A. Time course of the experiment: all animals were imaged at P28. The period of dark rearing is represented as a black line, while the white-dotted pattern represents the period of exposure to a normal light environment. DR animals were maintained and anesthetized before imaging in complete darkness. DR/2dL animals were dark-reared until P26 and then exposed to light. DR/7dL animals were dark-reared until P21 and then exposed to light. Control animals were reared in a normal 12 hour light/12 hour dark environment.
B. Representative images of the cortical intrinsic signal in response to a visual stimulus in individual mice from the different groups imaged. Red hues indicate strong activation, according to color key at right depicting the change in reflectance, dR/R. In dark-reared mice, cortical activity in response to light is low and increases as mice are exposed to light. Scale bar = 0.5 mm.
C. Representative retinotopic maps of visual field elevation in individual mice with different visual experience. The map is highly disorganized in dark reared animals and the level of organization progressively increases together with the duration of light exposure. Color key at right depicts visual elevation.
D. Quantification of the visually evoked optical signal across animals with different visual experience. The strength of the signal (normalized change in reflectance, dR/R) is low in dark reared animals and recovers with longer light exposure. *=p<0.05 when compared to control.

A. In vivo two-photon image of the apical tuft of a layer 5 pyramidal neuron in visual cortex of a P28 mouse. The image is a collapsed z stack showing the extent of the apical tuft dendritic arbor. Scale bar = 100 μm.
B. 3D reconstruction showing the dendritic arbor from the side. Scale bar = 100 μm.
C. Two-photon image of dendritic spines in vivo from dendrite shown in A (boxed area). Scale bar = 5 μm.
D. Following imaging, red tracer was injected into the imaged area based on the blood vessel pattern. The animal was perfused and the imaged area identified in fixed section. The injection site is shown in red and resides in visual cortex as identified using coordinates of the mouse brain atlas.
E. Time lapse image of dendritic spines in the visual cortex of a control mouse in vivo. Images shown were taken 30 minutes apart. Scale bar = 2 μm. Spines are motile at these ages (notice spine 3 withdraws into the dendrite during the first hour of imaging).
F. Lengths of the 4 spines at left are shown plotted over 2 hours.
G. The motility index for the same four spines showing the approximate range of motilities observed in control animals (filopodia are not shown in this figure – filopodia were generally more motile and were rarely observed in control animals).
H. Spine motility indices for P28 mice exposed to different visual environments. Analysis included all spine classes and filopodia. There is a significant increase in spine motility in visually deprived animals (DR) as compared to controls. 2 days of exposure to a normal light-dark cycle does not affect motility but after seven days of exposure to normal dark-light conditions the motility index is no longer significantly different from that in control animals. *=p<0.05 compared to control.

A. High magnification two-photon images of representative dendrites in the visual cortex of animals reared in different visual environments. Scale bar = 5 μm.
B. Image showing the classification of dendritic spines into morphological classes. M= mushroom; S=stubby; T=thin; F= filopodium.
C. Variations in the morphologies of dendritic spines across animals with different visual experience. Animals reared in normal conditions show a significantly higher percentage of mushroom and stubby spines compared to animals that have been visually deprived, which show increased numbers of thin spines and filopodia (*=p<0.05, comparing control to other conditions). 7 days of re-exposure to light after dark-rearing restores the morphological profile of dendritic spines to control levels.

A. Time course of the experiment: all animals were imaged at P28. Five groups of animals were used: DR animals were maintained and anesthetized before imaging in complete darkness; DR/7dL animals were dark-reared until P21 and then exposed to light; DR/7dDIA animals were maintained in the darkness but injected with a daily dose of diazepam starting at P21; one DR/7dSAL animal received saline daily instead of diazepam starting at P21; control animals were reared in a normal 12 hour light/12 hour dark environment.
B. Representative images of the cortical intrinsic signal in response to light in individual mice from the different groups imaged. Red hues indicate strong activation, according to the dR/R scale at right. Mice treated with diazepam (DR/7dDIA) show strong visually driven responses similar to control mice and dark reared mice exposed to light for 7 days (DR/7dL). Scale bar = 0.5 mm.
C. Representative retinotopic maps of elevation in individual mice, as per key at right. Diazepam treatment results in an increase in the level of organization of the visual map similar to light exposure. Scale bar = 0.5 mm.
D. Quantification of the visually evoked optical signal across animals with different visual experience. The strength of the signal (normalized change in reflectance, dR/R) is low in dark reared animals and increases in animals exposed to light for 7 days as well as in animals treated in the dark with diazepam (n=3 animals). Only animals in the DR group had ΔR/R values that were significantly different than control animals (*=p<0.05).
E. Image showing a dendritic branch in an animal treated with diazepam (DR/7dDIA). Scale bar = 5 μm.
F. Diazepam treatment reduced the motility of spines in dark reared animals (n=7 animals) to control levels.
G. Spine morphology was not affected by diazepam treatment and these animals had increased numbers of thin spines and filopodia when compared to control animals (*=p<0.05).

A. Schematic of experimental timeline. Three groups of animals were used: animals reared in the darkness from birth (DR; same as Fig. 1,3,4), DR animals exposed to light for two hours before imaging (DR/2hL) and DR animals in which the NMDA antagonist CPP was injected systemically 30 minutes before two hours of light exposure (DR/2hL+CPP).
B. Representative images of the cortical intrinsic signal in response to light in individual mice from the different groups imaged. Red hues indicate strong activation, as per dR/R scale at right. Mice exposed to light for two hours show strong visually driven responses similar to control mice. Mice pretreated with the NMDA receptor antagonist CPP do not show increased visual responses after brief light exposure. Scale bar= 0.5 mm.
C. Representative retinotopic maps of elevation in individual mice, as per key at right.
D. Quantification of the amplitude of visually-evoked cortical activation in the three groups of animals. Brief light exposure significantly increased visually-evoked cortical activation (*=p<0.05 compared to dark reared animals). This effect was prevented by systemic injection of CPP before light exposure.

A. Time lapse image of dendritic spines in the visual cortex of a dark reared mouse in vivo following light exposure for 2 hours at P28. Images shown were taken 20 minutes apart. Scale bar = 2 μm. Dendritic protrusions are highly motile in this group of animals (DR/2hL).
B. Lengths of the 4 protrusions labeled in A are shown in the left panel plotted over 2 hours. Notice the large changes observed in length over this timescale. The right panel shows the motility index for the same four protrusions. Protrusion 1 was classified as a filopodium due to its length. Not all spines are highly motile. Spine 3 exhibits length changes and a motility index typical of spines in control mice at this age.
C. Brief light exposure rapidly increases spine motility in dark-reared animals (*=p<0.05). This effect is prevented when CPP is administered systemically before light exposure.
D. High magnification two-photon images of representative dendrites in the visual cortex of animals exposed to light for 2 hours following dark rearing. Notice the increased numbers of thin spines and filopodia in dark reared animals briefly exposed to light. Animals pretreated with CPP before light exposure have fewer thin spines and filopodia than untreated animals, but similar in proportion to dark reared animals. Scale bar = 5 μm.
E. Brief light exposure increases the proportion of thin protrusions (thin spines and filopodia) and decreases the proportion of mushroom and stubby spines compared to DR animals (*=p<0.05). The re-organization of dendritic spine morphology is prevented by administration of CPP before light exposure.

A. Top panel shows a dendritic arbor imaged over 2 days in a dark reared animal exposed to light after the first imaging session. Bottom panel shows a higher magnification of a dendrite bearing dendritic spines. Notice the outgrowth of a filopodium (arrow head) and spine (arrow). The filopodium withdraws after 2 days of light exposure but the spine is maintained. Scale bar = 30 μm (top); 5 μm (bottom).
B. The formation of dendritic protrusions is enhanced after two hours of light exposure in dark reared animals relative to animals maintained in the dark and relative to normal light reared controls (*=p<0.05), while the rate of elimination is not altered.
C. The percentage of new protrusions formed between the end of the dark rearing period and after two days of light exposure is not different than that over a 2 day period in light reared animals. However, the elimination of spines is significantly enhanced suggesting a pruning of pre-existing synapses.