Physiological Society. We performed chronic optical imaging repeatedly twice or three times for 12 among 35 goggle-reared kittens to see the recovery or consolidation of altered orientation maps, and for 15 among 23 normally reared kittens to see the age- dependent changes of orientation representation bias. We made efforts to minimize the animals’ suffering and to reduce the number of animals examined as much as possible. We refrained from performing optical imaging experiments within 7 days after surgery, and from repeating optical imaging on identical animals at an interval shorter than 7 days. After experiments, we sacrificed animals injecting the overdose of pentobarbital (50–100 mg/kg, i.v.). Goggles for single-orientation exposure We used goggles fitted with planoconvex acrylic cylindrical lenses (lens thickness, 10.0 mm; lens aperture diameter, 15.0 mm; lens power, +67 D), through which the animals were able to see elongated images of their environments [24]. We used two types of goggles: v- and h-goggles, which elongated visual images vertically and horizontally, respectively. Surgical procedure Surgery was conducted according the procedure described in our previous papers [14,15]. Initial anesthesia was induced using ketamine hydrochloride (5.0 mg/kg, i.m.) following sedation with medetomidine hydrochloride (0.1 mg/kg, i.m.). The animals were fixed on a stereotaxic apparatus and were artificially ventilated with a 60:40% mixture of N2O and O2 containing 0.5–1.0% isoflurane. Heart rate, end-tidal CO2 concentration, and rectal temperature were continuously monitored during surgery. A metal head holder for fixing the goggles and a metal chamber for optical imaging were cemented on the animal’s skull using dental resin, and the skull and dura mater covering the recording area of the lateral gyrus were removed. The cranial window (17 mm612 mm) was positioned approximately from P5 to A12, spanning the midline. Next, the chamber was filled with 2% agar and sealed with a polyvinylidene chloride thin film and a plastic plate. Finally, the frame of the goggles was fixed to a head holder and the position of the goggles was calibrated so that the cylindrical lenses covered the visual field as widely as possible. Optical imaging Animals were anesthetized as in surgery and paralyzed with pancuronium bromide (0.1 mg/kg/h). They were artificially ventilated. Contact lenses with appropriate curvatures were used to prevent the drying of eyes. The cortex was illuminated with a 700-nm wavelength light. The focal plane was adjusted to 500 mm below the cortical surface using a tandem-lens macroscope arrangement [18]. Intrinsic optical signals were measured while the animals were exposed to visual stimuli displayed on a 20-inch CRT monitor placed 30 cm in front of the animal. Images were obtained with a CCD video camera, and digitized and stored using CAPOS (3206240 pixels) [43] or Imager 3001 (7446480 pixels) (Optical imaging Inc. New York). For each stimulus presentation, the intrinsic signal was recorded for 1.0 s before and 5.0 s after the stimulus onset. A blank stimulus was presented for 15 s between successive captures of intrinsic signals. Each visual stimulus was presented once in a pseudorandom sequence in a single trial of recordings. Twenty-six to 30 trials were collected in each recording session. As visual stimuli, we used full-screen square- wave gratings, which drifted in two directions at six equally spaced orientations (interval, 30u). To functionally identify area 17, we used gratings of a spatial frequency of 0.5 c.p.d., which is optimal for area 17 neurons [20,44,45]. The temporal frequency of the Cat Orientation Plasticity gratings was fixed at 2.0 Hz. The optical imaging in one session was completed within 5 hours. Cortical infusion of D-AP5 During 1-week GR, D-AP5 was continuously infused into the visual cortex of 3 kittens using an osmotic minipump system. The method has been described in detail elsewhere [46]. In brief, D- AP5 (Tocris Cookson Inc.) was prepared, using sonication, as a 50- mM solution with sterilized distilled water or PBS (pH 7.4). We made a cannula of 33 G hypodermic needle, connected to an osmotic minipump that contained D-AP5 solution, and inserted the cannula stereotaxically at a depth of 1.0 mm around AP0, aiming at the border of areas 17 and 18. They were fixed on the skull with dental cement. The infusion rate was 1 ml/hr for a duration of 1 week. In two kittens, the right visual cortex was infused with saline as control. In optical imaging, we used stimulus gratings of a 0.15 c.p.d. spatial frequency for activating both areas 17 and 18 [20,45], because D-AP5 spread widely over the lateral gyrus including the two areas. Analysis of optical imaging data The analysis methods that we used were described in a previous paper [15]. It is noteworthy to explain the methods in some detail here to show that observed map changes are attributable to biological changes rather than artificial changes originating from our analysis methods. One trial of optical imaging was composed of six frames (duration of each frame, 1 s). To extract stimulus-related intrinsic signals, we subtracted signals recorded in the first frame (without stimulus presentation) from those signals recorded in succeeding frames with stimulus presentations. Then, we averaged the subtracted signals over the 4th to 6th frames for each trial. Next, we applied the generalized indicator function method to these averaged signals [22], which efficiently excluded noisy signals originating from volume and oxygenation changes in thick blood vessels and spatially slowly varying fluctuations of signals inherent in the recorded intrinsic signals. It should be noted that the image data processing based on the generalized indicator function method underestimates the effects of over-representation of exposed orientation induced by single-orientation exposure, because the data processing method eliminates spatially slowly varying point-spread components of intrinsic signal [21], which may partially contain responses to the exposed orientation. Having excluded the spatially slow noise components, we summed the stimulus-related signals over all trials for each stimulus orientation and applied Gaussian low-pass filtering with a 150-mm standard deviation to eliminate high-frequency noise. In this way, we constructed a single-orientation map for each stimulus orientation. We also defined an integrated response-strength map by summing single-orientation maps over all stimulus orientations. To determine the preferred orientation at each pixel inside the recorded area, we used the vector sum method [19], which is based on the Fourier analysis in the circular symmetric orientation dimension. Thus, at each pixel, we obtained the preferred orientation and the modulation amplitude in the second harmonic components, which is regarded as orientation selectivity. The orientation polar map was constructed with the preferred orientation and orientation selectivity as color and brightness, respectively. For further analysis, we discarded pixels eliciting response strengths lower than a half of the response strength averaged over all pixels inside the recorded area. According to this criterion, the domains containing the remaining pixels nearly lined up with functionally defined area 17, which was exclusively activated by PLoS ONE | www.plosone.org 8 April 2009 | Volume 4 | Issue 4 | e5380