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HOME :: CHAPTER 13 :: HORSERADISH PEROXIDASE STAINING :: CELL MARKING WITH HORSERADISH PEROXIDASE |
Cell Marking with Horseradish Peroxidase
Adapted from D. Purves and J. W. Lichtman, 1985. Principles of Neural Development. Sinauer Associates, Inc., Sunderland.
In recent years the technique of marking neurons with the enzyme horseradish peroxidase (HRP) has become a major tool. In its brief history, this method has probably been used by more neurobiologists than have used the Golgi stain since its discovery in 1870.
Although the Golgi technique was unequivocally the invention of a single person, the widespread use of peroxidase is difficult to attach to the efforts of any individual. Perhaps this is because several different advances led to its current applications. First was the observation by P. Weiss and H. B. Hiscoe in 1948 that materials are continually transported along axons. This observation led to work on the now well-established, but incompletely understood, phenomenon of axonal transport. Second was the idea that transported macromolecules might act as markers. The notion of using horseradish peroxidase for this purpose probably arose from the use of this molecule in extracellular marking studies pioneered by W. Straus, M. Karnovsky, and R. C. Graham, who showed that a reaction product catalyzed by this enzyme forms a dense precipitate that can be seen in both light and electron microscopes (Mesulam, 1982). In 1971, K. Kristensson and Y. Olsson first used HRP to label neurons retrogradely in the peripheral nervous system (Kristensson and Olsson, 1971). J. H. and M. M. LaVail then demonstrated the efficacy of HRP in the central nervous system and worked out much of the cell biology of its transport (LaVail and LaVail, 1972).
Horseradish peroxidase is a heme protein found in the roots of the horseradish and has a molecular weight of about 40,000 daltons. The advantages of this particular agent are striking. It is readily taken up by nerve cells; because of its size, HRP tends to remain inside the cells and their axons and dendrites. Most important is the amplification that occurs because this protein is an enzyme. It is not the transported molecule itself that is seen in the microscope but rather a reaction product produced by incubation with an appropriate substrate and hydrogen peroxide. In consequence, a relatively small number of enzyme molecules can create an easily visualized precipitate. Interestingly, the sequence of reactions involved is similar to the reactions that produce melanins, a large family of plant and animal pigments.
Numerous variations in HRP methodology have now been described (see, for example, reviews in Heimer and Robards, 1981; Mesulam, 1982). In general, the enzyme is used in three ways by neurobiologists. The most common use is as a retrograde label. In this approach, HRP (as crystals or solution) is placed on or near injured axons. Retrograde axonal transport then carries the enzyme molecules to the cell bodies, which are made visible by catalysis of an appropriate substrate such as diaminobenzidine, tetramethylbenzidine, or pyrocatechol. This simple, yet powerful approach shows the location of nerve cells that project to a given region and has radically changed the ease with which one can obtain information about neuronal projections. A variant of this approach is uptake of the enzyme by intact axon terminals; for example, intraocular injections retrogradely label cells in the superior cervical ganglion. Terminal uptake can be augmented by electrical stimulation and can also be used to distinguish active from inactive terminals (Holtzman et al., 1971).
A second way of using the enzyme is to introduce it near injured nerve cell bodies. The enzyme is then taken up by cell somata (presumably by endocytosis) and transported distally by anterograde axonal transport. This approach can demonstrate the projection site and the anatomy of terminal arborizations and thus provides information complementary to the retrograde transport approach (see Figure 1). Finally, HRP can be introduced into the electrolyte solution filling a micropipette, a technique that permits intracellular marking of individual cells after electrophysiological studies. This approach was first used to mark crustacean neurons with the fluorescent dye Procion Yellow by A. O. Stretton and E. A. Kravitz (1968); in the mid-1970s a number of workers adapted HRP to this intracellular method (see Mesulam, 1982). Intracellular injection has remarkable power because it provides a Golgi-like picture of cells whose properties and connections can be studied in detail prior to staining.
It is hard to overestimate the importance of this technique in neurobiology generally and developmental studies in particular.
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| Figure 1 (A) Preganglionic neurons in the embryonic rat spinal cord (E14) retrogradely labeled after HRP injection into the stellate ganglion (T1-T3 indicate thoracic segments). (B) Anterograde transport of HRP into motor neuron axons after injection intothe spinal cord of an embryonic chick. (C) Mammalian sympathetic neuron after intracellular injection of HRP through a micropipette. (A from Rubin, 1985; B after Hollyday, 1983; C from D. Purves, unpublished.) |
Literature Cited
Heimer, L. and Robards, M. J. 1981. Neuroanatomical Tract Tracing Methods. Plenum Press, NY.
Hollyday, M. 1983. Development of motor innervation of chick limbs. In Limb Development and Regeneration, Part A. Alan R. Liss, NY. Pp. 183-193.
Holtzman, E., Freeman, A. R., and Kashner, L. A. 1971. Stimulation dependent alterations in peroxidase uptake at lobster neuromuscular junctions. Science 173: 733-736.
Kristensson, K. and Olsson, Y. 1971. Retrograde axonal transport of protein. Brain Res. 29: 363-365.
LaVail, J. H. and LaVail, M. M. 1972. Retyrograde axonal transport in the central nervous system. Science 176: 1416-1417.
Mesulam, M.-M. (ed.) 1982. Tracing Neural Connections with Horseradish Peroxidase. John Wiley and Sons, NY.
Rubin, E. 1985. Development of the rat superior cervical ganglion: Ingrowth of preganglionic axons. J. Neurosci., published in D. Purves and J. W. Lichtman, op. cit.
Stretton, A. O. W. and Kravitz, E. A. 1968. Neuronal geometry determination with a technique of intracellular dye injection. Science 162: 132-134.
Weiss, P. and Hiscoe, H. B. 1948. Experiments on the mechanism of nerve growth. J. Exp. Zool. 107: 315-396.
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HOME :: CHAPTER 13 :: HORSERADISH PEROXIDASE STAINING :: CELL MARKING WITH HORSERADISH PEROXIDASE |