Colour Vision Threshold Figure 1. The relative number of colours (n colours) that a dichromat (dashed black line) and a trichromat (grey solid line) can discriminate in dim light. Due to the high noise levels in dim light intensities a dichromat should theoretically discriminate more colours than a trichromat. Adapted from [6]. doi:10.1371/journal.pone.0003711.g001 the optical properties of the eye and determined the optical sensitivity of the horse eye. This allows us to compare the colour vision thresholds both with regards to absolute light intensity and to the optical sensitivity of the eyes. Results We trained three horses in a dual choice experiment with one positive rewarded stimulus and one negative unrewarded stimulus (Fig. 2). Chap and Rosett were trained to blue as the positive stimulus and Rex was trained to green as positive stimulus. When the horses had reached a specified learning criterion of at least 70% correct choices for a number of days, the light intensity was lowered. All three horses chose correctly at significantly high frequencies, for all intensities higher than or equal to 1.2 cd/m2 (Fig. 3a), which is comparable to light intensities at sunset. At lower light intensities, Rex totally lost the motivation for the experiment, and after that 2 chose randomly even in tests in brighter light (15 cd/m ). He was therefore excluded from further testing and his data are not presented in the result figure. Rosett lost interest in comparing the two stimuli at a light intensity of 0.02 cd/m2 and developed a preference for the right side. After this point, she preferred the right side and performed poorly, even when the light intensity was increased to the highest intensity used in this experiment. The last three days she chose the left side in only 6 out of 30 choices. After this point she was excluded from further testing. Chap still chose the positive blue stimulus in a total 31 out of 40 presentations, at 0.02 cd/m2 (binominal tests, P,0.05, note that only the last 30 choices are presented in Figure 3). However, at the next lower intensity, 0.007 cd/m2, he failed to choose correctly and made only 30 correct choices in 60 presentations. Directly after the last day of tests in the dimmest light intensity, Chap was tested in bright light again (15 cd/m2) to determine whether he had lost motivation. In this control, he confidently chose the positive blue stimulus nine out of ten times (P,0.05), ensuring that his motivation still was high. Chap’s results thus suggest that at least some horses can discriminate colours at a light intensity 2 similar to moonlight intensities (0.02 cd/m ). The results in Figure 3a include choices starting with the second day the horses reached the learning criterion of at least 70% correct choices. All choices were examined both with respect to calculated quantum catches for the long wavelength-sensitive cone and the rods (see methods) to assure that no brightness cues had Figure 2. The dual choice apparatus wall. The door presenting the rewarding stimuli is unlocked and enables the horse to reach the pieces of carrots behind the door. doi:10.1371/journal.pone.0003711.g002 guided the horse in its choices. Of Chap’s correct choices, 52% were made when the positive stimulus was darker than the negative stimulus with regard to the quantum catches in the long- wavelength-sensitive cone. The same number was found for the rods. Thus, Chap did not use brightness in his choices. Six human subjects were tested with the same stimuli and experimental apparatus as a comparison to the horse result. Only twenty presentations at the intensities around threshold were performed. At 0.02 cd/m2, the results varied from 70–95% correct choices and choices by all but one subject (RF, 70%) were significantly different from chance (binominal tests, P,0.05; Fig. 3b). At 0.007 cd/m2 the choice frequency varied between 35 and 70% for five of the six human subjects. Thus, all five subjects failed to discriminate the colours at this dim light intensity. However, the sixth human subject (AB) chose correctly in 80% of the twenty choices (P,0.05) at 0.007 cd/m2 and did not fail to choose correctly 2 until the light intensity was lowered to 0.003 cd/m . Optical sensitivity Horse eye dimensions were obtained by sections through freshly frozen eyes (Fig. 4a). Assuming a well-focused image on the retina, a posterior nodal distance (here also called focal length) of 25 mm was found for the horse (Fig. 4a). The cones of a different horse eye were examined under the light microscope. They measured 1 mm in diameter and the outer segments were 4 mm long (Fig 4b), which is similar to the cone dimensions measured in the bovine retina [17]. The measurements taken from the horse eyes are summarized in Table 1 together with the human data that were obtained from the literature [18–20]. The optical sensitivity that we determined for cone vision of horses and humans corresponds to the ability of a cone to capture photons from an extended source of white light through the opened pupil in light intensities corresponding to moonlight (See methods section, Eqn. 2). This equals the sensitivity of the eyes without any signal summation, at the lowest light intensity at which colour discrimination was possible in our experiments. Even though the horse eye is very large and has a relatively short focal length the calculated sensitivity for cone vision is similar to that of humans, due to the short outer cone segments in the horse retina. Using obtained data and Eqn. 2, the eye of the horse yields a sensitivity of 0.1 mm2sr when the reflecting tapetum lucidum in the back of the horse retina is taken into account. Due PLoS ONE | www.plosone.org 2 November 2008 | Volume 3 | Issue 11 | e3711