Patch-clamp Recordings and Single Fiber Labeling from Spiral Ganglion Somata in Acutely Prepared Semi-intact Cochleae from Neonatal Rats

Biochemical, physiological, pharmacological, and more recently enzyme histo- chemical data have indicated that cholinergic circuits exist in the hypothalamus. Ultrastructural correlates of these pathways such as acetylcholinesterase (AchE) positive neurons in the arcuate nucleus (ARC) and stained terminals in the median eminence (ME) have yet to be described. Initial studies in our laboratories utilizing chemical lesioning and microdissection techniques coupled with microchemical and light microscopic enzyme histo- chemical studies suggested the existence of cholinergic neurons in the ARC which project to the ME (1). Furthermore, in adult male rats with Halasz deafferentations (hypothalamic islands composed primarily of the isolated ARC and the ME) choline acetyltransferase (ChAc) activity, a good marker for cholinergic neurons, was not significantly reduced in the ME and was only somewhat reduced in the ARC (2). Treatment of neonatal rats with high doses of monosodium 1-glutamate (MSG) results in a lesion largely restricted to the neurons of the ARC.

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High-voltage electron microscopy of patch-clamped membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127402 ◽

1987 ◽

Vol 45 ◽

pp. 582-583

Author(s):

F. Sachs ◽

M. J. Song

Keyword(s):

Electron Microscopy ◽

Cell Membrane ◽

Patch Clamp ◽

High Voltage Electron Microscopy ◽

Small Patch ◽

Cellular Electrophysiology ◽

Voltage Electron ◽

Electron Microscopes ◽

Patch Technique ◽

Membrane Patch

Cellular electrophysiology has been revolutionized by the introduction of patch clamp techniques. The patch clamp records current from a small patch of the cell membrane which has been sucked into a glass pipette. The membrane patch, a few micons in diameter, is attached to the glass by a seal which is electrically, diffusionally and mechanically tight. Because of the tight electrical seal, the noise level is low enough to record the activity of single ion channels over a time scale extending from 10μs to days. However, although the patch technique is over ten years old, the patch structure is unknown. The patch is inside a glass pipette where it has been impossible to see with standard electron microscopes. We show here that at 1 Mev the glass pipette is transparent and the membrane within can be seen with a resolution of about 30 A.

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Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141238 ◽

1995 ◽

Vol 53 ◽

pp. 972-973

Author(s):

R H. Selinfreund ◽

A. H. Cornell-Bell

Keyword(s):

Membrane Potential ◽

Confocal Microscopy ◽

Patch Clamp ◽

Electrical Activity ◽

Time Lapse ◽

Neuronal Firing ◽

Neuronal Membrane ◽

Pituitary Cells ◽

Patch Clamp Recording ◽

Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

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Polarized microbeam FT-IR analysis of single fibers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163496 ◽

1996 ◽

Vol 54 ◽

pp. 206-207

Author(s):

Liling Cho ◽

David L. Wetzel

Keyword(s):

Molecular Orientation ◽

Transition Point ◽

Polymer Fibers ◽

Single Fiber ◽

Wire Grid ◽

Polyester Fibers ◽

Ft Ir ◽

Ir Analysis ◽

The Relationship ◽

Wire Grid Polarizer

Polarized infrared microscopy has been used for forensic purposes to differentiate among polymer fibers. Dichroism can be used to compare and discriminate between different polyester fibers, including those composed of polyethylene terephthalate that are frequently encountered during criminal casework. In the fiber manufacturering process, fibers are drawn to develop molecular orientation and crystallinity. Macromolecular chains are oriented with respect to the long axis of the fiber. It is desirable to determine the relationship between the molecular orientation and stretching properties. This is particularly useful on a single fiber basis. Polarized spectroscopic differences observed from a single fiber are proposed to reveal the extent of molecular orientation within that single fiber. In the work presented, we compared the dichroic ratio between unstretched and stretched polyester fibers, and the transition point between the two forms of the same fiber. These techniques were applied to different polyester fibers. A fiber stretching device was fabricated for use on the instrument (IRμs, Spectra-Tech) stage. Tension was applied with a micrometer screw until a “neck” was produced in the stretched fiber. Spectra were obtained from an area of 24×48 μm. A wire-grid polarizer was used between the source and the sample.

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