frequency, i.e. horses did not choose the brighter or darker stimulus but the correct colour. The dual choice apparatus (Fig. 2) was described in Roth et al. [16] and was similar to the one used by Macuda and Timney [15] and Geisbauer et al. [13]. In short, the apparatus consisted of a light grey wall with two lockable doors on either side of a divider, to force the horse to make a choice. Stimuli were presented on both doors. The door with the negative stimulus was locked and the door presenting the positive stimulus was unlocked such that the horse could reach pieces of carrots as reward. Behavioural experimental procedure The experiment took place in a windowless barn between October 2007 and January 2008. Horses were trained in several steps to associate a colour with a reward of carrots. The horses were first made familiar with the experimental apparatus by leading them to the open apparatus doors by the halter, rewarding them with carrots. In the next step, they were taught to open the doors and reach for the carrot pieces by themselves. At this stage both doors showed positive stimuli (blue for Chap and Rosett, and green for Rex). Depending on the performance of the horses it took 2 to 4 days before we started to release the horses three meters in front of the apparatus and introduced them to the training sessions with both negative and positive stimuli. Normally they were presented with 20 combinations each day, five days a week. However, at the three lowest light intensities, we allowed the horses to dark-adapt for five to fifteen minutes prior to testing and then, to avoid loss of motivation, we only presented ten stimuli per day. For initial training we used the fluorescent tube that was present in the barn (50 cd/m2) as illumination source. A learning criterion was set to 75% correct choices for 20 combinations, which is significant according to the binominal test (P,0.05). When the horses had reached the learning criterion during three days, we lowered the light intensity and started using two halogen spot lights 2 (15 cd/m ). Lower light intensities were achieved by adding neutral density filters (0.6 Neutral density filters, 210 Lee filters) in front of the halogen spots, and the achieved intensities were measured using a radiometer (International light IL 1700). When the horses reached the learning criterion at one intensity level for a number of days we dimmed the light the following day. When a horse was tested at 0.08 cd/m2, at least five minutes were allowed for dark adaptation before testing started. When we tested the horses at 0.02 cd/m2 or lower light levels, we allowed them to 2 dark-adapt five minutes at 0.08 cd/m . The horses were then presented with five stimulus combinations to test adaptation before we lowered the intensity further to 0.02 cd/m2 and waited five more minutes. By then even the human experimenters were well dark-adapted and the horses were presented ten stimulus combinations during the session. The learning criterion was 70% during three days when presenting only ten combinations. Six human subjects (including the authors) were asked, and gave their written consent, to perform the very same experiment; three women, which included both the experimenters, and three males. Experiments were conducted according to Swedish ethical regulations and due to the uncomplicated nature of the experiments no special permission was required. The human subjects were tested with 20 stimulus combinations at two light levels, 0.02 cd/m2 and 0.007 cd/m2. One of the female experimenters (AB) was also tested at 0.003 cd/m2. References Colour Vision Threshold Optical sensitivity of the eye The eyes of one twelve years old mare were frozen and sectioned sagitally in a cryostat. Pictures were taken every 0.5 mm to obtain the largest eye dimensions (Fig. 4a). For calculations of the posterior nodal distance (from nodal point to retina, here also called focal length), we applied the Gullstrand model (for calculations see [2]). As refractive index of the aqueous humour and the vitreous humour a value of 1.335 was used, which is similar to values previously used for horses and other mammals [25,26]. Since the horse has a lens with different concentric zones of different refractive indices [27] the effective lens index will be higher if treated as one homogenous lens. We assume that the optical system of the horse is well focused. Therefore, to achieve a calculated focused image on the retina a total refractive index of 1.507 was used for the lens, which is close to the index used in Nicolas’ study (1.49) [28] but much higher than the index used in Sivak and Allen’s study (1.43) [26]. In our study, assuming a well- focused image on the retina, a posterior nodal distance of 25 mm for the horse was found (Fig. 4a). Pieces of retina from a one year old half blood mare were fixated 2% paraformaldehyde, 2% glutaraldehyde with cacodylate buffer over night followed by post fixation in 1% osmium tetroxid, dehydration and embedding in Epon. Semithin sections dyed with methylene blue were used for light microscopy. From the histological sections we obtained the diameter d and the length l of the cone outer segments. The pupil diameter A of the horse was determined from photographs of living horses at a light intensity corresponding to moonlight. To calculate the optical sensitivity Sw for white light for horses we used the obtained measurements on pupil size, focal length (posterior nodal distance) and cone dimensions (Eqn. 2) [18,20]. The opical sensitivity Sw gives the number of photons absorbed by a photoreceptor when looking at an extended source of white light (Eqn 2): Sw~ðp=4Þ2A2ðd=f Þ2ðkl=ð2:3zklÞÞ 2 ð Þ where k is the absorption coefficient of the receptor, and A is the pupil diameter. A typical vertebrate photoreceptor absorption coefficient k of 0.035 was taken from Warrant and Nilsson [18]. Acknowledgments We are deeply grateful to the horse owners, Gerda och Magnus Wissler, and the 4H stable at O¨stra Torn, Lund, that allowed us to perform our experiments every morning during three months. We also would like to thank Eva Landgren, Rita Walle´n and Stefan Sydoff for technical help and Jan Skidell and colleagues at the Helsingborg’s Animal Hospital who kindly helped us to get hold of the horse eyes. We specially thank the extra human volunteers, Andreas Olsson, Rikard Frederiksen, and Olle Lind, who behaved very well during the experiment. We are grateful for financial support from the Crafoord Foundation, Stockholm, and the Swedish Research Council (VR). Author Contributions Conceived and designed the experiments: LSVR AB AK. Performed the experiments: LSVR AB. Analyzed the data: LSVR. Wrote the paper: LSVR AK. 1. Warrant EJ (1999) Seeing better at night: Life style, eye design and the optimum strategy of spatial and temporal summation. Vision Res 39: 1611–1630. 2. Land MF, Nilsson D-E (2002) Animal eyes. Oxford: Oxford University Press. 3. Lythgoe JN (1979) The ecology of vision. Oxford: Clarendon Press. PLoS ONE | www.plosone.org 5 November 2008 | Volume 3 | Issue 11 | e3711