Cell, Vol. 14, 741-759.
July 1978,
Copyright
0 1978 by MIT
Functional Connections between Cells as Revealed by Dye-Coupling with a Highly Fluorescent Naphthalimide Tracer Walter W. Stewart National institute of Arthritis, Metabolism, and Digestive Diseases Bethesda, Maryland 20014
Summary This report describes a method of marking nerve cells which is approximately 100 times more sensitive than those previously available. The method depends upon intracellular injection of a new, highly fluorescent dye, Lucifer Yellow CH, which can be viewed both in living tissue and after fixation and embedding. The intense fluorescence of the dye makes injected neurons visible in cleared wholemounts, where the complex three-dimensional structure of neurons is readily apparent. Three new observations have been made with Lucifer Yellow. First, many of the invertebrate neurons studied possess an extensive and complex array of fine processes not visible with other techniques. Second, dye spreads rapidly within an injected cell. Third, dye frequently spreads from the injected cell directly to certain other cells. The movement of dye from cell to cell, termed “dye-coupling,” occurred primarily, but not exclusively, between cells known to be electrically coupled. Dye-coupling in the turtle retina revealed striking and distinctive patterns of connections. Type I horizontal cells appear to be multiply connected to each other in an extensive net. Type II horizontal cells are often connected to each other in a hexagonal array. Individual type I and type II cells, widely separated, are frequently dye-coupled; in one case, they were connected by a dyefilled axon. Dye-coupling, readily observed because of the low molecular weight and the intense fluorescence of the new dye, may serve as a general method of tracing certain functional connections by morphological means, and of studying the transfer of small molecules between cells. Preliminary results suggest that systems of dye-coupled cells are substantially more common than was previously believed. Introduction The electrical activity of single nerve cells is usually studied by means of a fine glass micropipette, which is advanced gradually into neural tissue until electrical monitoring of the micropipette indicates that its tip has penetrated a nerve cell. The electrical behavior of that cell can then be recorded, but
its shape and its location relative to other nerve cells are usually unknown. Since the shape and location of nerve cells are strong clues to their function, it is clearly desirable to mark the cell so that it can be seen. Marking nerve cells has proved difficult. All methods depend upon injecting a marker substance, usually a dye, into the cell through the micropipette. The first attempts, made during the 1950s and the early 1960s resulted in a deposit of stain up to several hundred microns in diameterfar beyond the boundaries of the injected cell. Often the cell injected could not even be identified. Later methods gave true intracellular staining. The agents used included potassium ferrocyanide followed by ferric chloride; Aniline Blue; a mixture of Fast Green FCF and Orange G; and Methyl Blue (Kerkut and Walker, 1962; Behrens and Wulff, 1965; Thomas and Wilson, 1966; other early work has been reviewed by Nicholson and Kater, 1973). All these methods had two drawbacks. First, the stains were not covalently bound to the tissue; to minimize leakage of stain from the injected cell, the tissue had to be processed in ways that resulted in serious distortions of structure. Second, the methods were all relatively insensitive, since they all depended upon light absorption for detection of the marked cell; a cell had to contain large amounts of dye to be visible. Stretton and Kravitz (1968) solved the first of these problems by introducing the tissue-reactive fluorescent dye Procion Yellow M-4RS. This dye remains in place during fixation and dehydration, presumably because it combines covalently with tissue components. It is not generally recognized that Procion Yellow did not solve the second problem-the low sensitivity of earlier methods. Because of its low fluorescence efficiency, Procion Yellow is only slightly easier to detect than dyes which depend upon light absorption. Other disadvantages of Procion Yellow include its uncertain structure, the lack of a published synthesis, and the unknown and probably variable purity of the commercial material. Most recent methods of intracellular marking (Pitman, Tweedle and Cohen, 1972; Christensen, 1973; Gillette and Pomeranz, 1973) share the basic limitation of Procion Yellow: they lack sensitivity. A tracer to which this generalization may not apply is horseradish peroxidase, which can be used for both light and electron microscopy (Muller and McMahan, 1976; Snow, Rose and Brown, 1976). The method is promising, but there is not yet enough experience to indicate how generally useful it will be. Procion Yellow injection, in spite of its low sensitivity, has played a decisive role in correlating the structure of nerve cells with their electrical func-
Cell 742
tion. The dyes described below possess most of the good features of Procion Yellow but in addition have a much higher fluorescence efficiency. The intense fluorescence of the new dyes allows a marked neuron to be seen in the living state as well as in cleared wholemounts, where the entire structure of the neuron can often be ascertained at a glance. The spread of Lucifer Yellow CH was observed not only throughout single neurons, but often from one neuron to another. The movement of dye from cell to cell, termed “dye-coupling,” occurred primarily but not exclusively between cells known to be electrically coupled. My preliminary observations on dye-coupling suggest that this type of functional connection between cells is substantially more common than was previously believed.
with proteins and with other compounds containing amino or sulfhydryl groups. The absorption and corrected emission spectra of Lucifer Yellow CH in water are shown in Figure 2. As expected, the excitation spectrum (not shown) was the same as the absorption spectrum within the limits of experimental error. The absorption spectrum was not significantly different in 0.1 N KHCOs or in 10 N HCI, which strongly suggests that the 4-amino group is unprotonated in aqueous solution and that its pK is
July 1978,
Copyright
0 1978 by MIT
Functional Connections between Cells as Revealed by Dye-Coupling with a Highly Fluorescent Naphthalimide Tracer Walter W. Stewart National institute of Arthritis, Metabolism, and Digestive Diseases Bethesda, Maryland 20014
Summary This report describes a method of marking nerve cells which is approximately 100 times more sensitive than those previously available. The method depends upon intracellular injection of a new, highly fluorescent dye, Lucifer Yellow CH, which can be viewed both in living tissue and after fixation and embedding. The intense fluorescence of the dye makes injected neurons visible in cleared wholemounts, where the complex three-dimensional structure of neurons is readily apparent. Three new observations have been made with Lucifer Yellow. First, many of the invertebrate neurons studied possess an extensive and complex array of fine processes not visible with other techniques. Second, dye spreads rapidly within an injected cell. Third, dye frequently spreads from the injected cell directly to certain other cells. The movement of dye from cell to cell, termed “dye-coupling,” occurred primarily, but not exclusively, between cells known to be electrically coupled. Dye-coupling in the turtle retina revealed striking and distinctive patterns of connections. Type I horizontal cells appear to be multiply connected to each other in an extensive net. Type II horizontal cells are often connected to each other in a hexagonal array. Individual type I and type II cells, widely separated, are frequently dye-coupled; in one case, they were connected by a dyefilled axon. Dye-coupling, readily observed because of the low molecular weight and the intense fluorescence of the new dye, may serve as a general method of tracing certain functional connections by morphological means, and of studying the transfer of small molecules between cells. Preliminary results suggest that systems of dye-coupled cells are substantially more common than was previously believed. Introduction The electrical activity of single nerve cells is usually studied by means of a fine glass micropipette, which is advanced gradually into neural tissue until electrical monitoring of the micropipette indicates that its tip has penetrated a nerve cell. The electrical behavior of that cell can then be recorded, but
its shape and its location relative to other nerve cells are usually unknown. Since the shape and location of nerve cells are strong clues to their function, it is clearly desirable to mark the cell so that it can be seen. Marking nerve cells has proved difficult. All methods depend upon injecting a marker substance, usually a dye, into the cell through the micropipette. The first attempts, made during the 1950s and the early 1960s resulted in a deposit of stain up to several hundred microns in diameterfar beyond the boundaries of the injected cell. Often the cell injected could not even be identified. Later methods gave true intracellular staining. The agents used included potassium ferrocyanide followed by ferric chloride; Aniline Blue; a mixture of Fast Green FCF and Orange G; and Methyl Blue (Kerkut and Walker, 1962; Behrens and Wulff, 1965; Thomas and Wilson, 1966; other early work has been reviewed by Nicholson and Kater, 1973). All these methods had two drawbacks. First, the stains were not covalently bound to the tissue; to minimize leakage of stain from the injected cell, the tissue had to be processed in ways that resulted in serious distortions of structure. Second, the methods were all relatively insensitive, since they all depended upon light absorption for detection of the marked cell; a cell had to contain large amounts of dye to be visible. Stretton and Kravitz (1968) solved the first of these problems by introducing the tissue-reactive fluorescent dye Procion Yellow M-4RS. This dye remains in place during fixation and dehydration, presumably because it combines covalently with tissue components. It is not generally recognized that Procion Yellow did not solve the second problem-the low sensitivity of earlier methods. Because of its low fluorescence efficiency, Procion Yellow is only slightly easier to detect than dyes which depend upon light absorption. Other disadvantages of Procion Yellow include its uncertain structure, the lack of a published synthesis, and the unknown and probably variable purity of the commercial material. Most recent methods of intracellular marking (Pitman, Tweedle and Cohen, 1972; Christensen, 1973; Gillette and Pomeranz, 1973) share the basic limitation of Procion Yellow: they lack sensitivity. A tracer to which this generalization may not apply is horseradish peroxidase, which can be used for both light and electron microscopy (Muller and McMahan, 1976; Snow, Rose and Brown, 1976). The method is promising, but there is not yet enough experience to indicate how generally useful it will be. Procion Yellow injection, in spite of its low sensitivity, has played a decisive role in correlating the structure of nerve cells with their electrical func-
Cell 742
tion. The dyes described below possess most of the good features of Procion Yellow but in addition have a much higher fluorescence efficiency. The intense fluorescence of the new dyes allows a marked neuron to be seen in the living state as well as in cleared wholemounts, where the entire structure of the neuron can often be ascertained at a glance. The spread of Lucifer Yellow CH was observed not only throughout single neurons, but often from one neuron to another. The movement of dye from cell to cell, termed “dye-coupling,” occurred primarily but not exclusively between cells known to be electrically coupled. My preliminary observations on dye-coupling suggest that this type of functional connection between cells is substantially more common than was previously believed.
with proteins and with other compounds containing amino or sulfhydryl groups. The absorption and corrected emission spectra of Lucifer Yellow CH in water are shown in Figure 2. As expected, the excitation spectrum (not shown) was the same as the absorption spectrum within the limits of experimental error. The absorption spectrum was not significantly different in 0.1 N KHCOs or in 10 N HCI, which strongly suggests that the 4-amino group is unprotonated in aqueous solution and that its pK is
