Publisher Summary

This chapter discusses that the isolation of characterized neurons, nervous tissue parts, and populations of cells is the most critical step in the biochemical analysis of the nervous component. When working with vertebrate tissue, the most hazardous period is usually between the moment the blood supply is cut off and the dissection. It cannot be overstressed that either the functional integrity of the nerve cell should be maintained after dissection, or the metabolism of the neuron should be stopped abruptly by means of rapid freezing. The chapter explains that the first alternative is essential for direct neurophysiological and neuropharmacological correlations and it can be achieved comparatively easily when using invertebrate neurons. The second alternative is often necessary when analyzing vertebrate nervous tissue, especially labile metabolites. A couple of pairs of fine forceps together with a microscalpel are all that is required to free a characterized cell from the surrounding nervous tissue.

1 Isolation of Characterised Invertebrate Neurons

The problems encountered in dissecting individual neurons from the various isolated ganglia are generally caused by the illumination of the ganglion, the identification of the neuron involved and its removal, intact, from the ganglion. An essential first step is to pin the ganglion down in a relatively stretched out position. It should not be stretched too much, otherwise the cells will tend to ‘pop out’ when the connective tissue layers are removed. In the instance of the snail (Helix pomatia) circumoesophageal ganglia, a small bath (volume 0.7 ml) containing a nylon sheet at the bottom and filled with snail saline is most suitable. This stiff plastic sheet is necessary to retain the insect pins used for holding the ganglia down. Either transillumination, reflected light, or dark-field illumination can then be used for the identification and dissection of individual neurons, which is carried out under microscopic vision. The most useful method by far is dark-field illumination. One simple approach is to focus a pencil of light on to the cell concerned. This can be done by attaching a tapered glass rod to a microscope lamp, and bringing the glass tip (about 3 mm diameter) very close to the preparation, thus illuminating the cell alone. An alternative to this is the Dark-field Dissecting Stand obtainable from Brinkmann Instruments Company (Westbury, New York). Proper illumination is essential for identifying individual neurons within the ganglia.

The localisation of characterised neurons will depend upon the familiarity of the experimenter with the ganglia, though a number of published maps of ganglia are available, e.g. Aplysia californica (Coggeshall et al., 1966; Frazier et al., 1957; Kandel et al., 1957), Helix pomatia (Kerkut, 1969; Osborne, 1973), the cockroach (Cohen and Jacklet, 1965), Hirudo medicinalis (Nicholls and Baylor, 1969) and Tritonia (Willows, 1967, 1968). It should be kept in mind that these maps are based on a variety of physiological characteristics such as electrical activity, neurohormone response, sensitivity to drugs, etc., as well as on morphology. Morphology, which depends on size, colour and position, can be used to identify cells, though often variations do occur which can be due to age differences among different animals. For example, in Aplysia more small cells appear as the animal gets larger, often making the giant neurons difficult to identify. When identification by morphology alone is found difficult, it is advisable to use neurophysiological methods to provide confirmation. Generally it is preferable to make the identification of characterised neurons before cutting through the final connective tissue sheath, and so prevent disturbance of the morphology.

A couple of pairs of fine forceps together with a microscalpel are all that is required to free a characterised cell from the surrounding nervous tissue. A constant check should be made that the tips of the forceps fit exactly together and are sharply pointed, best achieved by filing them with emery stone. Using two pairs of forceps, one in each hand, connective tissue surrounding the cell can be gently teased away until the cell ‘pops out’. The microscalpel together with a single pair of forceps are then used to cut the cell’s axon, so freeing it entirely (see Fig. 3). Needless to say, considerable caution has to be taken throughout the dissection to avoid any damage to the cell. The cell can then be transferred to a microtube by means of a constricted pipette (see page 18) attached to the mouth by rubber tubing, which allows complete control of the transfer process. Alternative methods have been used to transfer cells which include merely using the scalpel tip, not a method to be advocated. Another procedure is to expose the cell without cutting the axon. A loop of fine wire (enamelled michrome ‘Trophet C’, size 001, Wilbur Driver Co., Newark, New Jersey, USA) is then dropped over the cell and drawn tightly around the axon at the base of the cell. The axon is severed below the wire knot and the wire and cell transferred to a microtube (McCaman and Dewhurst, 1970).

2 Isolation of Cell Components from Fresh Tissue

Giacobini (1956), working with spinal and sympathetic ganglion cells and anterior horn cells of spinal cord, was one of the first to dissect nerve cell bodies from fresh tissue. Subsequently Hydén (1959) used the same procedure on mammalian brain. Such free-hand dissections are normally carried out on slides with a layer of about 1 mm paraffin wax (histological quality) in a drop of a suitable Ringer medium (see Edström and Neuhoff, 1973). The surface tension of a water drop on a paraffin layer is high enough to prevent the drop spreading, and the evaporation is therefore low. The microdissection is carried out under a stereomicroscope, and where this lasts for longer periods the stereomicroscope should be equipped with a cooling stage to give temperatures of between 0°C and 10°C.

Small pieces of tissue approximately 1 mm square containing the cells to be studied are placed in the drop of suitable Ringer solution on top of the paraffin wax-covered slide. The uppermost surface of the piece of tissue should be observed under a stereomicroscope and differences in colour and texture noted. Then a drop of solution of methylene blue in isotonic Ringer solution (1 part in 10,000) is applied to the surface. As soon as the dye is taken up, preferentially by the neurons (see Fig. 4), the methylene blue solution is washed away and substituted by Ringer alone. Single cells can then be dissected free from the tissue with fine steel wires fixed to a glass rod (see page 18). The free nerve cells which float in the drop of Ringer can be seen clearly under the stereomicroscope and can be transferred by use of hair loops (see page 18). Once the technique has been mastered, nerve cells with many dendrites can be isolated individually in less than a minute. Some neurons isolated from human brain tissue are shown in Figs. 1 and 2.

Clumps of glial and neuropile can also be prepared by this procedure. A piece of fresh nervous tissue is placed on a drop of medium on a slide and single nerve cell bodies adhering to the neuropile/glial may then be lifted out of the tissue with a stainless steel microspatula and forceps. Giacobini (1959) isolated glial cells of spinal and sympathetic ganglia through such an approach.

Lasek and Dowler (1971) have developed a useful technique for obtaining nuclei from freshly dissected invertebrate (Aplysia) neurons. Their method is as follows. Single characterised neurons are...