Monocular deprivation was initiated by suturing shut the right eyelid of C57BL/6 mice early in the critical period (P22–24) and was continued to 4 to 5 months of age. The right eye was then re-opened to allow for binocular vision (BV), and baseline cortical responses through the two eyes were recorded using intrinsic signal imaging. Both eyes remained open afterward, and changes in responsiveness were measured over the next 3 weeks (Figure 1A). For 4 hr each day during these 21 days of BV (21d-BV), experimental animals viewed a visual stimulus (VS) while being permitted to run on a freely rotating spherical treadmill with their heads fixed. We used contrast-modulated stochastic noise matched to the spatiotemporal frequency response of the mouse as the visual stimulus because it drives nearly all cells in the primary visual cortex to some extent (Niell and Stryker, 2008).

Figure 1 with 3 supplements with 3 supplements see all Download asset Open asset Visual stimulation during locomotion enhances recovery of cortical responses through the deprived eye after prolonged MD. (A) Experimental schedule to examine changes in visual cortical responses over 21d-BV following prolonged MD started at postnatal day (P) 22–24. (B and C) Examples of intrinsic signal responses to the closed eye in the binocular visual cortex during 21d-BV in a home-cage control mouse (B) and in a mouse viewing contrast-modulated noise as VS during daily runs (VS+run, C). (D and E) Changes in intrinsic signal responses evoked by the noise through the closed (D) and open (E) eyes in home-cage (n = 8) and VS+run mice (n = 8). (F) Ocular dominance index (ODI) computed from response amplitude to contralateral (closed) and ipsilateral (open) eyes shown in D and E. ODI represents normalized difference in response magnitude between two eyes with 0 being equal amplitude to two eyes; the higher the number, more contralateral eye dominant. (G and H) Changes in intrinsic signal responses evoked by the noise through the closed (G) and open (H) eyes in run-only (n = 7) and VS-only mice (n = 7). (I) Ocular dominance index (ODI) computed from response amplitude to contralateral (closed) and ipsilateral (open) eyes shown in G and H. Gray area in D–I indicates the range of response amplitude or ODI in age-matched mice with normal visual experience. **p<0.01, *p<0.05, between groups. https://doi.org/10.7554/eLife.02798.003

In control mice that were kept in the standard housing condition (home-cage), cortical responses to the closed eye in binocular visual cortex slowly increased over 21d-BV, at which point they were still well below the range of age-matched mice with normal visual experience (Figure 1B,D). In contrast, mice that experienced the noise stimulus during their daily running (VS+run) showed a remarkable increase in closed-eye responses after only 7 days of BV (Figure 1C,D). Examined in a separate set of animals, this effect of VS+run was already significant after 3 days of training (Figure 1—figure supplement 1). Responses through the open eye did not change significantly in either group (Figure 1B,C,E). As a result, the ocular dominance index (ODI), computed as a normalized difference between responses to two eyes, recovered much more rapidly in animals with visual experience during running than in controls (Figure 1F). Recovery using reverse occlusion (switching the eye closure) instead of BV was similarly enhanced by VS+run (Figure 1—figure supplement 2).

To determine whether VS or running alone enhances recovery, we tested two more groups of mice: one that ran on the ball without VS (run-only) and one that viewed the visual noise stimulus in the home cage but did not run on the ball (VS-only). Closed-eye responses and ODI in both of these groups increased only slowly, similar to those of the home-cage control group (Figure 1G,I). Running velocity and duration were similar between run-only and VS+run groups (Figure 1—figure supplement 3).

VS+run enhanced recovery of monocular visual areas as well (Figure 2). This effect was rapid (Figure 2—figure supplement 1) and was particularly prominent for a secondary monocular area, where the home cage and the run-only groups showed no significant improvement even after 21 days of BV.

Figure 2 with 1 supplement with 1 supplement see all Download asset Open asset Visual stimulation during locomotion enhances recovery of cortical responses in the monocular visual cortex after prolonged MD. (A) Examples of intrinsic signal images of monocular visual areas. (B and C) Changes in intrinsic signal magnitudes (mean ± SEM) through the closed eye in response to the noise in the monocular V1 (B) and monocular secondary visual cortex (C) (same animals shown in Figure 1D–F). **p<0.01, between groups. https://doi.org/10.7554/eLife.02798.007

Perception in humans can be strengthened by training in an experience-specific manner (reviewed in Sagi, 2011), and responses in mouse visual cortex are reported to do so as well (Frenkel et al., 2006). We next tested whether recovery of closed-eye responses is preferentially enhanced to the particular visual stimuli presented during running. Mice viewed either square bars drifting in eight evenly-spaced directions (barVS) or the contrast-modulated noise (noiseVS) during running each day for 3 weeks. Intrinsic signal responses to both bar and noise stimuli were measured weekly. Mice that experienced barVS+run showed significantly greater recovery of closed-eye responses to bars than to noise (Figure 3). Likewise, mice that experienced noiseVS+run showed significantly greater recovery of response to noise than to bars (Figure 3). Responses to the stimulus that was not experienced during running were similar to those in control mice presented with a blank screen during running. These observations reveal that enhanced recovery is stimulus-specific and suggest that only the specific visual cortical circuits that are active during locomotion recover.

Figure 3 Download asset Open asset Preferential enhancement of recovery of closed-eye responses to the visual stimuli experienced during locomotion. (A) Peak intrinsic signal amplitude in response to bar stimuli through the closed eye in mice that experienced noise (noiseVS+run, red, n = 6), drifting bars (barVS+run, n = 6), or a blank screen during running (blue, n = 6). (B) Peak intrinsic signal amplitude in response to bar stimuli in same mice as in A. Data are show mean ± SD. **p<0.01 and *p<0.05 compared with the blank-screen control (blue). https://doi.org/10.7554/eLife.02798.009

To reveal changes in response properties of individual neurons, we made extracellular single unit recordings from neurons in layers 2/3 and 4 of binocular V1 contralateral to the closed eye. The use of 16-site silicon probes and spike-sorting methods (Niell and Stryker, 2008) enabled unbiased sampling from both responsive and unresponsive units. We examined five groups of mice with different treatments (Figure 4A). We first describe results from the broad-spiking, presumed excitatory cells that constitute ∼80% of the recordings.

Figure 4 with 2 supplements with 2 supplements see all Download asset Open asset Response magnitude of individual broad-spiking cells to drifting gratings and contrast-modulated noise. (A) Experimental schedule for single unit recording. Color-coded bars for different treatment groups apply to all panels. (B) Response magnitude to drifting gratings. Left panel: median (±s.e.) response rates to optimal drifting gratings through the deprived eye. Right panel: cumulative frequency distribution of ocular dominance index (ODI) in cells that were responsive (>2 spikes/s) through either deprived or open eye. ODI was computed from response magnitude to optimal gratings through each eye as shown in Figure 4—figure supplement 1A. (C) Response magnitude to contrast-modulated noise. Left: median F1 response (±s.e.) to the noise stimulus through the deprived eye. Right: cumulative frequency distribution of ODI calculated for each cell that were responsive to the noise (F1 response >0.2) through either eye from the data shown in Figure 4—figure supplement 1B. Data in left panels of B and C are from same populations of all cells isolated, as numbers are indicated below bars in B. Horizontal lines above bars; black: p<0.01, gray: p<0.05. Results of Kolmogorov–Smirnov tests for cumulative frequency distributions are shown in Table 1. https://doi.org/10.7554/eLife.02798.010

Consistent with the intrinsic signal observations as described above, closed-eye responses in mice that viewed visual stimuli while running during 7d-BV recovered significantly from their profoundly decreased level after long-term monocular deprivation (LTMD), in a stimulus-specific manner (Figure 4, Figure 4—figure supplements 1, 2). Closed-eye responses to the optimal grating (preferred orientation and spatial frequency) were most increased in grating+run mice among groups (Figure 4B), while responses to contrast-modulated noise were improved most in noise+run mice (Figure 4C). Open-eye responses did not differ among four groups of monocularly deprived mice (Figure 4—figure supplement 1). As a result, ocular dominance recovered to normal levels with similar stimulus specificity (Figure 4B,C).

In normal animals, the majority of cells in layers 2–4 are orientation-selective (OSI >0.5, Figure 5A,B) and the OSI for the two eyes is similar in each binocular cell (Figure 5—figure supplement 1B). Closed-eye OSIs and the binocular correlation of OSI were significantly improved only in grating+run mice but not in other groups (Figure 5A,B, Figure 5—figure supplement 1B). Prolonged MD caused mismatch of the preferred orientation of two eyes (Figure 5C,D), consistent with requirement of binocular visual experience during the critical period to develop orientation matching (Wang et al., 2010). 7d-BV improved binocular matching almost to normal levels in grating+run but not in home-cage or noise+run, mice (Figure 5C,D). In addition, the distribution of the preferred spatial frequency in closed-eye responses was almost normalized in grating+run mice but not in home-cage or noise+run mice. (Figure 5E,F). Such shifts in preferred spatial frequency may account for improvements in behaviorally measured acuity (Mitchell and Sengpiel, 2009).

Figure 5 with 1 supplement with 1 supplement see all Download asset Open asset Tuning properties of individual broad-spiking cells in response to drifting gratings. (A and B) Orientation tuning of deprived-eye responses, expressed as mean (±SEM) orientation selectivity index (OSI) (A) and cumulative frequency distribution of OSI (B). (C and D) Binocular matching of preferred orientation. Absolute differences in preferred orientation between two eyes in binocularly responsive cells are presented as the median (±s.e.) (C) and the cumulative frequency distribution (D). (E and F) Spatial frequency tuning. Preferred spatial frequencies at the preferred orientation of drifting gratings through the deprived eye are shown as mean (±SEM) (E) and cumulative frequency distribution (F). Sample sizes are indicated below bars. Horizontal lines above bars; black: p<0.01, gray: p<0.05. Results of Kolmogorov–Smirnov tests for B, D, F are shown in Table 1. https://doi.org/10.7554/eLife.02798.017

Table 1 Results of Kolmogrov-Smirnov tests for cumulative frequency distributions https://doi.org/10.7554/eLife.02798.016 Groups compared Non-deprived Non-deprived Non-deprived Non-deprived LTMD LTMD LTMD Home cage Home cage noise + run LTMD Home cage noise+run grating+run Home cage noise+run grating+run noise+run grating+run grating+run Broad-spiking cells Spontaneous firing <0.00001 <0.0001 <0.001 <0.05 <0.01 <0.01 <0.001 <0.01 <0.05 <0.01 Grating response: closed <0.00001 <0.00001 <0.01 n.s. n.s. <0.0001 <0.05 <0.05 <0.0001 <0.01 Grating response: open <0.01 <0.05 <0.05 n.s. n.s. n.s. n.s. n.s. n.s. n.s. Grating response: ODI <0.00001 <0.00001 <0.0001 n.s. <0.01 <0.001 <0.00001 <0.01 <0.00001 <0.001 |dO| <0.00001 <0.00001 <0.00001 <0.01 n.s. n.s. <0.001 n.s. <0.001 <0.001 OSI: closed <0.00001 <0.00001 <0.00001 <0.00001 n.s. <0.05 <0.001 <0.001 <0.001 <0.01 OSI: open <0.01 <0.01 <0.05 <0.05 n.s. n.s. n.s. n.s. n.s. n.s. Orientation tuning width: closed <0.0001 <0.05 <0.05 <0.05 n.s. n.s. <0.05 n.s. n.s. n.s. Orientation tuning width: open n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. Preferred SF: closed <0.001 <0.00001 <0.001 n.s. n.s. n.s. <0.05 <0.05 0.001 <0.05 Preferred SF: open <0.00001 <0.001 <0.001 <0.001 <0.01 <0.01 <0.01 n.s. n.s. n.s. Noise response (F1): closed <0.00001 <0.0001 <0.01 <0.001 <0.05 <0.00001 <0.001 <0.00001 <0.05 <0.0001 Noise response (F1): open <0.0001 <0.0001 <0.0001 <0.00001 n.s. n.s. n.s. n.s. n.s. n.s. Noise response: ODI <0.00001 <0.00001 n.s. <0.0001 <0.0001 <0.00001 <0.0001 <0.00001 <0.001 <0.001 Narrow-spiking cells Spontaneous firing <0.00001 <0.00001 <0.00001 <0.00001 n.s. n.s. n.s. n.s. n.s. n.s. Grating response: closed <0.00001 <0.0001 <0.0001 <0.001 n.s. n.s. <0.01 n.s. <0.05 <0.05 Grating response: open n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. Grating response: ODI <0.00001 <0.001 <0.0001 <0.05 <0.01 n.s. <0.001 n.s. n.s. <0.05 OSI: closed <0.001 <0.0001 n.s. n.s. n.s. n.s. n.s. <0.01 <0.01 n.s. OSI: open <0.01 <0.01 <0.01 <0.01 n.s. n.s. n.s. n.s. n.s. n.s. Noise response (F1): closed <0.001 <0.001 <0.01 <0.01 <0.05 <0.01 <0.05 <0.01 n.s. n.s. Noise response (F1): open n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. Noise response: ODI <0.00001 <0.00001 <0.001 <0.01 <0.001 <0.0001 <0.0001 <0.001 <0.001 <0.05

In response to the contrast-modulated noise stimuli, the peak firing of a cell occurs at the frequency of contrast modulation (F1, at 0.1 Hz). We assessed contrast sensitivity by calculating average value of contrast that elicits half-maximal response (C 1/2 ). Contrast sensitivity was significantly impaired after LTMD and was restored almost completely in noise+run mice but not in gratings+run mice (Figure 6).

Figure 6 Download asset Open asset Change in contrast sensitivity in broad-spiking cells. Average values of contrast that gives half-maximal response are shown. Horizontal lines above bars; black: p<0.01, grey: p<0.05. https://doi.org/10.7554/eLife.02798.019

Narrow-spiking cells are thought to correspond to inhibitory, predominantly fast-spiking, interneurons (McCormick et al., 1985; Bartho et al., 2004). Although a minority population, inhibitory cells play an important role in plasticity (Espinosa and Stryker, 2012). We observed several notable differences between broad- and narrow-spiking cells in the changes following LTMD and 7d-BV. First, spontaneous activity of narrow-spiking cells was greatly reduced after LTMD and did not change significantly after 7d-BV regardless of the treatment (Figure 7C,D), whereas those of broad-spiking cells were elevated after LTMD and significantly decreased toward normal level in mice after 7d-BV (Figure 7A,B). These opposite changes in spontaneous firing between broad- and narrow-spiking cells may reflect a homeostatic mechanism that alters excitatory–inhibitory balance to maintain cortical activity during prolonged deprivation. Second, recovery of closed-eye responses and ocular dominance to the gratings in narrow-spiking cells was only modest even in grating+run mice (Figure 8A, Figure 8—figure supplement 1A), whereas it was nearly complete in broad-spiking cells (Figure 4B). Third, responses of narrow-spiking cells to contrast-modulated noise through the deprived eye and ocular dominance were incompletely restored both in noise+run and grating+run mice to similar extents, showing no preference for the experienced stimulus (Figure 8B, Figure 8—figure supplement 1B). Fourth, responses of narrow-spiking cells through the open-eye were not elevated after LTMD either to the noise or grating stimulus, and did not change significantly in any of 7d-BV mice (Figure 8—figure supplement 1A,B). This lack of potentiation of open-eye responses during LTMD in narrow-spiking cells may contribute to potentiation of those in broad-spiking cells. The responses of narrow-spiking cells after LTMD suggest that deprivation reduces intracortical inhibition. Such a reduction may provide a starting point that allows meager deprived-eye excitatory pathways of the cortical circuit to drive activity upon re-opening. The incomplete recovery of orientation tuning of broad-spiking cells in spite of nearly full restoration of the magnitudes of responses may result from the very limited recovery in narrow-spiking cells, as inhibition is critical for generation of sharp orientation tuning in upper layer neurons in the primary visual cortex (Liu et al., 2011).