goggles for vertical orientation exposure. The persistent, modest over-representation of the exposed orientation observed after long- term GR is supported by recordings using the subspace reverse- correlation-mapping technique [42]. They have shown that a substantially increased number of neurons in cat areas 17 and 18 responded optimally to the exposed orientation after long-term (.45 days) GR started at P21–28. This effect was robust, irrespective of the length of normal rearing intervals (0–50 days) before single-unit recording. Comparisons with previous studies There have been two hypotheses about the effect of visual experience on orientation plasticity. One is the selection hypothesis and the other the instruction hypothesis. In the former hypothesis, after single-orientation exposure, neurons innately selective for unexposed orientations just decrease their responses to the unexposed orientations without changes of preferred orientations. In the latter hypothesis, neurons selective for unexposed orientations change their preferred orientations towards the exposed orientation. Blakemore and Cooper [9] supported the instruction hypothesis, because they found preferred orientations of recorded units were strongly biased toward the exposed orientation in single-unit recording in cats that had experienced striped environment. Later, Stryker et al. [11] found in cats that had experienced parallel lines with opaque goggles [10] that single-orientation exposure changed a large portion of units nonselective or unresponsive, although responsive units preferring for the exposed orientation relatively increased. Particularly, they observed an orderly arrangement of selective units according to preferred orientation along the electrode tracks, as found in normal cats, but clustering of nonselective or unresponsive units frequently appeared. In our optical imaging on kittens exposed to a single orientation, stimulus-related intrinsic signals in response to unexposed orienta- tions were reduced in cortical domains originally selective for the unexposed orientations (Fig. 3b), and a proportion of pixels without orientation selectivity tended to increase [15], consistently with single-unit recording by Stryker et al. [11]. However, stimulus- related intrinsic signals in response to the exposed orientation tended to increase in these domains (Figs. 3c and d), resulting in the changes of orientation preference. As seen in Fig. 5C of Stryker et al. [11], it seems that nonselective/unresponsive units were rather distributed randomly, even if they might be weekly clustered along the track. In addition, this figure showed a wide range of a constant preferred orientation around the exposed horizontal orientation from 3–6 mm in the track distance. Therefore, this figure does not definitely support that neurons innately selective for unexposed orientations decrease selectivity or responsiveness without changes of their preferred orientations. In the single-unit recordings by Ohzawa et al. (SFN Abstract 2007-A-109015) on cats reared with our goggles for vertical orientation exposure, when they penetrated electrodes into the medial bank, they also recorded responsive units in these cats fewer than in normal cats, as in Stryker et al. [11]. Among the responsive units, most units were selective for the exposed orientation, but intriguingly the number of units selective for the orthogonal horizontal orientation was larger than that of units selective for oblique orientations (Fig. S3 online). The presence of the orthogonally oriented units may not be accounted for by the selection hypothesis alone, because horizontally oriented units should be most unlikely detected if the lack of experience decreases selectivity or responsiveness. On the contrary, in the instruction hypothesis, preferred orientations closer to the exposed orientation may be shifted more effectively towards the exposed orientation, and the orthogonally oriented units can possibly remain with their Cat Orientation Plasticity preferred orientations unchanged due to the largest separation in the preferred orientation from the exposed orientation, as we have previously postulated a possible mechanism for orientation modification (Fig. 8 in [15]). Differences of experienced patterns during single-orientation exposure may be worth noting. Stryker et al. [11] exposed kittens to stationary lines through their goggles. Carlson et al. [16] also presented stationary stripe patterns with various spatial frequencies to monocularly deprived infant monkeys. To examine the effect of exposure to stationary oriented stimuli, we have tried to rear 4 kittens chronically with spherical-lens-fitted goggles for exposure to a stationary stripe with a spatial frequency of about 0.5 and 0.15 c. p. d [24]. Although the exposed orientation was over-represented at the first optical imaging experiments after 2- or 3-week GR in 3 kittens, the under-representation of the exposed orientation occurred in the other kitten, in which the orthogonal orientation was over-represented (Fig. S4 online). Even in the kittens showing the over-representation of the exposed orientation, the layouts of orientation preferences were labile during prolonged GR. In 3 of the 4 kittens, the over-representation disappeared or changed to the under-representation after long-term GR. Such labile alteration of orientation maps is characteristic of exposure to a stationary oriented pattern. This is contrasted with the finding that orientation maps altered by exposure to a dynamic single orientation through cylindrical-lens-fitted goggles are consolidated preserving the modest over-representation of the exposed orientation (Fig. 7). It should be noted that the instability in orientation map alteration for stationary stripe pattern exposure was not due to the repeated optical imaging, because orientation maps altered by rearing with cylindrical-lens-fitted goggles changed gradually in each optical imaging, and were finally stabilized at the moderate over- representation of the exposed orientation. The fact that Carlson et al. [16] recorded units selective for the orthogonal orientation to the exposed orientation in the open eye may be such labile modification of orientation selectivity induced by the stationary stripe pattern exposure. The disappearance of the orientation selectivity modification for prolonged exposure to stationary oriented stimuli is suggested to have a weak impact on the structural modification of orientation maps. This may be consistent with the fact that Stryker et al. [11] observed gradual and progressive changes of the preferred orientation of selective units along the electrode track in kittens exposed to stationary lines, which are similar to those observed in normal cats. Conclusion Orientation plasticity appears to have two phases: First, the visual cortical circuit is rendered highly modifiable in orientation selectivity during a brief postnatal critical period for 6 weeks; and second, modified orientation selectivity consolidates during continuous long-term single-orientation exposure. The combined application of GR and optical imaging to not only cats but also rodents would be instrumental to further analyses of molecular and cellular mechanisms underlying this unique system phenom- enon, orientation plasticity. Materials and Methods Preparation of animals We used 58 kittens (normal: 23; goggle-reared: 35) obtained from the institutional colony. The procedures of surgery and optical imaging were approved by the Institutional Animal Research Committee at RIKEN (No. H13-B040), and performed in accordance with the ‘‘Guiding Principles for the Care and Use of Animals in the Field of Physiological Science’’ of the Japanese PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5380