FUENTE: RESULTADOS DE LA TOMA DE MUESTRA ECOLAB.SRL (DICIEMBRE DE 2015 Y ENERO DE 2017) ELABORADO: TESISTA.

All procedures were conducted under Home Office Project licence numbers PPL-60/00353 and PPL-60/01453 and Personal licence number PIL- 60/02475.

Stewtaxic implant

The implant contained two stainless steel David Kopf wells and two restraining head bai' holders. The position, in sterotaxic co-ordinates of the wells typically were 8-15mm anterior of the interaural line, and 12-14 mm lateral of the midline. Once the precise positional measurements had been decided, a to scale diagram (using graph paper marked out with a 1mm grid) was made, indicating where the restraining bars would be positioned. The elements of the implant were then placed in position on a glass sheet and joined using dental acrylic.

Pre-operation proceclure

The subject was put into the travelling cage and given a .sedating injection of Ketamine (0.5-1.0 ml of Vetelar). Liquid paraffin oil (Vaseline in liquid form) was dropped into the eyes to protect and prevent drying. The head was also shaved then swabbed down with alcohol and tincture of iodine. Atropine (1 ml of 600 micro-gram/ml) was injected to reduce secretions. A general purpose wide spectrum anti-biotic (1 ml ampicilin) was given as a precautionary measure. An intravenous cannula was inserted allowing direct administration of the anaesthetic

(Sagatal).

The operation

The monkey was placed in a stereotaxic frame and rested on a diathermy base plate. A breathing counter and rectal thermometer probe were put in position and linked to a heating plate to maintain body temperature. The operation involved one incision, running along the midline of the skull from just above the eye ridges to the back of the crown. The skin and underlying membranes were reflected away from the skull and held away using the haemostats. Any local bleeding was quarterised using a diathermy needle.

General Methods (2.40) Drilling of the skull was done with constant irrigation to keep the bone

temperature down. [If the skull tissue rises above 50 degrees centigrade, then it dies.] Two wells were drilled out as circular plugs. The implant was then positioned to allow for the localization of the T bars and screw placement. The stainless steel T bars and screws were inserted into small slots drilled in the skull. Dental acrylic was then placed around the implant to secure it in place. After allowing time for recovery from the operation (2-4 days), the subject was retrained until pre-operative performance was reached. There after the dura underneath the well caps was swabbed clean every 2-3 days.

RECORDING

Behavioural task

Before recording began, the subjects were retrained to discriminate between the red or green colour of an LED light (see Training section above). The LED was typically situated level with the monkey's line of sight on a blank white wall at a distance of 4 m, but could also be placed +/- 15 degrees to the left or right or +/- 10 degrees above or below this central position. Head movement of the subjects in the primate chair was restrained by passing restraining rods through the restiaining tubes in the implant. Recording sessions lasted for periods of 2-4 hours. The LED and test visual stimuli were presented from behind a large aperture (6.5 cm diameter) electromechanical shutter (Compur) or an alternative (20 cm square) liquid crystal shutter (Screen Print Technology Ltd.). Both types of shutter had rise times of < 15 ms. When open the shutter allowed the monkey to view only the central 30 (Compur) or 100 (liquid crystal shutter) degrees of visual space. On each trial the shutter was opened under computer control (after a 0.5 s signal tone) to reveal the stimulus and remained open for a period of 1 s. The LED light became visible at the time of shutter opening (stimulus presentation) and was randomly red or green on different trials. The monkey's

task was to lick for a fruit Juice reward when the colour of the LED was green and to refrain from licking when the LED was red to avoid delivery of a weak salt solution. The LED colour was changed in a pseudo-random order under computer control. Subjects were deprived of water for periods of up to twenty-four hours before training and recording sessions to motivate task performance.

Although the subjects did not have to fixate the LED throughout the trial period, the monkeys attended to the LED at the beginning of trials in order to lick several times for multiple juice rewards in the 1.0 s trial period. Once they had judged the colour of the LED they were then free to move their eyes. The 2D test stimuli were projected onto the wall on which the LED was located, 3D test stimuli were presented in front, below or to either side of the LED. The monkeys performed the task at a high level of accuracy during the recording sessions independent of simultaneously presented test stimuli. On trials where the monkey licked for fruit juice, normally two and occasionally three licks were completed in the 1 second period available.

Stimulus presentation

Various types of visual stimuli were presented while the monkeys performed the behavioural task (see above). Trials were initiated by the experimenter but thereafter under computer control and consisted of a 0.5 second warning tone, followed by the shutter opening for 1 second to reveal the stimulus. Slides, video disc frames or real 3-D objects were presented either to the side of the LED or projected to cover the LED at each trial. Presentations to the side of the LED were within 2 degrees of the LED. Presentation was for 1 second after a 0.5 s warning tone. The stimuli were either real 3-D static presentations of an experimenter (or control object), or 2-D slides, or still frames on a video disc. Each stimulus was presented 5 or more times in computer controlled pseudo­ random order. In addition, a 'no stimulus' condition was also used, where only the

General Methods (2.42)

LED and wall could be seen. The inter-trial interval was varied between 0.5 and 5 seconds.

Recording techniques

For each recording session, topical anaesthetic (lignocaine hydrochloride, Xylocaine 40 mg/ml) was applied to the dura and a David Kopf micro-positioner fixed to the recording well. A trans-dural guide tube was inserted 3-5 mm through the dura and a tungsten in glass microelectrode (Merrill and Ainsworth 1972) advanced with a hydraulic micro-drive to the temporal cortex. Position of the cannula was varied using a micro-positioner (David Kopf Instruments) adapted to permit up to 20 degrees of tilt. The target area for recording was area STPa in the anterior part of the upper bank of the STS (which includes areas TPO, PGa of Seltzer and Pandya 1978). The electrode was advanced using a micro-drive (David Kopf 607W) and the depth of each cell recorded noted.

The electrical signals were amplified (Neurolog NL104) and then filtered with a 50 Hz notch filter together with low (300 Hz) and high (20 KHz) pass filters (Neurolog NL125). Spikes from individual cells were discriminated using a threshold voltage window (Modified Digitimer DM130), set for each cell tested and by the visual appearance of the spike on a fast time base oscilloscope. The spikes were converted to TTL signals and these were used to form peri-stimulus time histograms (PSTHs) with 250 bins usually of size 5.2 ms but 5.0 or 4.8 ms in some recordings. The PSTHs had a 1 second post-stimulus period and either 200, 250 or 300 ms pre-stimulus sample periods, depending on the bin size. Data were stored using CED 1401 (Cambridge Electionic Design) and custom software.

Measurement of cell responses

The time at which the shutter became transparent or was fully open was recorded. Subsequent analysis was linked to this, the true stimulus onset time. Neuronal firing rates were measured using standard techniques for a period of 250

ms beginning 100 ms after stimulus presentation. This analysis period was selected because most cells in the STS have latencies of 100-150 ms and because few eye movements occur in this period (see below). A 500 ms sample period was occasionally used for cells with small or late responses. These data were analysed on-line by a microcomputer (AT compatible PC (Hyundai, Dell)).

Eye movement recording

Horizontal and vertical eye movements were monitored using an infra-red corneal reflection system (ACS, modified to allow recording of both signals from one eye) to determine whether any response differences reflected differential patterns of fixation. Differentiating the eye position information allowed assessment of whether speed or velocity of eye movements affected response magnitudes. The output was sampled at the same rate as single cell signals and stored with each trial on the CED 1401 with 8 bit accuracy.

It has been reported that STP cells show differential responses depending on eye movements (Colby and Miller 1986; see Colby 1991 for an example of such a cell). As the effects of eye movements on cell responses were not expressly tested, and eye position was not monitored for all the cells tested, the possibility that some of the responses observed were influenced by eye movements cannot be excluded. However the effects of eye movements are likely to be small, as only 20% (18/90) of cells in STP were found to be related to eye movements. Of these 18 cells, 9 were visual (responding to the onset of the target stimulus) and 4 were visuomotor (firing from target stimulus onset until the saccade was made), while only 5 cells were related exclusively to the saccade (C.L. Colby, personal communication). Thus, only 6% of cells in the present study would be expected to be related solely to saccadic movements and not the stimulus. Further, it is not deal' if the cells reported by Colby and colleagues were recorded from the posterior (STPp) or anterior portion (STPa) of STP. Given the large proportion of eye movement related responses in MST and that the input to STPa from MST is

General Methods (2.44) via STPp, one would expect there to be more eye movement related responses in

STPp than STPa. For several other reasons given below it is unlikely that the cell responses reported in this thesis reflect differential eye movements.

The experiments described here used a task in which the subject was not required to maintain steady fixation throughout the whole trial period. The monkey performed the LED colour discrimination task with a high level of accuracy, and more importantly obtained multiple rewards by repeatedly licking. The short period during which reward was available meant that the monkey had to be attending the LED from the trial onset in order to lick more than once. Examination of eye position records showed that this was indeed the case, and that fixation was only broken some 400 ms after stimulus onset (see Figures 3.2, 5.2, 6.6, and 7.4). The analysis of the response magnitudes was based on spike counts between 100 and 350 ms (post-stimulus) and therefore during the period of maintained fixation. More than 90% of the cells responding to pursuit eye movements in MT and MST had the eye movement related response starting after

the onset of the pursuit eye movements (Newsome et al. 1988). This would suggest eye motion 'contamination' of response less likely in the analysis period.

For the few trials where fixation was broken before 350 ms post-stimulus no clear change in either the response latency or response magnitude was observed (see Figures 3.2, 5.2, 6.6, 7.4). Furthermore, as can tdso be seen in these Figures, even when eye movements were comparable between two stimuli, only one (the preferred) stimulus would give a clear response.

Studies of MST neurons that responded with directional selectivity during pursuit eye movements (where there was no stimulus motion on the retina) also show clear directional responses to retinal motion when the animal was fixating throughout the trial period (stimulus motion but no eye motion). More important the preferred directions obtained under pursuit and fixation tasks were coincident (Erickson and Thier 1991; Komatsu and Wurtz 1988a; Thier and Erickson 1992). Even if the directional selectivity observed in STPa was reflected cell tuning for

direction of pursuit eye movements the findings in MST suggest that the directional selectivity would be the same under stimulus motion with maintained fixation.

It is also relevant to note that many cells recorded in MST do not respond to self-induced retinal motion produced by eye movements (Erickson and Thier 1991). Cells in STP receive a direct input from MST, so they may reflect other response characteristics of MST cells. Indeed it has been argued that there is a trend for cell responses to become less sensitive to self-induced retinal motion at higher in the motion processing hierarchy (Erickson and Thier 1991). It has been shown that STPa cells responsive to motion (and similar to those reported here) tu'e not responsive to equivalent self-induced motion (Hietanen and Perrett 1992). If this is the case then the effects of eye movements on measurements of preferred directions in STP is likely to be small.

It is also relevant that the receptive field size of cells in STPa is very large and typically covering the fovea (Bruce et al. 1981). Similar selectivity for static stimuli at different positions within the huge receptive fields has been reported for cells in infero-temporal cortex and STPa (Desimone et al 1984; Gross 1992, Tovee and Roils 1993). Although the receptive fields for all cells were not expressly checked, of those cells which receptive fields were mapped, similar positional invaiiance was observed within the STPa. Selectivity for static and moving stimuli was maintained to eccentricities of 10-20 degrees either side of the fovea (Perrett et al. 1989b; unpublished studies Perrett, Harries and Oram). Thus with large receptive fields and positional invariance, difference in eye positions (+/-20 degrees) is unlikely to have effected response selectivity in this study.

In summai'y these arguments indicate that it is unlikely that the observed selectivity for biological motion stimuli was due to eye movements. First, the available evidence suggests that cell responses in STPa are generally unrelated to eye movements. Second, given the size of STPa cell receptive fields and

General Methods (2.46) positional invariance, any small variation in eye position would not account for

differential responses. Third, direct measurements of eye position indicated that differences in eye position/velocity across stimulus conditions were indeed small. Finally and more importantly there was no consistent relation between eye position/velocity and neural responses reported here.

Localization o f recording session.

Frontal and lateral X-radiographs were taken of the position of microelectrodes at the end of each recording session.