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Properties of the demarcation membrane system in living rat megakaryocytes.

ABSTRACT: The demarcation membrane system (DMS) is the precursor of platelet cell membranes yet little is known of its properties in living megakaryocytes. Using confocal microscopy, we now demonstrate that demarcation membranes in freshly isolated rat marrow megakaryocytes are rapidly stained by styryl membrane indicators such as di-8-ANEPPS and FM 2-10, confirming that they are invaginations of the plasma membrane and readily accessible from the extracellular space. Two-photon excitation of an extracellular indicator displayed the extensive nature of the channels formed by the DMS throughout the extranuclear volume. Under whole-cell patch clamp, the DMS is electrophysiologically contiguous with the peripheral plasma membrane such that a single capacitative component can account for the biophysical properties of all surface-connected membranes in the majority of recordings. Megakaryocyte capacitances were in the range of 64-694 pF, equivalent to 500-5500 platelets (mean value 1850). Based upon calculations for a spherical geometry, the DMS results in a 4- to 14-fold (average 8.1-fold) increase in specific membrane capacitance expressed per unit spherical surface area. This indicates a level of plasma membrane invagination comparable with mammalian skeletal muscle. Whole-cell capacitance measurements and confocal imaging of membrane-impermeant fluorescent indicators therefore represent novel approaches to monitor the DMS during megakaryocytopoiesis and thrombopoiesis.

SUBMITTER: Mahaut-Smith MP

PROVIDER: S-EPMC1302831 | biostudies-other | 2003 Apr

REPOSITORIES: biostudies-other

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Properties of the demarcation membrane system in living rat megakaryocytes.

Mahaut-Smith Martyn P MP Thomas David D Higham Alex B AB Usher-Smith Juliet A JA Hussain Jamila F JF Hussain Jamila F JF Martinez-Pinna Juan J Skepper Jeremy N JN Mason Michael J MJ

Biophysical journal 20030401 4

The demarcation membrane system (DMS) is the precursor of platelet cell membranes yet little is known of its properties in living megakaryocytes. Using confocal microscopy, we now demonstrate that demarcation membranes in freshly isolated rat marrow megakaryocytes are rapidly stained by styryl membrane indicators such as di-8-ANEPPS and FM 2-10, confirming that they are invaginations of the plasma membrane and readily accessible from the extracellular space. Two-photon excitation of an extracell ...[more]

PMID: 12668473

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Project description:The mammalian olfactory bulb (OB) has a vast population of dopamine (DA) neurons, whose function is to increase odor discrimination through mostly inhibitory synaptic mechanisms. However, it is not well understood whether there is more than one neuronal type of OB DA neuron, how these neurons respond to different stimuli, and the ionic mechanisms behind those responses. In this study, we used a transgenic rat line (hTH-GFP) to identify fluorescent OB DA neurons for recording via whole-cell electrophysiology. These neurons were grouped based on their localization in the glomerular layer ("Top" vs. "Bottom") with these largest and smallest neurons grouped by neuronal area ("Large" vs. "Small," in μm2). We found that some membrane properties could be distinguished based on a neuron's area, but not by its glomerular localization. All OB DA neurons produced a single action potential when receiving a sufficiently depolarizing stimulus, while some could also spike multiple times when receiving weaker stimuli, an activity that was more likely in Large than Small neurons. This single spiking activity is likely driven by the Na+ current, which showed a sensitivity to inactivation by depolarization and a relatively long time constant for the removal of inactivation. These recordings showed that Small neurons were more sensitive to inactivation of Na+ current at membrane potentials of -70 and -60 mV than Large neurons. The hyperpolarization-activated H-current (identified by voltage sags) was more pronounced in Small than Large DA neurons across hyperpolarized membrane potentials. Lastly, to mimic a more physiological stimulus, these neurons received ramp stimuli of various durations and current amplitudes. When stimulated with weaker/shallow ramps, the neurons needed less current to begin and end firing and they produced more action potentials at a slower frequency. These spiking properties were further analyzed between the four groups of neurons, and these analyses support the difference in spiking induced with current step stimuli. Thus, there may be more than one type of OB DA neuron, and these neurons' activities may support a possible role of being high-pass filters in the OB by allowing the transmission of stronger odor signals while inhibiting weaker ones.

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Properties of the demarcation membrane system in living rat megakaryocytes.

Publications

Properties of the demarcation membrane system in living rat megakaryocytes.

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