GENERAL METHODS
HISTOLOGICAL AND ANATOMICAL METHODS
After all recording sessions were finished, and any anatomical tracers had been administered, the monkey was sacrificed for histological reconstruction of recorded cell position and anatomical studies. The methods by which this was done can be broken down into 3 stages. These are (i) perfusion fixing and removal of the brain, (ii) sectioning and staining of the brain tissue and (iii) the reconstruction process itself.
Peffusion, Fixing and Removal o f the Brain
The monkey was given an anti-coagulant (Heparin, 5000 units for every 1.5 Kg of body weight) then left for approximately 30 minutes to allow the anti coagulant to pass throughout the body. A ketamine injection (1.0 ml Vetalar) was followed by a lethal intravenous injection of barbiturate (Sagatal). The animal was assessed to be in a deep coma by the absence of a gabel la reflex (gently tapping the eye produced no reflexive closing of the eyelid).
Perfusion was performed trans-cardially, using first a pre-fixative wash to flush the blood from the brain, then passing the fixative through the tissue. After
this, the brain was removed and kept refrigerated in phosphate buffer until sectioned and stained. The fixative used was phosphate buffered 4% paraformaldehyde, 0.5% giutaldehyde.
In more detail, the animal's thorax was opened to reveal the heart and a large bore cannula (internal diameter 2 mm) was inserted into the left ventricle . The right atrium was also punctured to allow the exit of the blood / pre-fixative.
After cannula placement the pre-fixative wash (Hartmann’s solution, pre- warmed to 37 degrees centigrade) was passed through the animal (approximately 5 litres of pre-fixative for 5-8 Kg animals) with a centrifugal pump to assist the flow. After the pre-fixative wash, the perfusing fluid was changed to the fixative. To ensure good fixation of the brain, the perfusing cannula was pushed gently up through the left ventricle and into the ascending branch of the aorta. Similar quantities of fixative and pre-fixative were used. The brain was then removed from the skull using bone cutters and sunk in successively higher concentrations (10, 20 and 30%) of sucrose solution or 2% Dimethylsulphoxide (DMSO) and 20% glycerol (Rosene et al. 1986). These solutions were designed to avoid freezing artifacts in histology. The durai tissue was left intact and surrounding the brain as much as possible. The isolated brain was then placed in 0.1 M phosphate buffer and refrigerated prior to the histological work.
Sectioning and Staining
The posterior part of the fixed brain tissue was cut away to leave a stereo- taxically vertical (coronal) surface. Immediately prior to sectioning, the fixed brain tissue was immersed in pre-cooled isopentane. The temperature of the isopentane was maintained between -70 and -90 degrees centigrade using dry ice (CO2). The tissue was then mounted in a freezing microtome (Bright Instruments Company Ltd) pre-cooled to -30 degrees centigrade. Coronal sections of 25 microns were talten every 250 microns. During sectioning, photographs were taken of the tissue just before a section was taken. With each photograph the
General Methods (2.48) section number and anterior/posterior position was included, as was a small
mai'ker of known length (to allow scaling).
Reconstruction
Reconstruction of electrode position was achieved by reference to the positions of micro-lesions (10 micro amp DC for 30 s) made at the end of some electrode tracks which were subsequently identified using standard histological techniques. In 3 monkeys additional markers used in calibration of electrode position were provided by micro-injection of anatomical tracers (horseradish peroxidase and fluorescent dyes true blue and diamadino yellow) at the site of cell recording on 3 recording tracks. For these markers the position of injection, recorded in X-radiographs, could be compared to the anatomical location of injection revealed through normal or fluorescence microscopy. Further probes were inserted into the perfused brain prior to removal from the skull. The position of these probes was also recorded with X-radiographs. Two horizontal alignment probes were also inserted per hemisphere along the full anterior-posterior extent of the brain to allow alignment of each section.
From each frontal and lateral X-radiograph, the electrode tip was measured relative to the interaural plane and midline. The position of the electi'ode at a known height (25 mm above the interaural plane) was also measured. The 3-D trajectory of each track was calculated from these X- radiograph coordinates. Cell positions along each track were then mapped onto the sections (See Harries & Perrett 1991 for full details). Recently software was developed that allowed entry using a mouse of the photographed brain tissue sections. These maps were stored on PC and allow the supersition of the electrode ti'acks and cell position.
Table 2.1 gives a breakdown of the number of cells recorded from each monkey and the number of cells included in each chapter.
Table 2.1 Number of ceils recorded MONKEY Chapter Number
3 3 6 7+8 9 Total cells recorded
B 0 36 29 64 0 1300
D 22 48 14 30 4 3182
H 9 4 5 9 5 364
J 13 33 34 58 35 1613
TOTAL 44 216 82 161 44 6549
Table 2.1. In addition to the cells shown, 95 cells from money F were included in the analysis of chapter 5 (directional tuning).
The nature of neurophysiological recordings of the type described in this thesis is such that a collaborative effort is required for data collection. My collaborators for the work presented here have been D. Perrett, and over the years, M. Harries, J. Hietanen, P. Benson, S. Thomas, R. Bevan, H. Ortega, and W. Dittrich. I participated in the collection of the data (i.e. in the laboratory) approximately half the time with the exception of monkeys F, B and the first third of the data from monkey D.
About two thirds of the way through the data collection for monkey D (and. for the subsequent monkeys), I wrote the software that was used for data collection, analysis and display. The computerization of data collection formalized and led to the final design of the testing. Without the data collection program the detailed examination of the time course of the neural responses
General Methods (2.50) would not have been possible. I also wrote the software and entered the brain slice
data that is used for the histological reconstruction for monkey J.
I have assisted Dave Perrett with 2 operations, and been responsible for maintaining anaesthesia in another one (and an observer in another). I have performed perfusions and helped with the sectioning of the brain for three monkeys.