Experimental Settings

2.1.1 Electrophysiological Specification

Extracellular recordings were obtained with tungsten microelectrodes (impedance 0.5-1.5 MΩ), insulated with parylene or epoxyte (FHC Inc. or Microprobe Inc).

Transdural penetrations were done with custom made guide tubes. Single-electrode recording was based on either of two systems with similar filtering and gain regimes but different spike discrimination methods. With the single electrode BAK system, raw signals were amplified in the range of 20000-60000 and filtered at 100Hz/5kHz (Low/High-Cut). The second system from Plexon Inc. provided a gain range of up to 32000 with a 150Hz/5kHz filtering. About half of the cells were isolated with a window discriminator (BAK Electronics Inc.). The other half of the cells were isolated under computer control with box discrimination, or a template matching algorithm provided by software packages from Plexon Inc.

2.1.2 Confirmation of Recording Sites

To access area MT we used a vertical approach with a recording chamber implanted on the skull over the superior temporal sulcus. Anatomical information from the lit-erature and knowledge from previous experiments (Treue and Maunsell, 1996, 1999) guided the exact stereotactic placement of the chamber of the left hemisphere at 8mm posterior to the AP-reference center and 13mm lateral from midline. At the beginning of the study several penetrations were done to ensure that electrodes were in appropriate parts of area MT in the superior temporal sulcus. The physiological criteria for cells to be attributed to area MT were (i) directional selectivity to uni-form motion with an indexed tuning strength (see below for details) that exceeded tuning strength for spiral motion components (expansion, contraction, rotation) (see below for details) and (ii) receptive fields that were confined to circumscribed regions within the visual field (Gattass and Gross, 1981; Maunsell and van Essen, 1983; Des-imone and Ungerleider, 1986; Logothetis, 1994; Graziano, Andersen, and Snowden, 1994). The experiment did not consider units with properties suggestive of other areas than area MT, including (i) RFs spanning large regions of a visual quadrant and including the fovea, (ii) with strong spiral motion tuning, (iii) stronger tuning to oriented bars than to moving RDPs, (iv) bimodal direction tuning curves (indicative of orientation tuning), and (v) eye-movement and fixation related responses.

As already noted we performed the main experiment only on neurons which could be assigned to area MT based on the physiological criteria mentioned above. This assignment can also be based on the spatial topography of the physiological criteria.

The following two paragraphs will briefly (i) compare the observed topography of RFs within and surrounding the superior temporal sulcus with that reported in the literature, and (ii) then provide the results of the anatomical reconstruction obtained after recording the main experiment.

Characteristics of RF Topography

The majority of neurons assigned to area MT had RFs in the lower right visual quadrant, while a small sample of cells had RF centers in the upper right quadrant close to the vertical meridian. The anatomical distribution of these RFs followed nicely the expected topography of area MT. The representation of the lower visual field has been described to be at the posterolateral part of area MT, while the upper visual field representation is reported to lay anteromedially (Gattass and Gross, 1981; van Essen and Maunsell, 1983; Desimone and Ungerleider, 1986). This is in general agreement with the observed distribution of RF centers. Furthermore, neuronal response properties surrounding the area where most neurons were sampled for the experiment matched reports from the literature: We found large neuronal RFs crossing the vertical meridian antero-lateral to our recording site (Desimone and Ungerleider, 1986). These neurons could be located in area MST, which lies

lateral-to-anterolateral to MT and whose neurons have larger RFs that typically include the fovea and are responsive to more complex optical flow patterns (’spiral motion’, see Graziano, Andersen, and Snowden 1994) such as expansion and rotation (Desimone and Ungerleider, 1986). We tested neurons with such spiral motion at two different speeds and found RFs tuned to spiral motion (expansion, contraction, or rotation) as expected for area MST antero-laterally to our main recording site.

Furthermore, we recorded neurons with properties expected for area V4/V4t at the posterior border and posterior-lateral to our main recording site. At these sites the majority of neurons were orientation selective, few were direction selective to a manual moving bar, and even less were direction tuned to motion of a random dot pattern. Both, the proportion of tuning and the anatomical location relative to area MT match descriptions of area V4 and V4t in the macaque cortex (Desimone and Ungerleider, 1986; Logothetis, 1994). It should be noted that the described neuronal properties were predominantly arrived at by manually sweeping a bar and/or RDP of different orientation/direction of motion over the visual field and are thus primarily subjective reports. No main experiment was run on cells not classified to lie within area MT (which can also be seen in the list of recorded cells in appendix B, p. 127).

Anatomical reconstruction of recording sites.

Recording sites were histologically reconstructed after the experiment based on ref-erence coagulation lesions and lesions with electrodes with blunt tips applied im-mediately prior to perfusion. After initial Ketanest anaesthesia and an overdose of pentobarbital the monkey was perfused intracardially with 5L heparinized, physio-logical saline (0.9%) followed by 5L phosphate buffer (PB) solution (pH 7.4) with 4%

paraformaldehyde. After three months in paraformaldehyde, the brain was stereo-tactically blocked to contain the full extent of the recording site with four coagula-tion sites that demarcated the borders of the recording region and including three sites with marker tracks to reconstruct the chamber site. After refixation within 4% paraformaldehyde PB solution the brain was cryoprotected with a 20% sucrose solution and frozen to -60^o celsius. Brain blocks were cut in 40µm sections at -20^o celsius with a Reichart-Jung Microtome (2800 Frigocut E) and mounted on gela-tine coated slides. We stained sections for Nissl substance with cresyl violet. The positions of recording tracks were reconstructed based on the spatial reference from coagulation sites, stereotactic coordinates, and anatomical landmarks. Figure 2.1 illustrates selected sections along the anterior-posterior (AP) axis (cf. figure legend for details). Our main recording site could be reconstructed based on the refer-ence coagulation lesions and landmark-pin lesions (cf. section 99 in figure 2.1). It was found to correspond well to the region where area MT was expected from the anatomical literature (cf. fig. 2.2, p. 49). It should be noted, that the superior temporal sulcus (STS) of the left hemisphere of the studied brain appears to have a

A-P -11.1 99 MTIPS - intraparietal sulcus STS - superior temp. sulcus V4tMST STS

IPS

main (A-P) recording site

section ID mm: A-P mm: from main site recording site

59

73

91 114128

144

617691118133148163 -7.7-8.9-10.1-11.3-12.5-13.7-14.9-16.3 -2.2-3.4-1.00.21.642.844.045.24

106

Figure 2.1: Summary of histological reconstruction. Spatial axis (top rows) from anterior (left) to posterior (right) for coronal sections of the brain covers a range of levels that could be targeted by electrodes from within our recording chamber.

Our main recording site was found to lie at about -11mm posterior from the AP-Zero level (section number 99), which is shown in the middle axis. The bottom axis plots distance in mm relative to the identified main recording site to facilitate comparisons between sections. Selected sections allow to track the superior temporal sulcus, which becomes particular evident in sections 91, 99 and 114. Labels in the subsection of section 99 are derived from the literature (Ungerleider and Desimone, 1986; Gattass et al., 1997). The black rectangular region corrresponds to the medial-to-lateral and dorsal-to-ventral extent in which all neurons were recorded that went into the analysis of the attentional mapping experiment.

IPS - intraparietal sulcus to right hemisphere slices from literature (see left)

Figure 2.2: Confirmation of our main recording site with functionally characterized anatomical drawings from two studies concerned with cortical connectivity (and localisation) of area MT (Ungerleider and Desimone, 1986; Gattass et al., 1997). All drawings come from a scientific group which frequently determined the anatomical location of area MT based on a particular strong myelination and a high proportion of neurons selectively tuned to motion direction (Desimone and Ungerleider, 1986).

A: Coronal section selected from case one of Ungerleider and Desimone (1986) with the outline of the brain indicating the AP level at which the section had been taken.

The selected subsection had to be rotated by -65^oto fit well to the anatomical form of the superior temporal sulcus in our sections (shown inC).B: A further example from a more recent study by Gattass et al. 1997. The subsection covering the superior temporal sulcus had to be rotated by -75^oto match with the superior temporal sulcus of the cresyl violet section in C. C: Subsection selected from section 99 at AP -11.2 and flipped vertically to ease comparison with the right hemisphere drawings in A and B (cf. figure 2.1). Comparison of the outline of the superior temporal sulcus of this section with those in A and B shows a close match after rotation of -65 to -75^o of the cases shown in A and B . The need for rotation reflects the finding that the STS in the cresyl violet stained section is far less extensive in the lateral-to-medial axis than would have been expected. This confirms our recording experience before histological sectioning. Labels in A and B are those from the original drawings.

Rotated subsections and brain views were slightly modified versions of the original drawings.

peculiar angle and arrangement (cf. fig. 2.2,C) . Compared to anatomical descrip-tions and drawings of the STS in the literature, the cresyl violet stained STS extends

from dorsal to ventro-medial in the brain and forms a particular strong medial-to-dorsal curvature when compared to reports in the literature (cf. fig. 2.2, and for an overview of anatomical drawings of comparable brain sections cf. Ungerleider and Desimone 1986; Boussaoud, Ungerleider, and Desimone 1990; Gattass et al. 1997;

Bisley and Pasternak 2000). The consequence of this peculiarity for recording from area MT with electrodes advanced vertically is a restricted lateral-to-medial extent of the region covering area MT, which matches the experience of the experimenter in recovering direction tuned neurons and functionally deleinating area MT.

2.1.3 Stimulus Presentation & Behavioral Control

Visual stimuli were presented with a custom computer program running on an Apple Macintosh G4 Computer. The same software monitored eye position and behavioral responses of the animal, and recorded the behavioral and neuronal data. During the experiment the monkey was seated in a primate chair 57cm in front of a monitor with a resolution of 32/33 pixel per degree allowing 960/1280 and 1024/1600 pixel in the vertical and horizontal dimension. The monitor covered a visual field region of about 32 vertical and 39 horizontal degrees in visual angle. Monitors were run with a refresh rate of 75/76Hz, providing a duration of 13.33/13.16msec for each frame of the stimulus. Motion stimuli consisted of random dot patterns (RDPs) presented within a non-moving circular aperture with motion induced by dot displacement within the aperture. Dots moving outside of the aperture were replaced by dots at random positions at the opposite side of the RDP aperture. At the beginning of each experimental session dot positions of the movie frames were generated. Once generated RDPs of the movie frames remained constant throughout the experimen-tal session. RDPs contained 10 dots per square degree of visual angle with indivdual dots extending 2 square pixel. Dots were always white presented on a black monitor background which had a luminance of 0.7 candela during experimental recording.

Luminance of the dots was 46.9 candela for all stimuli used in the current project with the exception of the behaviorally relevant target and distractor stimuli of the mapping experiment (see below) which had a luminance of 18.6 candela. These lu-minance values are averages from fluctuations due to slight variations in background luminance across the duration of the experiment.