A.ppendix Brain Slice Methods BRADLEY E. ALGER, s. s. DHANJAL, RAYMOND DINGLEDINE, * JOHN GARTHWAITE, GRAEME HENDERSON, GREGORY L. KING, PETER LIPTON, ALAN NORTH, PHILIP A. SCHWARTZKROIN, T. A. SEARS, M. SEGAL, TIM S. WHITTINGHAM, and JOHN WILLIAMS 1. INTRODUCTION The purpose of this Appendix is to provide the prospective slicer with a convenient package of methods; where choices exist among a number BRADLEY E. ALGER • Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201. S. S. DHANJAL and T. A SEARS • Sobell Department of Neurophysiology, Institute of Neurology, The National Hospital, Univer­ sity of London, London, England. RAYMOND DINGLEDINE and GREGORY L. KING • Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27514. JOHN GARTHWAITE • Department of Veterinary Physiology and Pharmacology, The University of Liverpool, Liverpool, England. GRAEME HENDER­ SON • Department of Pharmacology, University of Cambridge, Cambridge CB22QD, England. PETER LIPTON • Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706. ALAN NORTH and JOHN WILLIAMS • Neuro­ pharmacology Laboratory, Department of Nutrition and Food Science, Massachusetts In­ stitute of Technology, Cambridge, Massachusetts 02139. PHILIP A SCHWARTZ­ KROIN • Department of Neurological Surgery, University of Washington, Seattle, Washington, 98195. M. SEGAL • The Weizmann Institute of Science, Rehovot 76100, Israel. TIM S. WHITTINGHAM • Laboratory of Neurochemistry, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205. * To whom correspondance should be directed. 381 382 B. E. Alger et al. of approaches, this section also provides a discussion of those areas of agreement and disagreement among current practitioners. By collecting this information into one narrative, it should be easier for the newcomer to choose a set of procedures appropriate to slicing his favorite brain region. As is the case for all experimental preparations, the brain slice should be chosen only if it is an appropriate way to approach particular problems. The prospective slicer should be warned that it is unrealistic to consider sliced tissue as "normal," no matter how skillful and careful one is with the preparation. Slices are isolated tissue (without normal inputs) immersed in an artificial environment. This environment can be manipulated to ''bias'' the tissue however the investigator desires, but it is certainly never "normal." Investigators must weigh the potential advantages of the preparation (e.g., technical accessibility) with the ob­ vious disadvantages (e.g., loss of normal input pathways, shearing of dendritic processes and axons) as they apply to a particular problem. For supplemental reading, a number of reviews on brain slice meth­ odology are available (Dingledine et al., 1980; Hatton et al., 1980; Lynch and Schubert, 1980; Teyler, 1980; Kerkut and Wheal, 1981). 2. PREPARATION OF SLICES 2.1. Slicing the Brain It is perhaps on this topic more than others where myths, un­ founded dogmas, and notions based on intuition or anecdotal evidence tend to influence the choice of method. The following comments reflect our own experiences and are not meant to discourage the enterprising investigator from testing alternatives. Regardless of the method em­ ployed, the goal is to prepare a slice of tissue having those neurons, glia, and synapses that are important to the experiment in a viable con­ dition. Although the procedure used to dissect the brain will depend somewhat on whether a tissue chopper or Vibratome is used to prepare the slice, some general comments can be made. A gentle brain dissection may be the single most important factor in producing healthy slices. It appears that younger animals (e.g., guinea pigs less than 400 g, rats less than 175 g) produce better results than older animals. This may be in part because their thinner skulls are easier to remove, allowing a less traumatic dissection. If female animals are used, it is well to keep in mind the findings of Teyler et al. (1980), who showed sex-linked dif­ ferences, and differences across the estrous cycle, in the responses of hippocampal neurons to estradiol and testosterone. Brain Slice Methods 383 In general, the speed of dissection seems not nearly so important as the care taken in removing and slicing the tissue. The method of slicing the tissue is undoubtedly important as documented by Garth­ waite et al., (1979). They found that careful chopping and careful hand­ cutting of cerebellum did not appear to differ outwardly in the "trauma" inflicted on the tissue, yet there was a huge difference in the ultimate quality of the slices, as judged by electrophysiological and histological criteria. These findings point to the actual cutting of the tissue as being a critical step. Brains taken from lightly anesthetized rats are more often mushy or sticky during the slicing procedure. Animals that are deeply anes­ thetized with ether may yield better slices than marginally anesthetized ones, possibly due to the protective effects of ether anesthesia against spreading depression (van Harreveld and Stamm, 1953). In place of ether anesthesia, decapitation or killing by a blow to the neck has also been employed. Tissue in contact with stagnated blood (or deoxygenated blood) may suffer, not so much because of the hypoxia but because of effects of the blood itself. This impression is supported by the following comparison (P. A. Schwartzkroin, unpublished superstition): if one wants to make slices from adult rabbit or cat hippocampus (or cortex), one can either (1) anesthetize the animal, expose the relevant structure, and excise a piece for study, or (2) decapitate the animal and then do the dissection. Decapitation tends to be slower but less bloody, and it seems to yield superior results. 2.1.1. Hippocampus. 2.1.1a. The Tissue Chopper. A variety of methods can be em­ ployed to dissect out the hippocampus; the following is one technique found to be reliable. After removal from the skull case, the brain is placed into a beaker of cold oxygenated artificial CSF (ACSF) for a minute or so. The brain is then bisected sagittally with a razor blade, and one hemisphere placed on a flat surface with its medial face up. The tissue should be kept wet with chilled ACSF during dissection. The cerebellum and lower brainstem are lifted with forceps to expose the choroid fissure. Fine forceps can then be used to gently strip away the bulk of the tough choroid plexus. At this point, the ventral surface of the hippocampal formation is visible as a curved structure. Spatula cuts are made to free the septal and temporal ends of the hippocampus, and the spatula then inserted into the ventricular slit to gently roll the hippocampus out of the surrounding tissue. It is sometimes helpful to free the fimbria with forceps cuts as the hippocampus is rolled out. The entorhinal area is then trimmed away with a minimum of spatula cuts to completely free the hippocampus, which has the appearance of a small cashew nut. The 384 B. E. Alger et al. ~--SEPT AL AREA Figure 1. Dorsal view of the rat hippocampal formation. The ventricular surface (alveus) is exposed by removing the overlying neocortex. The hatched bar represents the approx­ imate orientation for preparing hippocampal slices. hippocampus can be stored in fresh, chilled ACSF while its counterpart from the other half of the brain is removed. Either or both hippocampi are placed on a layer of moistened filter paper glued to the chopping block, and the block rotated to adjust the tissue to the desired orientation for slicing (see hatched area in Figure 1). It is usually possible to view striations on the alvear surface with oblique lighting; these striations can be conveniently placed parallel to the blade. Excitatory pathways are better preserved when an angle of 15 to 30° from the transverse axis is used (Andersen et al., 1971; Rawlins and Green, 1977). Inhibitory pathways might be better preserved by a more longitudinal orientation (Struble et al., 1978; Dingledine and Lang­ moen, 1980), although this is not the impression of Schwartzkroin and co-workers in studying interneurons. It seems prudent to clean the chop­ per blade with ether to dissolve oils, etc. that are added by the manu­ facturer for lubrication and rust prevention, although no obvious dif­ ficulties have arisen when this step was occasionally forgotten. Slices of 350 to 500 f.Lm are serially cut and gently removed in a rolling motion with a fine sable brush into a Petri dish containing chilled oxygenated ACSF. The slices are placed individually in the recording chamber (or in a holding chamber) with a wide bore glass tube and suction bulb. The transfer pipette can be conveniently made by flaring and polishing the end of a broken Pasteur pipette in a Bunsen flame. Several different tissue choppers have been used successfully. Com­ mercial models include the Sorvall and McIlwain choppers. Of these two, Brain Slice Methods 385 the Sorvall, although 3 to 4 times more expensive, is easier to use since it allows fine adjustment of the blade height and blade angle, as well as rotation of the chopping block to orient the tissue. Duffy and Teyler (1975) have described a robust chopper, the blade of which falls by grav­ ity into the tissue block. This design can be modified so that a solenoid with adjustable force is used to drive the chopping arm. Whatever ma­ chine is used, it appears important that the blade have minimal lateral movement and that the vertical position of the blade be adjusted so that it does not slam into the surface of the chopping block. A blade height that just dimples the surface of the wet filter paper on the chopping blocks works well. 2.1.1b. The Vibratome. Currently two vibrating knives are avail­ able commercially: the Oxford Vibratome (Oxford Laboratories, U.s.A.) and the Vibroslice Oscillating Tissue Slicer designed by Jefferys (1981) (available from Campden Instruments, U.K.