anaesthetics were investigated on spike
afterhyperpolarizations (AHPs). The AHPs which are produced specifically by a CaÂČâș -activated Kâș -conductance were suppressed by the anaesthetics in a
dose-dependent manner under conditions where contaminating IPSPs had been
blocked by bicuculline. Since the passive membrane properties were
unaffected, an interference with a transmembrane Caâș -influx may be involved
in the anaesthetic actions.
The effects of anaesthetics on glutamate-induced and voltage-dependent increases in intraneuronal CaÂČâș ([CaÂČâș]i) were determined in cultured
hippocampal neurons with a Ca-sensitive probe (Fura-2) and microspectro-
fluorometric techniques. Isoflurane application depressed the increases in [CaÂČâș]i. produced by application of glutamate under conditions where its
actions would be favoured at NMDA- and quisqualate-subtypes of receptors. Kâș -induced increases in [CaÂČâș]i also were reduced by application of isoflurane, probably due to actions on voltage-dependent Ca-channels in the membrane.
These investigations have provided evidence for the first time that excitatory transmitter actions in neocortex are selectively depressed by anaesthesia. A plausible mechanism would include suppression of the postsynaptic Ca-conductances associated with the AHPs and glutamatergic, as well as cholinergic interactions at pre- and post-synaptic sites on neurons involved in neocortical arousal.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat