BruinRcmwch Bulletin, Vol. 11, pp. 31-42, 1983.QAnkho lnteznationalInc. Printed in the U.S.A.

A Method For Dissection of Discrete Regions of Rat Brain Following Microwave Irradiation
JAMES P. OCALLAGHAN,* KEWIN L. LAVIN,* QUINTUS CHESSY AND DORIS H. CLOUETt *Neurotoxicology Division, Health Efects Research Laboratory U.S. Environmental Protection Agency, Research Triangle Park, NC 2771 I and tNew York State Division of Substance Abuse Services, Testing and Research Laboratory Rrook~y~, NY 11217 Received 3 December

1982

OCALLAGHAN, J. P., K. L. LAVIN, Q. CHESS AND D. H. CLOUET. A ~eth~for dissection ofdiscrete reglows of rot bruin f~~~lowing~ic~owuve irrodiution. BRAIN RES BULL 11(l) 31-42, 1983.---A simple microdissection technique

for obtainingdiscrete areas from rat brainsexposed to microwaveirradiationis describedand illustrated.Usingthe atlas of Pellegrinoer of. 1101 a guide, stereotaxically defined areas were removed from coronal sections prepared with sectiouing as
stages constructed from microscope slides. The dissection of sixteen discrete regions is shown in photographic and schematic form. This technique may prove useful for examining neurochemical processes in discrete areas oftbe rat central nervous system and may aid in establishing the distribution of pharmacological and toxicological agents at a neuroanatomicat level. Microwave i~diation Rat Central nervous system BraiN

EXPOSURE of experimental animals to high intensity microwave irradiation is widely employed for the rapid inactivation of brain enzymes to permit the steady-state measurement of heat-stable neurochemicals. As with fresh tissue, brains fixed by microwave irradiation can be dissected into gross areas [ 1,111 or sectioned with a cryostat [7] or vibratome [6] for removal of discrete nuclei. Gross dissection procedures are simple, inexpensive and can be performed rapidly, but do not result in the delineation of discrete brain nuclei. The removal of discrete btain areas can be accomplished with techniques that rely on mech~ic~ s~tio~ng but these procedures are difI5cult to perform on microwaved tissue [6] and require a considerable investment of time and equipment. We have observed that exposure to high intensity microwave scion results in the preservation of brain tissue to a degree that it can be microdissected without the use of expensive sectioning equipment. In this paper, we describe a rapid and simple technique for removing discrete areas from the rat central nervous system following microwave irradiation. This procedure relies on the coordinate system of Pellegrino et al. [ 101for the free-hand dissection of stereotaxitally defined regions of the brain.

METHOD

Materiels Stereotaxic atlas. The atlas of Pellegrko et al. [IO] was used as a guide for performing the dissection technique described in this paper. To achieve a satisfactory level of stereotaxic control we recommend the use of this or similar atlas of the rat brain as a reference guide. This permits the preparation of coronal sections with deBned rostral-caudal dimensions and allows for the accurate removal of discrete regions from a given section. S&jects_ We used male, Long-Evans hooded rats (Charles River, Wilmington, MA) weighing 280-320 grams. Rats of this strain and weight range coincide with those used by PeIlegrino et al. [lo]. In our experience other strains and weight ranges can be succesklly employed to perform the technique outlined below. This can be readily accomplished by modifiying the sectioning process in a fashion that yields coronal sections that correspond to the atlas plates containing the particular structures of interest (see below). Additionally, a mathematical formula has been devised [13] that permits the use of the Pellegrino et al. [lo] atlas with rats ranging in weight from 161-782 grams.

ITo whom reprint requests should be addressed.

*Present address: Department of Pathology, Memorial-Sloan-Kettering Institute, New York, NY 10021.

31

F F

FIG.

I Photographic

(top) and illustrative

(bottom)

guide

to the placement

of initial

reference

cuts.

DISSECTION OF MICROWAVED RAT BRAIN

~icrawave irradiatqr. Rats were killed by exposure of the head to microwave eon generated by a GerlingMoore Metabostat, Model 4095 (Gerling Moore Inc. Santa Clara, CA) (2450 MHz, 3.5 Kw maximum output power) for 2.5-2.8 seconds. Core brain temperatures were obtained by inserting a thermocouple probe into the hypothahunic area within 5 seconds of microwave exposure. The average core temperature obtained (95C) was used to determine the total energy absorbed by the brain. Assuming a mean brain weight of 1.9 grams and a core brain temperature of 3YC, microwave exposure for 2.8 seconds results in 445 Joules supplied to the brain which is equivalent to 4.5% of the available energy (9IUXl Joules). For a sample calculation see Schneider er ai. [ 111.where measurements of specific neurochemicals are desired, by aecessity, microwave irradiation must be of sufftcient intensity and duration to achieve effective enzyme denaturation [l lf, however, care must be taken to avoid conditions that result in overheating of brain tissue since this may cause disruption of cell structure and subsequent diiusion artifacts [8,12]. Microwave irradiators that deliver less energy than the unit we employed have been used successfully to both inactivate enzymes aud preserve morphological integrity of rat brain. For example, microwave-Sxatior using cheaper, less powerful instruments will render brain tissue suitable for microdisse&m f9] aud result in steady-state levels of cyclic nucleotides [4,9], neurotransmitters [4] and energy metabolites [4] that are equivalent to those observed by freezeblowing (see [4]). However, the suavity of a given irradiator for producing the degree of enzyme inactivation and tissue preservation needed for a particular application must be determined on a case-by-case basis. Slicing wedge and dissection instruments. A wedge made from a large cork or other suitable material was used to support the brain for placement of the initial reference cuts (see the Procedure section and Fii. 1). The surface of the wedge was angled at 20 degrees to perpendicular in order to match the stereotaxic plane of de Groot [3] as described [IO]. Prior to sectioniug the brain, microdissecting tweezers (Dumont, Roboz Surgical Instrument Co., Washington, DC) were employed to carefully remove the meninges. A singleedged razor blade was embedded in the cutting block at an angle 90 to horizontal in order to serve as a stop to prevent the brain from sliding off the wedge and as a guide for the placement of the reference cuts. Stainless steel double-edged razor blades were used to make the reference cuts and coronal slices (see the Procedure section). Due to the consistency of microwaved brain tissue, razor blades do not need to be particularly sharp to produce reproducible sections. Specific brain areas were dissected with a microdissection knife (Roboz Surgical Instrument Co., Washington, DC). Sectioning stages. Microscope slides and coverslips were glued together (Kraxy Glue, Krazy Glue, Inc., Itasca, IL) to form a series of sectioning stages for obtaining coronal sections of varying thicknesses (see Fig. 3 for an example). Slides of 0.4, 0.6, 0.8, 1.0, 1.6, 2.4, 2.6 and 3.0 mm were constructed for obtaining sections of the desired widths. To insure accuracy, a micrometer (Abbeon Cal., Inc., Santa Barbara, CA) was used to verify the thickness of each sectioning stage. Procedure Removal of brain. Following microwave irradiation the heads were severed from the body and intact brains were obtained from the skull by a careful piece-by-piece removal

33 of the tones comprising the cranial cavity. Since the meninges tend to be toughened foRowmg microwave irradiation, they must be removed with tweezers to prevent interference with the sectioning process. Placement ofinitiuf cuts. In order to produce stereotaxitally defined coronal sections, the brains were bissected in cross-section at either of the two positions identified by surface landmarks. The caudal edge of the wedge of grey matter between the optic tracts was used as a landmark for one cut while the most caudal extent of the cerebral bemispheres was used as a landmark for the other. The rostral-caudal coordinates (based on iuteraural0) [lOI that correspond to these initial cuts are 6.4 and -2.2, respectively, A photographic aud illustrative guide to the placement of these cuts is presented in Fig. 1. Both reference cuts were made at an angle parallel to the razor blade embedded in the cutting wedge. The placement of these initial cuts provides the proper stereotaxic plane and reference points for the preparation of all succeedii coronal sections. If it is determiued that the reference cut has been placed in an iucormct position, slight adjustments can be made by increasing or decreasing the thickness of the subsequent coronal sections as necessary. The placement of initial cuts and the performauce of all subsequent dissection procedures can be done at room temperature. Because prolonged exposure to air (greater than I5 minutes), tends to make microwaved brain difllcult to section, it is advisable to keep the tissue moistened with water or isotonic saline. Preparation of coronal sections. After placing either reference cut, sequential coronal sections of varying thicknesses can be prepared from the brain halves using the sectioning stages. The preparation of a 1.0 mm thick section is illustrated in Fig. 2. The cut surface of the brain is placed in the trough of a sectiouing stage moistened with water to facilitate adhesion (Fig. 2a). A section is then prepared by drawing a water-moistened razor blade through the brain in a ventral to dorsal direction while steadying the brain segment with light pressure from the f-r tips (Fig. 2, b-d). This procedure can then be repeated to produce sequential sections throughout the rostral-caudal axis of the brain. The sectioning procedure can be initiated with either the anterior or posterior portion of the brain following the placement of either reference cut. For ease of handlii and to prevent the loss of proper sectioning angle that may result from the preparation of sections several centimeters apart (from the same brain), we recommend beginning with the reference cut nearest to the area of interest. Alternately, if dissection of regions from both the anterior and posterior poles of the brain are desired, these might best be obtained from separate brains. Two sample schematics for preparing sequential coronal sections are presented in Fig. 3. For illustrative purposes we chose to remove stereotaxically defined areas from sections obtained throughout the brain. The thickness dimensions and atlas coordinates [IO] corresponding to specific sections obtained from each of 2 brains are shown (Fig. 3). The discrete brain regions removed from specific coronal sections are also represented in Fig. 3 and are shown in the photographic and diagrammatic plates that follow (see below). We found that the sectioning stages could be used to prepare reproducible sections as thin as 0.4 mm. Removal of discrete nuclei. The procedure for removing discrete areas from coronal sections is illustrated photographically in Fig. 4. By using a metric rule in combination
with a stereotaxic atlas, specific brain nuclei or areas can be excised from the appropriate section using the microdissec-

34

OCAI,I.A(.iHAN

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FIG.

2. a-d:

Photo~aphie

guide

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tion knife (Fig. 4). Neuro~atomic~ landmarks (see Table 1) serve as references points. Stereotaxicaliy defined brain areas as small as 0.5 mm3 can be removed reliably using this technique. Stainless steel tubing of varying diameters (Small Parts inc., Miami, FL) can be substituted for the microdissection knife if the removal of even smaller areas is desired (procedure not shown). Consistent amounts of discrete brain areas can be obtained using either a microdissection knife or tubing punches as evidenced by the low variability of regional protein levels (see Table 1).
RESULTS AND DISCUSSION

The protein values, stereotaxic coordinates [lo] and visual landmarks corresponding to 16 discrete brain areas dissected as described above are presented in Table I. A photographic and diagrammatic presentation of the dissected areas is shown in Plates 1-16.

The dissection technique presented can be used to prepare stereotaxicaily defined brain areas throughout the medial-lateral, dorsal-ventral and rostral-caudal axes of the brain. Although we obtained discrete regions from coronal sections, the procedure can be modified for the removal of areas from sagittal sections of defined coordinates [IO]. The reproducible nature of this procedure is predicated on the accurate placement of initial cuts and the use of sectioning stages in combination with an atlas of the rat brain to achieve proper stereotaxic control. Where the use of microwave irradiation is required, this technique should serve as a simple and inexpensive alternative to either the removal of gross brain areas or the microdissection of discrete areas from sections prepared with mechanical sectioning equipment. In situations where microwave fixation is not normally employed (e.g., drug and toxicant distribution studies; biogenic amine measurements), its use may be of advantage ifthere is a need to obtained measurements at a discrete neuroanatomical level.

DISSECTION OF MICROWAVED RAT BRAIN

35

1.0

a4
7.4 44 a4 2.4

.,.....,........,..,

55ff............................,

................

..I

3.0

?A0
0.0 -0.8

1 POSTERIOR 1
* INITIAL m Dl2CAitD REPERENCE CUTS

FIG. 3. Sample schematic for preparingsequential coronai sections from each of two separate brains. The abbreviations used are as follows: CP, caudate putamen; GP, giobus pallidus; LH, lateral hypothalamus; VMH, ventromedial hypothalamus; PC, parietal cortex; AMYG, amygdala; HIPPO, hippocampus; PAG, periaqueductal gray; LC. locus coeruleus; N. ACC, nucleus accumbens; SN, substantia nigra; RAPHE N., raphe nucleus; V. LOBES, vermian lobes and RF. reticular formation.

Heffner et al. [5] have recently described a technique for the dissection of fresh rat brain which is similar to the method we describe above. It is based on the removal of brain areas from coronal sections prepared with a cutting block. Since several coronal sections can be prepared at once, this method is faster than our procedure, however, the thickness of each section can not be adjusted to achieve stereotaxic control. In addition, the consistency of fresh brain restricts the use of the Heffner it al. [5] technique to the dissection of large terminal tields rather than discrete nuclei. Nevertheless, if only major neuroanatomical regions are to be dissected, this method, when applied to microwaved tissue, may represent a useful alternative to the one described in this report.

ACKNOWLELIGEMENTS The authors wish to thank Drs. D. B. MiBer and R. S. Dyer for useful discussions and Mr. Norman WiIliams for excellent technical assistance.

PIG. 4. Procedure for removing discrete areas from coronal sections.

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TABLE

Coordinates Plate No. Brain Area Nucleus Accumbens Caudate Putamen Globus Pallidus 4 Lateral Hy~thalamus Ventromedial Hypothalamus Parietal Cortex Amygdala Dorsal Hippocampus CAI Dorsal Hippocampus CA3 Hippocampus Dentate Gyrus II Substantia Nigra Periaqueductal Gray Raphe Nucleus Locus Coeruleus Reticular Formation Vermian Lobules Protein+ (r*P) 209 :: 20.5 f 191 .f 199 t 4.3i 3.5 8,8 3.8 RostralCaudal 9.8, 8.4. 7.4. 6.4. 8.8 7.4 6.4 5.4 MedialLateral 1.0. 2.0 1.5. 3.5 2.0. 3.5 1.5. 2.5 DorsalVentral 0.0. 1.0. I .o 3.0 Landmarhs Anterior Commis~ur~ Halo of Anterior Commissure Optic Tracts, Rectangular Shaped Fornix internal Capsule, WinpShaped Hippocampal Commissure Same as above Same as above Same as above Hippocampal Fissure. Thickness of Fimbria of Hippocampus Same as above

1.0. -1.0 - 3.0, -4.0

f Dorsal Cut
Angled) 214 S87 + 206 i. 83 + 7.5 16.1 7.5 5.0 6.4. 6.4, 6.4. 3.8. 5.4 5.4 5.4 3.2 0.5, 1.5 6.0. 7.s 4.0, 5.5 2.0, 3.0 - 2.5, --3.5 -2.0. -3.0

s b 7 8

--2.0. -3.0 3.0, 2.0

53 r

1.9

3.8.

3.2

3.0, 4.0

2.0.

1.0

81 + I55 -c

5.8 7.2

3.8. 3.4.

3.2 2.6

1s, 2.5 2.0. 3.0

2.0. .. 3.5.

I.0

Same as above Rhinal Fissure, Thickness of Corpus Callosum at Midline Aqueduct, Extent of Periaqueductal Gray Same as above Same as above Extent of Ventricle Extent of Cerebellar Cortex

.4.0

12 13 I4 is 16

2126 r 170 r

850 5.0

2.8, -,O.? 0.0, -0.8 -0.8. -2.2. -2.2, - I .4 3.2 -3.2

1.0, 1.0 0.5, 0.5 0.5, 1.0 1.0, 2.0 2.0, 2.0

- 1.5, -2.5 fTriangular) - 2.5, -. 3.5 -2.5, --3.5

99 t 2.5 129 c II.1

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*The rostral-caudal, medial-lateral and dorsal-ventral coordinates for each area are based on those described in the atlas of Pellegrino CI ol. f IO]. All coordinates are in millimeters. The rostra&caudal coordinates are taken with respect to the interaural line. *Determined by the procedure of Bradford 121. SMean 2 SEM of 4 determinations.

FACING

AND

FOLLOWING

PAGES

Plates 1- 16. Photographic and diagrammatic presentation of the dissection of the discrete brain areas described in Table 1.The medial-lateral, dorsal-ventral and rostral-caudal coordinates for each area are described in Table I and coincide with those described in the atlas of Pelletino P[ af. [IO]. All coordinates are in millimeters. The number in the upper right hand comer for each schematic is the rostral-caudal coo&ate with respect to the interaural line (see Table 1). The regions delineated in each schematic (other than those dissected) can be identified by refering to the atlas of Peilegrino or nl. [IO].

DISSECTION OF MICROWAVED RAT BRAIN

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7. Jacobowitz,

D. M. and A. M. Goldberg. Determination of acetylcholine in discrete regions of the rat brain. Brcrin Res 122: 575-577, 1977. 8. Maruyama, Y., R. Nakamura and K. Kobayashi. Effect of microwave irradiation on brain tissue structure and catecholamine distribution. Psychophnrmuc,oloKy CBerlin) 67: 119-123,
1980.

9 OCallaghan, J. P., Q. Chess, C. McKimmey and D. H. Clouet. The effects of opiates on the levels of cyclic 35 guanosine monophosphate in discrete areas of the rat central nervous system. J Pharmucol Exp Ther 210: 361-367, 1979. 10. Pellegrino, L. J., A. S. Pellegrino and A. J. Cushman. A Sfereoraxic Arlas of the Rat Bruin, second edition. New York: Plenum Press, 1979. 11. Schneider, D. R., B. T. Felt and H. Goldman. On the use of microwave radiation energy for brain tissue fixation. J Neurothem 38: 749-752, 1982. 12. Sharpless, N. S. and L. L. Brown. Use of microwave irradiation to prevent postmortem catecholamine metabolism: evidence for tissue disruption artifact in a discrete region of rat brain. Bruin Res 140: 171-176, 1978. 13. Whishaw, I. Q., J. D. Cioc, N. Previsich and B. Kolb. The variability of the interaural line vs. the stability of bregma in rat stereotaxic surgery. Physiol Behav 19: 719-722, 1977.