Intracellular fluorescent staining with carboxyfluorescein: a rapid and reliable method for quantifying dye-coupling in mammalian central nervous system

doi:10.1016/0165-0270(86)90050-6

Journal of Neuroscience Methods

Volume 16, Issue 4, June 1986, Pages 251-263

https://doi.org/10.1016/0165-0270(86)90050-6 Get rights and content

Abstract

Previous studies investigating electrotonic coupling in mammalian central nervous system have used the fluorescent marker Lucifer Yellow as an indicator of the presence of intercellular junctions between neurons. The fluorescent dye 5,6-carboxyfluorescein is known to have approximately 5 times the fluorescent yield of Lucifer Yellow. We have investigated the use of this dye as a potential alternative fluorescent marker on two types of neurons in the rat hippocampus in vitro. Unfixed hippocampal slices were mounted in a mixture of n-propyl gallate in glycerol and viewed with epifluorescence optics. Injections of small, brief hyperpolarizing currents through carboxyfluorescein-filled glass pipettes reliably produced neuronal fills of good quality. Both dendritic spines and axonal arborizations (including the thin mossy fibers of the dentate gyrus) were frequently observable. In addition to single cell fills, clusters consisting of 2–6 neurons were observed. No correlation was found between the number of cells per cluster and the ejection time. In addition, even cells exhibiting poor electrophysiological characteristics, or cells impaled only briefly, frequently exhibited good quality dye filling. This method will be particularly useful when large sample sizes are necessary to compare regional variations in the extent of electrotonic coupling in the mammalian brain.

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The intrinsic membrane properties of perirhinal cortical neurons were studied by intracellular recording in in vitro rat brain slices. Gross morphology was also examined through injection of the fluorescent dye carboxyfluorescein. The cells encountered displayed a diversity of electrophysiological properties, and were similar to cells reported in other neocortical areas with regard to spiking patterns, afterpotentials, and morphology. However, very few (4%) intrinsically bursting neurons were encountered. Two pyramidal cells with thick apical dendrites were filled, and both fired doublets of action potentials for their first suprathreshold events. Of the filled pyramidal cells with thin apical dendrites, most (91/1) fired single action potentials for their first suprathreshold events. A variety of classification schemes were used to group the data, and several schemes were found to be equally successful. According to one of the schemes, cells recorded with carboxyfluorescein filled electrodes had significantly greater action potential widths at half-amplitude and more depolarized resting potentials than cells recorded without this dye.
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  5. It is hypothesized that it is a key neuron in coordinating whole animal responses to local stimuli.
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Changes in light sensitivity, response time-course and electrical coupling of rod driven horizontal cells (RHC) were examined in the dark and light adapted states by intracellular recording. Comparative analyses were made of two fishes from different photic environments. RHC of Cyprinus carpio and external horizontal cells (EHC) of Scyliorhinus canicula retinas showed similar changes of their electrical responses to light stimuli when the dark-adapted retina was light-adapted: a decrease in sensitivity, an acceleration in the response time-course and an increase in the coupling strength.

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Research paperIntracellular fluorescent staining with carboxyfluorescein: a rapid and reliable method for quantifying dye-coupling in mammalian central nervous system

Abstract

Brain Res. Bull.

Neuroscience

Neuroscience

TINS

Neuroscience

Brain Res.

Int. Rev. Cytol.

Neurosci. Lett.

Brain Res.

Brain Res.

J. Neurosci. Meth.

Cell

Brain Res.

Neuroscience

Dye transfer through gap junctions between neuroendocrine cells of rat hypothalamus

Science

Electrotonic coupling between neurons in the rat mesencephalic nucleus

J. Physiol. (London)

Electrical transmission: a functional analysis and comparison with chemical transmission

Electrotonic junctions between teleost spinal neurons: electrophysiology and ultrastructure

Science

Dye coupling in hypothalamic slices: dependence on in vivo hydration state and osmolarity of incubation medium

J. Neurosci.

Recent evidence for and possible significance of gap junctions and electrotonic synapses in the mammalian brain

Transmission at the giant motor synapses of the crayfish

J. Physiol. (London)

Modification of neuron properties by electronic synapses. I. Input resistance, time constant and integration

J. Neurophysiol.

Research paper
Intracellular fluorescent staining with carboxyfluorescein: a rapid and reliable method for quantifying dye-coupling in mammalian central nervous system