RESUMO
Fast-scan cyclic voltammetry (FSCV) is applied to the tip of a scanning electrochemical microscope (SECM) for imaging the distribution of chemical species near a substrate. This approach was used to image the diffusion layer of both a large substrate electrode (3-mm-diameter glassy carbon) and a microelectrode substrate (10-microm-diameter Pt). Additionally, oxygen depletion near living cells was measured and correlated to respiratory activity. Finally, oxygen and hydrogen peroxide were simultaneously detected during the oxidative burst of a zymosan-stimulated macrophage cell. These results demonstrate the utility of FSCV-SECM for chemical imaging when conditions are chosen such that feedback interactions with the substrate are minimal.
Assuntos
Imageamento por Ressonância Magnética/métodos , Microscopia de Varredura por Sonda/métodos , Animais , Linhagem Celular , Células Cultivadas , Eletroquímica , Eletrodos , Macrófagos/química , Estrutura Molecular , Oxirredução , Células PC12 , Ratos , Fatores de TempoRESUMO
Undifferentiated and differentiated PC12 cells were imaged with the constant-distance mode of scanning electrochemical microscopy (SECM) using carbon ring and carbon fiber tips. Two types of feedback signals were used for distance control: the electrolysis current of a mediator (constant-current mode) and the impedance measured by the SECM tip (constant-impedance mode). The highest resolution was achieved using carbon ring electrodes with the constant-current mode. However, the constant-impedance mode has the important advantages that topography and faradaic current can be measured simultaneously, and because no mediator is required, the imaging can take place directly in the cell growth media. It was found that vesicular release events do not measurably alter the impedance, but the depolarizing solution, 105 mM K+, produces a dramatic impedance change such that constant-distance imaging cannot be performed during application of the stimulus. However, by operating the tip in the constant-height mode, cell morphology (via a change in impedance) and vesicular release could be detected simultaneously while moving the tip across the cell. This work represents a significant improvement over previous SECM imaging of model neurons, and it demonstrates that the combination of amperometry and constant-impedance SECM has the potential to be a powerful tool for investigating the spatial distribution of neurotransmitter release in vitro.
Assuntos
Eletroquímica/métodos , Microeletrodos , Microscopia Eletrônica de Varredura/métodos , Neurotransmissores/metabolismo , Animais , Impedância Elétrica , Células PC12 , RatosRESUMO
Synapses between gustatory receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the CNS. Several transmitter candidates have been proposed for these synapses, including serotonin (5-HT), glutamate, acetylcholine, ATP, peptides, and others, but, to date, none has been unambiguously identified. We used Chinese hamster ovary cells stably expressing 5-HT2C receptors as biodetectors to monitor 5-HT release from taste buds. When taste buds were depolarized with KCl or stimulated with bitter, sweet, or sour (acid) tastants, serotonin was released. KCl- and acid-induced 5-HT release, but not release attributable to sweet or bitter stimulation, required Ca2+ influx. In contrast, 5-HT release evoked by sweet and bitter stimulation seemed to be triggered by intracellular Ca2+ release. These experiments strongly implicate serotonin as a taste bud neurotransmitter and reveal unexpected transmitter release mechanisms.
Assuntos
Serotonina/fisiologia , Papilas Gustativas/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Técnicas Biossensoriais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Feminino , Técnicas In Vitro , Mianserina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor 5-HT2C de Serotonina/fisiologia , Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Papilas Gustativas/metabolismoRESUMO
Living PC12 cells, a model cell type for studying neuronal function, were imaged using the negative feedback mode of a scanning electrochemical microscope (SECM). Six biocompatible redox mediators were successfully identified from a large pool of candidates and were then used for imaging PC12 cells before and after exposure to nerve growth factor (NGF). When exposed to NGF, cells differentiate into a neuron phenotype by growing narrow neurites (1-2 microm wide) that can extend > 100 microm from the cell proper. We demonstrate that carbon fiber electrodes with reduced tip diameters can be used for imaging both the cell proper and these neurites. Regions of decreased current, possibly resulting from raised features not identifiable by light microscopy, are clearly evident in the SECM images. Changes in the morphology of undifferentiated PC12 cells could be detected in real time with the SECM. After exposure to hypotonic and hypertonic solutions, reversible changes in cell height of <2 microm were measured.