A method for vibratome sectioning of Golgi–Cox stained whole rat brain
Introduction
There have been many variations in Golgi’s method of impregnating nerve cells since its publication in the late 1800’s (Golgi, 1873). It was Cajal, however, who applied the technique to demonstrate previously unimagined morphology in virtually every part of the nervous system (Cajal, 1909). Although Cajal’s use of the Golgi technique was largely for descriptive purposes, in the past three decades there has been a resurgence in interest in using Golgi methods to investigate behavioural–morphological relationships (e.g. Globus and Scheibel, 1967, Coleman and Riesen, 1968, Woolsey and van der Loos, 1970, Valverde, 1971, Greenough, 1976, Juraska, 1984, Withers and Greenough, 1989, Kolb and Gibb, 1991, Steward, 1991, Jacobs and Scheibel, 1993, Steward and Rubel, 1993, Jones and Schallert, 1994).Such studies normally involve significant behavioral manipulation and/or training prior to tissue analysis and this carries two important requirements. First, it is desirable to have a staining protocol that is reliable since it is often difficult, or even impossible, to replace individual animals or groups after the experiment. Capricious staining can mean that the entire experiment must be repeated, which is both time consuming and expensive. Second, since it is not always obvious a priori where one would like to search for morphological changes, it is ideal to have a method that allows for simple embedding of the entire brain so that every section can be saved for later analysis. In this case it is desirable too, that there be even shrinkage of the tissue so, at least for small brains, it is best to stain without blocking the brain. We have been experimenting with various Golgi procedures over the past decade and have developed a modified Golgi–Cox procedure that meets these requirements.
There has been a recent increase in the use of the vibratome for the sectioning of fresh tissue, especially for immunohistochemical procedures, which has resulted in an increased availability of the vibratome in neuroscience laboratories (Landas and Phillips, 1982). We have found that the vibratome can easily be used for sectioning of Golgi-impregnated tissue and allows us to impregnate and section the whole rat brain without the costly celloidin-embedding and a special microtome. The method below is based upon Glaser and van der Loos (1981)Golgi–Cox procedure that we had previously modified for cryostat-sectioning (Kolb and McLimans, 1986).
Section snippets
Materials and methods
Rats are administered an overdose of sodium pentobarbital and perfused with 0.9% saline. Brains are removed and placed in 20 ml Golgi–Cox solution (Glaser and van der Loos, 1981). The brains are stored in the dark for 14 days after which the Golgi–Cox solution is replaced with 30% sucrose. The brains are allowed to sit in the dark for 2–5 days before sectioning. The sucrose step allows the tissue to remain more pliable and less prone to fracture when it dries. Storing the brains in the dark
Results
Using this procedure we have found that dendrites and spines are evenly and consistently stained, as illustrated in Fig. 1. The method can be used effectively for brains of rats ranging in age from postnatal day 0 until old age, with only minor modifications of the impregnation time. In particular, small brains (less than 1 g) should be left in the Golgi–Cox solution for 6 days. We have also found the procedure to work well with other rodent brains including voles, gerbils and mice as well as
Discussion
Our procedure offers the advantages that: (1) the whole brain is impregnated and cut at once, which eliminates the problems of unequal shrinkage of blocks of a given brain; (2) the sections are stained after mounting on the slides, which obviates the need to handle the individual sections; (3) coverslipping is much easier than for celloidin-embedded tissue in which the sections tend to curl; (4) the results appear to provide more extensive staining of fine branches than celloidin-embedded
Acknowledgements
Bryan Kolb wishes to thank the Natural Science and Engineering Research Council and Medical Research Council of Canada for their support.
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