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When biochemical determinations on brain tissue were required animals were stunned by a blow to the cervical spine before decapitation by guillotine. Where histological analysis was required, cardiac perfusion was conducted prior to decapitation (see section 2.3.4.). The brain was rapidly removed by the following method:

The skin overlying the superior aspect o f the skull was cut using a No. 22 scalpel blade, along the midline and peeled outwards to expose the skuh. One blade o f a pair o f blunt-ended scissors, was carefully inserted into the exposed foramen magnum and two cuts through the skuh were made at 45° to the dorsoventral midline extending to the lamboid suture. This enabled the occipital bone to be removed by tilting the scissor blade upwards and outwards. Subsequently the scissor blade could be run between the cortical hemispheres and the inferior aspect of the parietal bone. Taking care to avoid damaging the underlying brain tissue, the skuh was spht along the sagittal suture, through bregma and sphtting the frontal bone midline. The right and left parietal plates were twisted and turned outwards to expose the brain. If the dura covering the brain remained intact fohowing the initial stages of dissection, it was removed at this point with the aid o f fine forceps. The animal’s snout was held and the skuh inverted at a 45° angle, to ahow a microspatula to be run anterior to the olfactory bulbs, thus severing the optic nerves. After carefuhy passing the microspatula between the brain and the endocranial surface o f the skuh, the brain was dropped into a 100 mL beaker o f ice-cold saline for biochemical analysis or into a 4% paraldehyde v/v saline solution for histological analysis.

The removal of the brain was achieved within 1 minute of decapitation and brains used for biochemical determinations remained in ice-cold saline for less than 2 minutes before dissection. Brains placed into a 4% paraformaldehyde in saline solution for

histological investigations were stored refiidgerated (circa 4° C) until sectioniug was performed.

2.4.1. Preparation for biochemical analysis

All procedures were undertaken rapidly and both tissues and instruments were kept on ice to minimise post-mortem changes.

The brain was removed from the beaker o f ice-cold saline, blotted, and placed into a coronal brain sheer (1.3 mm section thickness, see Figure 2.4.1.1., Harvard Apparatus Bioscience, Edenbridge, UK) for sectioning (Henry and Kashpal, 1984).

The aim of the dissection procedure was to obtain tissue regions with various characteristics of serotonergic innervation. To this end, six ‘regions’ were collected, designated: frontal cortex, raphe, hippocampus, hypothalamus, striatum, and the rest of the brain (excluding the cerebellum), using a combination o f tissue shce with subsequent tissue punch (3 mm ID) (Palkovits and Brownstein, 1983) or by freehand dissection (Glowinski and Iversen, 1966).

Figure 2.41.1. Rat Brain Sheer, for coronal sectioning with 1.3 mm between slots (Harvard Apparatus Bioscience, Edenbridge, UK)

The brain was sectioned as illustrated in Figure 2.4.1.2. and as described below: The brain was positioned within the sheer so that its inferior aspect was uppermost. The location of bregma was taken as the anterior tip o f the optic chiasma, a visually identifiable structure (triangular in shape with a pale white colouration) on the midline of the brain. The interaural line was assumed to be 9 mm posterior to the bregma (Paxinos and Watson, 1982, 270-310 g male rats, error in resolution < 0.5 mm). Shght adjustment to the position o f the brain within the coronal sheer enabled the insertion o f a razor blade to pass though bregma. A second razor blade was inserted seven slots posterior to the first blade (7 x 1.3 = 9.1 mm posterior to the bregma) which defined the interaural line. A third blade was inserted two slots ( 2.6 mm) anterior to this interaural line and a fourth inserted four slots (5.2 mm) anterior to the bregma.

The tissue lying posterior to the interaural blade (the posterior rhombencephalon) was removed fi*om the sheer and the superior portion, cerebeUum, was teased away fi*om the remaining 'pons medulla' (pons and medulla oblongata) with the aid of a microspatula and blunt-ended forceps, and discarded. The 2.6 mm shce, lying between the interaural and the anterior lying third blade, was also removed and laid on a glass dissection plate with its anterior surface uppermost. Using the bottom of the cerebral aqueduct or 4th ventricle, as the upper guide mark, the pontine nuclei as the lower mark, and centred on the midline, a 3 mm diameter tissue punch was made in a posterior facing direction. This punched section was designated 'raphe' although it contained considerable extraneous material as weU as the dorsal, median and caudal nuclei of the raphe. The remaining tissue fi'om this shce after removal o f the 'raphe' was coUected together with the remaining pons and medulla oblongata as part o f the ‘rest o f brain’ tissue.

The tissues lying anterior and posterior to the blade positioned on the bregma, were removed fi'om the sheer and laid on the glass dissection plate so that the surfaces closest to the bregma were uppermost. This exposed the anterior and posterior portions of the striatum which were scooped out by careful manipulation o f a microspatula. Material fi'om both aspects and fi'om both hemispheres were included.

Figure 2.4.1.2. Schematic diagram illustrating the dissection procedure for the rat brain: A.) Sagittal section near mid-line, dashed lines indicate positions of sectioning; B. & C.) Coronal slices, dotted lines indicate punch line and sections respectively. B, Bregma 0.48 mm C orpus callosum Striatum A. _{Bregma -10} 2nd blade -15 + 5 3rd blade Frontal cortex Oltactory bulb Hypottiolamus 10 4th blade 1st blade Interaural +15 + 10 +5 C. Bregm a -8.00

D orsal rapht M edial raphe

The tissue remaining in the slab posterior to the bregma blade, was further sectioned by gently peeling away the superior cortical tissue and then teasing off the hippocampal segments from both hemispheres.

The midbrain region was further dissected by making a lateral cut extending 1.5 mm on either side o f the midline (formed by the cerebral aqueduct) and just below the anterior commissure. Two further cuts were made perpendicular and inferior to the first cut. Material between these cuts was designated the hypothalamus. The remaining tissue containing much of the midbrain, was collected with the ‘rest o f brain’.

Finally, having separated the olfactory lobes from the remaining brain tissue on insertion o f the fourth coronal sectioning blade, the cortical material posterior to this blade could be removed from the sheer, and collected as frontal cortex.

These six regions which were dissected from a brain within 7 minutes, were immediately placed into pre-labelled 5 mL tissue storage pots or 1.5 mL Eppendorf vials cooled on dry ice, before being transferred to a -70°C freezer for storage prior to biochemical analysis. The brain sheer, tissue punch, microspatula and forceps were thoroughly cleaned before they were used to dissect the next brain.