Darkness Alters Maturation of Visual Cortex and Promotes Fast ...

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Feb 14, 2013 - The effect of dark rearing on neurofilament protein was exam- ined by .... (A) A schematic illustration of the visual experience of the three immediate darkness (ID) ... amazing visual recovery may in part be because MD was.

Current Biology 23, 382–386, March 4, 2013 ª2013 Elsevier Ltd All rights reserved

http://dx.doi.org/10.1016/j.cub.2013.01.017

Report Darkness Alters Maturation of Visual Cortex and Promotes Fast Recovery from Monocular Deprivation Kevin R. Duffy1,* and Donald E. Mitchell1 of Psychology and Neuroscience, Dalhousie University, 1459 Oxford Street, Halifax, NS B3H 4R2, Canada

1Department

Summary The existence of heightened brain plasticity during critical periods in early postnatal life is a central tenet of developmental sensory neuroscience and helps explain the enduring deficits induced by early abnormal sensory exposure [1, 2]. The human visual disorder amblyopia has been linked to unbalanced visual input to the two eyes in early postnatal visual cortical development and has been modeled in animals by depriving them of patterned visual input to one eye [3, 4], a procedure known as monocular deprivation (MD). We investigated the possibility that a period of darkness might reset the central visual pathways to a more plastic stage and hence increase the capacity for recovery from early MD. Here we show that a 10 day period of complete darkness reverses maturation of stable cytoskeleton components in kitten visual cortex and also results in rapid elimination of, or even immunity from, visual deficits linked to amblyogenic rearing by MD. The heightened instability of the cytoskeleton induced by darkness likely represents just one of many parallel molecular changes that promote visual recovery, possibly by release of the various brakes on cortical plasticity [2]. Results and Discussion The changing vulnerability with age of the visual cortex to monocular deprivation (MD) stands as the quintessential demonstration of a critical period in visual system development [1, 4]. In kittens, MD as short as a few days induces functional and/or structural changes in the dorsal lateral geniculate nucleus [3, 5] and visual cortex [4, 6] as well as severe impairments of vision when using the deprived eye [7, 8]. The effects of MD on the visual cortex are particularly robust, leading to functional shifts in the relative weighting of the two eyes among cortical cells [1, 4] and to changes in the amount of cortex dominated by each eye [6]. The susceptibility of kitten visual cortex to MD peaks at between 4 and 5 weeks of age [9] and is followed by a gradual decline to negligible levels at between 8 and 10 months [10, 11]. Modeling of the molecular events that underlie critical period plasticity [1, 12] suggests an alteration to excitatory-inhibitory (E-I) balance coincident with the emergence of specific inhibitory circuits that interact with preestablished excitatory connections. Changes that occur downstream of the plasticity engendered by the reset E-I balance result in consolidation of plasticity at low (adult) levels. A number of molecules with late emerging developmental profiles have been proposed as ‘‘brakes’’ that gradually reduce plasticity to negligible levels.

*Correspondence: [email protected]

Biochemical [12, 13] or experiential interventions [14] directed toward alteration of the E-I balance or manipulation of brakes on plasticity have been proposed as ways to rescue in adulthood functions lost from visual deprivation. Studies from kittens suggest that complete darkness imposed immediately after MD may lessen its effects. The effects on cortical ocular dominance of short periods of MD were attenuated by darkness [15] and, in the thalamus, prompt restoration of appropriate visual input after MD was mimicked by an equivalent period spent in darkness [16]. The learning rule of synaptic modification referred to as the BCM theory [17], predicts that recovery after MD would be optimized by a period of darkness interpolated prior to introduction of well-correlated visual input to both eyes [18–20]. To investigate whether a period of darkness might reset the visual cortex to a more structurally plastic state, we examined levels of neurofilament protein, on the basis of their link to stabilization of neuron structure [21] and their link to MD-mediated structural plasticity [22–25]. Neurofilament Levels Increase from Birth to Adulthood We first examined the developmental profile of the obligatory neurofilament light (NF-L) subunit to assess whether it contributes to an intracellular ‘‘braking’’ system to limit plasticity. Levels of NF-L in primary visual cortex (V1) were barely detectable at postnatal day (P) 0 and P24 (Figure 1A), though heavily contrasted images did reveal extremely faint bands at both time points (Figure 1B). NF-L increased only modestly to P35, and at P90 levels remained lower than in adults. Quantification of total protein fluorescence revealed little variability across ages shown in Figure 1A, whereas NF-L levels in the same lanes increased considerably (Figure 1C). Quantification of NF-L normalized to the total protein control revealed that, relative to adult, NF-L levels were stable and low until P40, after which levels rose to 60% of adult values by P90 (Figure 1D). The gradual rise in NF-L suggests a progressive increase in the stabilization of the cytoskeleton and is congruent with the notion that neurofilament acts in conjunction with other putative braking molecules [22] to constrain plasticity in adulthood. Neurofilament Protein Is Reduced by Darkness The effect of dark rearing on neurofilament protein was examined by labeling sections of V1 for NF-L (Figure 2A) in animals that were raised with normal vision until P30, at which time they were placed in darkness for either 5, 10, or 15 days. Following 10 and 15 days of darkness (Figure 2B), there was a significant reduction in NF-L immunopositive neurons within V1 to about 50% of normal; however, no change was observed in animals dark reared for 5 days. The effects of darkness appeared greatest in the superficial layers and in layer VI but were less apparent in layer V where there were still many immunopositive pyramidal cells (Figure 2A). Immunoblots of homogenized V1 also showed a decrease in NF-L to almost half of normal levels after 10 or 15 days of darkness (Figure 2C, red bands). Quantification of NF-L relative to a cytoskeletal loading control protein, a-tubulin (Figure 2C, green bands), confirmed a significant reduction of NF-L after dark rearing (Figure 2D).

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Figure 2. Dark Rearing for 10 or 15 Days Reduces Neurofilament Levels in the Visual Cortex

Figure 1. Neurofilament Levels in the Visual Cortex Increase 100-Fold from Birth to Adulthood (A) Multiplexed immunoblots of homogenate from right and left V1 at birth (P0) and across postnatal development to adulthood. Labeling for NF-L (red bands) was examined relative to a total protein stain (green bands) for each age and for each hemisphere separately. The total protein control was used to overcome the difficulty in identifying a single control protein present in the same amount from birth through to adulthood. (B) High-contrast images of NF-L bands taken from the two youngest ages shown in (A). (C) Measurement of NF-L band volume (red squares) and total protein band volume (green circles) from left and right visual cortex of the animals shown in (A), plotted for each marker as a percentage of the adult volume from the same hemisphere. Protein controls indicated that low NF-L levels early in development were not due to reduced loading of protein. (D) Quantification of NF-L in the left (triangles) and right (asterisks) hemispheres of V1, calculated as a percentage of total protein measured from the same lane and then normalized to the highest adult NF-L level from the same hemisphere.

Profound Recovery of Vision Promoted by Brief Periods of Darkness Behavioral studies were conducted on animals following MD for 1 week, beginning at the peak of the critical period (P30). A 10 day period of darkness was imposed immediately after

(A) Immunolabeling for NF-L in tissue from V1 of a normal P40 control animal and from animals kept in complete darkness for 5, 10, and 15 days starting at P30. Layers of the visual cortex are indicated with roman numerals; scale bar represents 100 microns. (B) Stereological estimates of the density of neurons immunopositive for NF-L in the left and right hemispheres of V1 for normal and dark-reared groups. A Kruskal-Wallis nonparametric ANOVA of this data was significant (H[2] = 8.83, p = 0.012), and post hoc tests revealed a reduction from normal after 10 and 15 days of dark rearing (data combined; Dunn’s multiple comparison test: p < 0.05) but no reduction from normal after 5 days (Dunn’s multiple comparison test: p > 0.05). (C) Multiplexed immunoblots of homogenate from the left and right visual cortex of a control animal and of animals placed in complete darkness for 10 or 15 days. Levels of NF-L (red bands) and a cytoskeletal control protein, a-tubulin (green bands), within the same immunoblots are shown. (D) Quantification of the level of NF-L protein in V1 calculated separately for both hemispheres of each animal, measured as a proportion of the level of a-tubulin and plotted as a percentage of normal. Statistical comparison of NF-L levels between normal animals and those that were dark reared (10 and 15 day data combined) revealed a significant reduction after dark rearing (Mann-Whitney test: U = 1, p = 0.008).

termination of the period of MD for three animals (immediate darkness [ID] kittens) but was delayed either 5 or 8 weeks for a second group (delayed darkness [DD] kittens; n = 4). Immediately after animals in the ID group were removed from the dark (Figure 3), they appeared blind in both eyes. The detrimental effect of darkness on the nondeprived eye predicts the previously unexpected result that a normal light reared kitten would emerge blind after a 10 day period of darkness

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Figure 3. Ten Days of Darkness Imposed Immediately after 1 Week of MD Prevented Development of Amblyopia (A) A schematic illustration of the visual experience of the three immediate darkness (ID) kittens as a function of postnatal age in days (PD). (B) Changes in the visual acuity of the two eyes of one kitten (C153) following termination of 1 week of MD. Open and filled circles depict the acuity of, respectively, the initially deprived eye or nondeprived eye. Filled squares show the acuity measured with both eyes open. Animals initially appeared blind after dark rearing for 10 days. The poor vision of the nondeprived eye is highlighted by comparison with the acuity of the nondeprived eye in the DD animals (horizontal arrow), as measured at the same time point. Once the ability to detect the open door was achieved (OD), kittens were shortly thereafter able to discriminate a vertical from a horizontal grating, thereby allowing measurement of their visual acuity. (C) Comparable data from two other kittens. The vision of the deprived eye of C156 was assessed and shown to be blind (open circle symbol at time zero) shortly after the eyelids of this eye were reopened and immediately before it was placed in the darkroom. The brackets to the right of the data for C153, in (B), and C156 illustrate the range of values for the acuity of a normal kitten at 3 to 4 months of age, as tested on the same discrimination task employed here.

initiated at P37, a result since confirmed (see Figure S1 available online). In prior studies [1, 26, 27], darkness was imposed near birth and lasted for months, so that the profound immediate effect of only 10 days of darkness starting at P37 was unexpected. Vision of the two eyes improved slowly in lockstep from blindness to normal grating acuity over a 7 week period. The crucial result was that amblyopia never developed. For the DD group (Figure 4A), the period of darkness was

Figure 4. Extremely Rapid Recovery from Amblyopia after 10 Days of Darkness Imposed Either 5 or 8 Weeks after 1 Week of MD (A) A schematic illustration of the visual experience of the delayed darkness (DD) kittens as a function of postnatal age in days (PD). (B) Changes in the visual acuity of the two eyes of one kitten following termination of 1 week of MD before and after a 10 day period of darkness. Symbols are as in Figure 3. (C) Data for three other animals, for which data are not shown for the first 20 days after termination of the period of MD, so as to emphasize the rapid changes in the vision of the deprived eye upon removal of the kittens from the darkroom. The brackets to the right of the data for C157, in (B), and C155 illustrate the range of values for the acuity of a normal kitten at 3 to 4 months of age, as tested on the same discrimination task as employed here.

delayed for 5 to 8 weeks after the period of MD, at which time substantial and stable amblyopia had developed in the deprived eye that was slightly worse than previously reported values from kittens tested by the same methods after similar periods of deprivation that began 5 days later [28–30]. Results of longitudinal measurements of the acuities of the two eyes are shown for one animal (C157) in Figure 4B. In contrast to the ID group, darkness had no immediate deleterious effect on the vision of the nondeprived eye, indicating that the critical period for the effects of darkness on vision (Figure 3) is short. The acuity of the deprived eye improved rapidly after the period of darkness to attain values equal to that of the fellow eye in just 5 to 7 days, a result confirmed by the other three animals (Figure 4C). Because the acuity of the nondeprived eye of C155 (placed in darkness at P71) was unimpaired by

Darkness Promotes Fast Recovery from Amblyopia 385

darkness, it would appear that the critical period for the effects of darkness on acuity ends by 10 weeks of age. The swiftness and the entirety of the recovery of the deprived eye in the DD group were startling because darkness was imposed at 3 months, when changes of the vision of the deprived eye following conventional manipulations are slow and limited [8]. The recovery after darkness is also impressive when compared with conventional recovery protocols; even when MD is terminated early (at 4 to 6 weeks of age), recovery of acuity in the deprived eye continues for 3 to 4 weeks during either reverse occlusion [31] or binocular recovery, where initial changes are fastest [29]. Only with reverse occlusion can the acuity of the deprived eye sometimes reach normal levels, which comes at the cost of the vision of the other eye, and, moreover, the acuity gained by the deprived eye is often lost after vision is restored to the other eye [8, 31–33]. The recovery of spatial acuity was remarkable in three respects. First, as illustrated in Figure S2, it was complete in all seven animals. Second, the recovery occurred when both eyes were open following MD, a situation where full recovery of vision in the deprived eye never occurs [8]. And third, recovery was observed whether the dark interval occurred immediately (ID group) or even long after (DD group) offset of the amblyogenic event (MD), though the speed of recovery to normal levels differed by a factor of 7 to 10 between the groups. It is unclear whether the discrepant speed of recovery is due to differences in the age when darkness was imposed, the length of the delay between the period of MD and darkness, or a combination of the two. It is possible that the amazing visual recovery may in part be because MD was imposed after binocular cortical architecture was well established, and it may be less pronounced in animals for which MD occurred earlier and lasted longer. Although the benefits for the final acuity outcomes of immediate or delayed darkness were identical, it is possible that one of these conditions provides greater benefits to other visual abilities such as stereoscopic vision. It remains to be seen how the effects of darkness change with age and whether benefits persist at or beyond the end of the critical period for cortical ocular dominance plasticity at 8 to 10 months of age. In this respect, it is noteworthy that darkness imposed on adult Long-Evans rats can promote recovery on some measures from the acuity losses consequent to MD, but behavioral measurements of acuity indicated substantial residual deficits in the situation of binocular recovery [18]. Nonetheless, the fact that darkness was so successful when imposed on MD kittens points to its possible application to amblyopic children in order to boost outcomes of existing therapeutic interventions and thereby enhance career and lifestyle choices. Molecular Mechanisms Although modification of the stable cytoskeleton through reduction of neurofilament is likely one of a multitude of changes that occurs near the end of a cascade of molecular events triggered by darkness, it nevertheless may introduce a level of cytoskeletal instability typical of an earlier and more malleable state that translates to a heightened capacity for structural plasticity. Preliminary behavioral results from one animal indicates that 5 days of darkness is ineffectual at improving vision, so it appears that a close temporal relationship may exist between the behavioral events and changes in neurofilament levels, which were also not different from normal

after 5 days of darkness. It is important to recognize that the changes in neurofilament may be mirrored by changes of varying degrees in the levels of other proteins that may serve as brakes on cortical plasticity. In other words, darkness may alter to varying extents the multitude of molecular correlates of cortical maturation that include chondroitin sulfate proteoglycans [34, 35], such as aggrecan [36], which are constituents of the extracellular matrix, Lynx 1 that binds to nicotinic acetylcholine receptors [37], and myelin-related factors [38]. Additionally, it will be of interest to learn how darkness imposed at different ages influences separately the various molecules that may alter the capacity for synaptic modification, such as the ratio of NMDA receptor subunits NR2a/NR2b, which returns to a juvenile state in dark-reared adult rats [39]. Although the mechanisms underlying the recovery in both the ID and DD conditions may overlap, an understanding of the cellular response to darkness imposed at progressively later ages might help to explain, in monocularly deprived animals, the reduced susceptibility of the nondeprived eye to darkness with age. Potential Clinical Application and Open Issues Our results suggest that dark exposure may be entertained as an adjunct to conventional as well as newly developed binocular behavioral therapies [40] for amblyopia that may allow for the acquisition of stereopsis. Before application of this intervention to humans, it would be beneficial to document the strictness of the requirement for darkness in kittens, because this information would have a large impact on the means by which it could be implemented. Also, precise knowledge of the profile of the critical period for the effects of darkness on vision in kittens would help guide the choice of the earliest age at which the manipulation can be applied. Demonstrations of restored plasticity in both the auditory [41] and somatosensory [42] cortex of mature animals by experiential manipulations suggest that heightened plasticity may be induced in sensory regions other than the visual cortex in adulthood. Although knowledge of the molecular changes that are precipitated by the period of darkness might be instructive, it is unlikely that manipulation of just one or a limited number of such molecules would duplicate the effects of darkness itself. The advantage of a simple nonpharmacological sensory manipulation, such as a period of darkness, is that it may initiate changes in a constellation of molecules in a beneficial temporal order and in appropriate brain regions. Supplemental Information Supplemental Information includes two figures and Supplemental Experimental Procedures and can be found with this article online at http://dx. doi.org/10.1016/j.cub.2013.01.017. Acknowledgments This research was supported by a grant from the Canadian Institutes of Health Research (102653) to K.R.D. and D.E.M. and by Discovery grants from the Natural Sciences and Engineering Research Council to K.R.D. (298167) and D.E.M. (7660). Additional support was provided by the Critical Period Revisited Network of the James S. McDonnell Foundation. We thank Kaitlyn Holman and Katelyn McNeil for their care of the animals and for assistance with behavioral testing. All 27 kittens used in these studies were born and raised in a closed laboratory breeding colony at Dalhousie University. The breeding and experimental procedures followed protocols approved by the Dalhousie University Committee on Laboratory Animals in accordance with the Canadian Council on Animal Care.

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