SISTEMA NACIONAL DE ABASTECIMIENTO

/ % ■ c~a

track

food

reward

Figure 2.2. A side-on view of the rat running on the track showing the disposition of the data collection apparatus.

rotated to predetermined orientations. W hen recording a full set of 16

directions across 360 degrees, 8 orientations were used at 22,5 degree intervals (each orientation yielded two motion directions), which were recorded in random sequence. N o change was made to the surface of the track between orientations, and thus smell cues on the track surface remained in place.

Each recording lasted two minutes. T he rat would be picked up from the

holding platform and placed at the centre of the track. W hen the rat began to run towards one of the two ends, the recording was started. T he experimenter stood approximately 0.5m from the middle of the track and his position relative to the track was invariant of the track's orientation. Brief movements were made to

replenish the bait at the ends of the track, otherwise the experimenter stayed still. Typically, two recording sessions would be run consecutively, after which the rat would be returned to the holding platform and allowed to rest.

Virtually all the rats demonstrated a tendency to turn in one direction only, although the preferred direction of turn varied among animals. This caused the connecting wires running from the headstage gradually to become twisted and the rat had to be rotated in the opposite direction on the holding platform after each set of recordings. This was done immediately on returning the rat to the holding platform and the rat was allowed to recover on the platform for several minutes afterwards in order to alleviate any disorientation.

T he rat was also allowed to drink at regular periods. A complete set of 16 recordings generally took around one and a half hours, after which the rat was returned to its home cage

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w in d o w

radiator

s h e l v e s

carrying

old e q u ip m e n t

baited track on

turntable

h o ld in g box

a m p lifiers and c o m p u te r s

d o o r

mm

m #

H i

F i^ r e 7. An overhead view o f the laboratory. T h e track on which the rat ran was baited at both ends and mounted on a turntable. T h is was oriented at random in one o f eight directions (alternatives shown in light grey) spaced at 22.5° intervals. T h e laboratory contained a large number o f surrounding environmental cues which were constantly visible. A busy road lies outside the window, which provided a set o f auditory cues.

H isto lo g y

After it was decided that no further material could be collected from the animal (usually when the electrode had been lowered to a depth of 7,5-8mm), the rat was left without moving the electrode for a period (typically 1 to 2 weeks) and then culled with an intramuscular injection of 0.7ml/kg body weight of

Lethobarb (Duphar Vetinary, Southampton). W hen deep anaesthesia was obtained, the animal's abdomen was opened, the diaphragm cut in the midline and the mediastinum exposed by cutting along the left costo-chondral joint lines. A 19g needle was inserted into the left ventricle and held in place with a clamp. T he descending aorta was clamped and the inferior vena cava cut to allow exsanguination. T he rat was then perfused transcardially with 0.9% saline for 5 minutes and then with a 4% formaldehyde/phosphate buffer solution for 30 minutes.

After perfusion, the head was removed, the skull exposed and cut away with small rongeurs and the brain was dissected free. Care was taken to ensure that the electrodes were disturbed as little as possible in this process and were removed vertically. T he brain was then fixed in 4% formaldehyde for a week and slices were mounted to examine for electrode tracks. In all cases, there was sufficient gliosis or local tissue damage around the electrode track for it to be found and charted.

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Data A nalysis

D ata from the recording com puter w ere dow nloaded onto a netw ork of Sun w orkstations for analysis using custom -w ritten software. The initial phase consisted of elim inating noise, m ost com m only caused by myogenic

potentials from chewing, groom ing and scratching, from the recording.

As the four-channel waveform of each data capture could be view ed on­ screen, noise rem oval was easily perform ed by elim inating those data

sam ples w hose wave shape did not conform to a biphasic pattern on at least three of the four channels. Most data sets contained w aveform s which clearly clustered in this pattern. In a small n u m b er of cases and particularly w hen the spike was small, however, no reliable distinction could be m ade betw een an action potential and noise. In these instances there was a

continuum of wave shapes varying betw een those w hich m ay be regarded as a spike and those which were clearly noise. Such data sets were not used for further analysis and were discarded.

A t the sam e tim e as rem oving noise, the w aveform s w ere grouped into clusters corresponding to different cells. This could be done w ith ease as the w ave shapes of different cells w ere highly dissim ilar. The initial distinction w as usually m ade regard to the size of the w aveform on each of the four channels, b u t cells often differed in term s of the wave's shape as well. A lthough the w ave's size could vary, especially during a burst, the basic shape of the wave usually rem ained static u n d e r such circumstances.

M ultiple cells were recorded sim ultaneously in approxim ately half (29/61) of the recording sessions. The m axim um n u m b er of cells recorded at one tim e w as 5. Figure 2.4 shows the interface used to separate cells and an

exam ple of the w ave representations corresponding to spikes from four cells recorded sim ultaneously. On one occasion, five cells could be separated out from one set of data, although this w as unusual, w ith recordings of tw o or three sim ultaneous cells being m ore typical.

The position data returned by the video tracking system w as converted into velocity data after having been passed through a boxcar filter to sm ooth it. The position data w ould occasionally be lost at the acquisition stage w hen the lam p on the rat's head was occluded by the signal cables and these m issing d ata points were interpolated from the surrounding data. Once sm oothed and interpolated, the position data w ere used to calculate the velocity and direction of motion.

Initial analysis consisted of calculating the cell's autocorrelation histogram (ACH) and the phase of hippocam pal theta rhythm at which each spike fired. The phase was determ ined by fitting the EEG data from the

hippocam pus to a sequence of sinusoids. For each cycle, the data set was com pared to a set of half-period inverted sine w aves varying in frequency from 11.1 to 20.3Hz in steps of 0.4Flz. The goodness of fit was m easured by determ ining the sum of the squares of the differences betw een the data and each m odel. A num ber of successive start points (corresponding to the m axim um acceptable half period) w ere used. The start position and period

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Figure 2.4.1 he interface screens of the software program used to analyse the data. The top window carries the controls for the various analysis functions. The bottom left window shows the position associated with each spike in the sub-window on die top left. The other three sub-windows show the maximum minus the minimum voltage of the spike waveform on each of the four channels (the axes are labelled here for clarity). In this case, the data contained four separate cells. The bottom right window shows the first 100 spikes from one of the cells.

of the w ave m odel achieving the best goodness of fit was stored and used to calculate the starting point for the next cycle.

In many cases further analysis was performed. These techniques will be described in the later chapters where relevant.