Rapid recovery from the effects of early monocular deprivation is enabled by temporary inactivation of the retinas.

A half-century of research on the consequences of monocular deprivation (MD) in animals has revealed a great deal about the pathophysiology of amblyopia. MD initiates synaptic changes in the visual cortex that reduce acuity and binocular vision by causing neurons to lose responsiveness to the deprived eye. However, much less is known about how deprivation-induced synaptic modifications can be reversed to restore normal visual function. One theoretically motivated hypothesis is that a period of inactivity can reduce the threshold for synaptic potentiation such that subsequent visual experience promotes synaptic strengthening and increased responsiveness in the visual cortex. Here we have reduced this idea to practice in two species. In young mice, we show that the otherwise stable loss of cortical responsiveness caused by MD is reversed when binocular visual experience follows temporary anesthetic inactivation of the retinas. In 3-mo-old kittens, we show that a severe impairment of visual acuity is also fully reversed by binocular experience following treatment and, further, that prolonged retinal inactivation alone can erase anatomical consequences of MD. We conclude that temporary retinal inactivation represents a highly efficacious means to promote recovery of function.

Modification of Peak Plasticity Induced by Brief Dark Exposure.

The capacity for neural plasticity in the mammalian central visual system adheres to a temporal profile in which plasticity peaks early in postnatal development and then declines to reach enduring negligible levels. Early studies to delineate the critical period in cats employed a fixed duration of monocular deprivation to measure the extent of ocular dominance changes induced at different ages. The largest deprivation effects were observed at about 4 weeks postnatal, with a steady decline in plasticity thereafter so that by about 16 weeks only small changes were measured. The capacity for plasticity is regulated by a changing landscape of molecules in the visual system across the lifespan. Studies in rodents and cats have demonstrated that the critical period can be altered by environmental or pharmacological manipulations that enhance plasticity at ages when it would normally be low. Immersion in complete darkness for long durations (dark rearing) has long been known to alter plasticity capacity by modifying plasticity-related molecules and slowing progress of the critical period. In this study, we investigated the possibility that brief darkness (dark exposure) imposed just prior to the critical period peak can enhance the level of plasticity beyond that observed naturally. We examined the level of plasticity by measuring two sensitive markers of monocular deprivation, namely, soma size of neurons and neurofilament labeling within the dorsal lateral geniculate nucleus. Significantly larger modification of soma size, but not neurofilament labeling, was observed at the critical period peak when dark exposure preceded monocular deprivation. This indicated that the natural plasticity ceiling is modifiable and also that brief darkness does not simply slow progress of the critical period. As an antecedent to traditional amblyopia treatment, darkness may increase treatment efficacy even at ages when plasticity is at its highest.

Fast Recovery of the Amblyopic Eye Acuity of Kittens following Brief Exposure to Total Darkness Depends on the Fellow Eye.

Recent studies conducted on kittens have revealed that the reduced visual acuity of the deprived eye following a short period of monocular deprivation imposed in early life is reversed quickly following a 10-day period spent in total darkness. This study explored the contribution of the fellow eye to the darkness-induced recovery of the acuity of the deprived eye. Upon emergence of kittens from darkness, the fellow eye was occluded for different lengths of time in order to investigate its effects on either the speed or the extent of the recovery of acuity of the deprived eye. Occlusion of the fellow eye for even a day immediately following the period spent in darkness blocked any recovery of the acuity of the deprived eye. Moreover, occlusion of the fellow eye two days after the period of darkness blocked any further visual recovery beyond that achieved in the short period when both eyes were open. The results imply that the darkness-induced recovery of the acuity of the deprived eye depends upon, and is guided by, neural activity in the mature neural connections previously established by the fellow eye.

The critical period for darkness-induced recovery of the vision of the amblyopic eye following early monocular deprivation.

Exposure of kittens to complete darkness for 10 days has been shown (Duffy & Mitchell, 2013) to reverse the loss of visual acuity that follows a prior period of monocular deprivation (MD). In that study, recovery of acuity in the previously deprived eye was fast despite the fact that darkness was imposed 2 months after the period of MD when kittens were 3 months old. In a later study (Holman, Duffy, & Mitchell, 2018), it was demonstrated that the same period of darkness was ineffective when it was imposed on cats about 1 year old, suggesting that dark exposure may only promote recovery when applied within an early critical period. To determine the profile of this critical period, the identical period of darkness (10 days) was imposed on kittens at various ages that had all received the same 7-day period of MD from postnatal day 30 (P30). Recovery of the acuity of the deprived eye as measured by use of a jumping stand was complete when darkness was imposed prior to P186 days, but thereafter, darkness induced progressively smaller acuity improvements and was ineffective in kittens when it began at or beyond P191 days of age. These data indicate a critical period for darkness-induced recovery with an abrupt end over a 5-day period.

Short periods of darkness fail to restore visual or neural plasticity in adult cats.

It has been shown that the visual acuity loss experienced by the deprived eye of kittens following an early period of monocular deprivation (MD) can be alleviated rapidly following 10 days of complete darkness when imposed even as late as 14 weeks of age. To examine whether 10 days of darkness conferred benefits at any age, we measured the extent of recovery of the visual acuity of the deprived eye following the darkness imposed on adult cats that had received the same early period of MD as used in prior experiments conducted on kittens. Parallel studies conducted on different animals examined the extent to which darkness changed the magnitude of the MD-induced laminar differences of the cell soma size and immunoreactivity for the neurofilament (NF) protein in the dorsal lateral geniculate nucleus (dLGN). The results indicated that 10 days of darkness imposed at one year of age neither alleviated the acuity loss of the deprived eye induced by an earlier period of MD nor did it decrease the concurrent lamina differences of the soma size or NF loss in the dLGN.

Recovery of visual functions in amblyopic animals following brief exposure to total darkness.

KEY POINTS: Occlusion of one eye of kittens (monocular deprivation) results in a severe and permanent loss of visual acuity in that eye, which parallels closely the vision loss characteristic of human amblyopia. We extended earlier work to demonstrate that amblyopic vision loss can be either blocked or erased very fast by a 10 day period of total darkness following a period of monocular deprivation that begins near birth and extends to at least 8 weeks of age. The parameters of darkness were strict because no visual recovery was observed after 5 days of darkness. In addition, short periods of light introduced each day during an otherwise 10 day period of darkness obliterated the benefits. Despite recovery of normal visual acuity, only one-quarter of the animals showed evidence of having attained normal stereoscopic vision. A period of total darkness may catalyse and improve treatment outcomes in amblyopic children. A 10 day period of total darkness has been shown to either block or erase the severe effects on vision of a prior short period of monocular deprivation (MD) in kittens depending on whether darkness is contiguous or is delayed with respect to the period of MD. We have extended these earlier findings from kittens for which the period of MD began at 1 month and lasted for 1 week to more clinically relevant situations where MD began near birth and lasted for ≥ 6 weeks. Despite the far longer MD and the absence of prior binocular vision, all animals recovered normal visual acuity in the previously deprived eye. As before, when the period of darkness followed immediately after MD, the vision of both eyes was initially very poor but, subsequently, the acuity of each eye increased gradually and equally to attain normal levels in ∼ 7 weeks. By contrast, when darkness was introduced 8 weeks after MD, the visual acuity of the deprived eye recovered quickly to normal levels in just 1 week without any change in the vision of the fellow (non-deprived) eye. Short (15 or 30 min) periods of illumination each day during an otherwise 10 day period of darkness obliterated all the benefits for vision, and a 5 day period of darkness was also completely ineffective. Measurements of depth perception indicated that, despite possessing normal visual acuity in both eyes, only about one-quarter of the animals showed evidence of having attained normal stereoscopic vision.

Ten days of darkness causes temporary blindness during an early critical period in felines.

Extended periods of darkness have long been used to study how the mammalian visual system develops in the absence of any instruction from vision. Because of the relative ease of implementation of darkness as a means to eliminate visually driven neural activity, it has usually been imposed earlier in life and for much longer periods than was the case for other manipulations of the early visual input used for study of their influences on visual system development. Recently, it was shown that following a very brief (10 days) period of darkness imposed at five weeks of age, kittens emerged blind. Although vision as assessed by measurements of visual acuity eventually recovered, the time course was very slow as it took seven weeks for visual acuity to attain normal levels. Here, we document the critical period of this remarkable vulnerability to the effects of short periods of darkness by imposing 10 days of darkness on nine normal kittens at progressively later ages. Results indicate that the period of susceptibility to darkness extends only to about 10 weeks of age, which is substantially shorter than the critical period for the effects of monocular deprivation in the primary visual cortex, which extends beyond six months of age.

Shrinkage of X cells in the lateral geniculate nucleus after monocular deprivation revealed by FoxP2 labeling.

The parallel processing of visual features by distinct neuron populations is a central characteristic of the mammalian visual system. In the A laminae of the cat dorsal lateral geniculate nucleus (dLGN), parallel processing streams originate from two principal neuron types, called X and Y cells. Disruption of visual experience early in life by monocular deprivation has been shown to alter the structure and function of Y cells, but the extent to which deprivation influences X cells remains less clear. A transcription factor, FoxP2, has recently been shown to selectively label X cells in the ferret dLGN and thus provides an opportunity to examine whether monocular deprivation alters the soma size of X cells. In this study, FoxP2 labeling was examined in the dLGN of normal and monocularly deprived cats. The characteristics of neurons labeled for FoxP2 were consistent with FoxP2 being a marker for X cells in the cat dLGN. Monocular deprivation for either a short (7 days) or long (7 weeks) duration did not alter the density of FoxP2-positive neurons between nondeprived and deprived dLGN layers. However, for each deprived animal examined, measurement of the cross-sectional area of FoxP2-positive neurons (X cells) revealed that within deprived layers, X cells were smaller by approximately 20% after 7 days of deprivation, and by approximately 28% after 7 weeks of deprivation. The observed alteration to the cross-sectional area of X cells indicates that perturbation of this major pathway contributes to the functional impairments that develop from monocular deprivation.

The case from animal studies for balanced binocular treatment strategies for human amblyopia.

Although amblyopia typically manifests itself as a monocular condition, its origin has long been linked to unbalanced neural signals from the two eyes during early postnatal development, a view confirmed by studies conducted on animal models in the last 50 years. Despite recognition of its binocular origin, treatment of amblyopia continues to be dominated by a period of patching of the non-amblyopic eye that necessarily hinders binocular co-operation. This review summarizes evidence from three lines of investigation conducted on an animal model of deprivation amblyopia to support the thesis that treatment of amblyopia should instead focus upon procedures that promote and enhance binocular co-operation. First, experiments with mixed daily visual experience in which episodes of abnormal visual input were pitted against normal binocular exposure revealed that short exposures of the latter offset much longer periods of abnormal input to allow normal development of visual acuity in both eyes. Second, experiments on the use of part-time patching revealed that purposeful introduction of episodes of binocular vision each day could be very beneficial. Periods of binocular exposure that represented 30-50% of the daily visual exposure included with daily occlusion of the non-amblyopic could allow recovery of normal vision in the amblyopic eye. Third, very recent experiments demonstrate that a short 10 day period of total darkness can promote very fast and complete recovery of visual acuity in the amblyopic eye of kittens and may represent an example of a class of artificial environments that have similar beneficial effects. Finally, an approach is described to allow timing of events in kitten and human visual system development to be scaled to optimize the ages for therapeutic interventions.

An examination of linking hypotheses drawn from the perceptual consequences of experimentally induced changes in neural circuitry.

Because targeted early experiential manipulations alter both perception and the response properties of particular cells in the striate cortex, they have been used as evidence for linking hypotheses between the two. However, such hypotheses assume that the effects of the early biased visual input are restricted to just the specific cell population and/or visual areas of interest and that the neural populations that contribute to the visual perception itself do not change. To examine this assumption, we measured the consequences for vision of an extended period of early monocular deprivation (MD) on a kitten (from 19 to 219 days of age) that began well before, and extended beyond, bilateral ablation of visual cortical areas 17 and 18 at 132 days of age. In agreement with previous work, the lesion reduced visual acuity by only a factor of two indicating that the neural sites, other than cortical areas 17 and 18, that support vision in their absence have good spatial resolution. However, these sites appear to be affected profoundly by MD as the effects on vision were just as severe as those observed following MD imposed on normal animals. The pervasive effects of selected early visual deprivation across many cortical areas reported here and elsewhere, together with the potential for perception to be mediated at a different neural site following deprivation than after typical rearing, points to a need for caution in the use of data from early experiential manipulations for formulation of linking hypotheses.

Critical Periods in Vision Revisited.

For four decades, investigations of the biological basis of critical periods in the developing mammalian visual cortex were dominated by study of the consequences of altered early visual experience in cats and nonhuman primates. The neural deficits thus revealed also provided insight into the origin and neural basis of human amblyopia that in turn motivated additional studies of humans with abnormal early visual input. Recent human studies point to deficits arising from alterations in all visual cortical areas and even in nonvisual cortical regions. As the new human data accumulated in parallel with a near-complete shift toward the use of rodent animal models for the study of neural mechanisms, it is now essential to review the human data and the earlier animal data obtained from cats and monkeys to infer general conclusions and to optimize future choice of the most appropriate animal model.

Protection against deprivation amblyopia depends on relative not absolute daily binocular exposure.

Short daily periods of binocular exposure (BE) can offset longer single daily episodes of monocular exposure (ME) to prevent the development of deprivation amblyopia. To determine whether the outcome depended upon an absolute daily amount of BE or its proportion of the daily visual exposure, daily mixed visual input of 3 different durations (3.5, 7, or 12 h) was imposed on 3 cohorts of kittens. Measurements of the visual acuity of the deprived eye at the end of mixed daily visual input revealed that the acuity of the deprived eye developed to normal values so long as the proportion of the total exposure that was binocular was 30% or more. By contrast, the development of functional ocular dominance domains in V1 revealed by optical imaging suggests that normal domains emerge with a fixed amount of daily binocular exposure. The latter result is consistent with the effects of any daily period of ME, or BE, or both, effectively saturating with a small dose so that the effects of ME of any length can be offset by a short period of BE. The different result for vision may reflect neural events at higher and/or multiple levels in the visual pathway.

Documentation of the Development of Various Visuomotor Responses in Typically Reared Kittens and Those Reared With Early Selected Visual Exposure by Use of a New Procedure.

A new procedure was used to study the development of gaze (responses to moving targets or laser spots in normal kittens, those that had been reared in total darkness to 6 weeks of age, and others that received a period of monocular deprivation (MD). Gaze responses were observed to all stimuli in normal kittens at between 25-30 days of age and striking responses occurred on the same day or the next. Despite slow acquisition of spatial vision in the dark reared kittens over 3 months, they were able to follow and even strike at moving visual stimuli within a day of their initial exposure to light. By contrast, for a week following a period of MD, kittens showed no gaze or striking responses to moving stimuli when using their previously deprived eye. The very different profiles of acquisition of visuomotor skills and spatial vision in visually deprived kittens point to a dissociation between the neuronal populations that support these functions.

Daily mixed visual experience that prevents amblyopia in cats does not always allow the development of good binocular depth perception.

Kittens reared with mixed daily visual input that consists of episodes of normal (binocular) exposure followed by abnormal (monocular) exposure can develop normal visual acuity in both eyes if the length of the former exposure exceeds a critical amount. However, later studies of the tuning of cells in primary visual cortex of animals reared in this manner revealed that their responses to interocular differences in phase were not reliable suggesting that their binocular depth perception may not be normal. We examined this possibility in 3 kittens reared with mixed daily visual exposure (2 hrs binocular vision followed by 5 hrs monocular exposure) that allowed development of normal visual acuity in both eyes. Measurements made of the threshold differences in depth that could be perceived under monocular and binocular viewing revealed a 10-fold superiority of binocular over monocular depth thresholds in one animal while the depth thresholds of the other two animals were poor and there was no binocular superiority. Thus, there was evidence that only one animal possessed stereopsis while the other two were likely stereoblind. While 2 hrs of daily binocular vision protected against the development of amblyopia, the poor outcome with respect to stereopsis points to the need for additional measures to promote binocular vision.

A special role for binocular visual input during development and as a component of occlusion therapy for treatment of amblyopia.

PURPOSE: To review work on animal models of deprivation amblyopia that points to a special role for binocular visual input in the development of spatial vision and as a component of occlusion (patching) therapy for amblyopia. METHODS: The studies reviewed employ behavioural methods to measure the effects of various early experiential manipulations on the development of the visual acuity of the two eyes. RESULTS: Short periods of concordant binocular input, if continuous, can offset much longer daily periods of monocular deprivation to allow the development of normal visual acuity in both eyes. It appears that the visual system does not weigh all visual input equally in terms of its ability to impact on the development of vision but instead places greater weight on concordant binocular exposure. Experimental models of patching therapy for amblyopia imposed on animals in which amblyopia had been induced by a prior period of early monocular deprivation, indicate that the benefits of patching therapy may be only temporary and decline rapidly after patching is discontinued. However, when combined with critical amounts of binocular visual input each day, the benefits of patching can be both heightened and made permanent. CONCLUSIONS: Taken together with demonstrations of retained binocular connections in the visual cortex of monocularly deprived animals, a strong argument is made for inclusion of specific training of stereoscopic vision for part of the daily periods of binocular exposure that should be incorporated as part of any patching protocol for amblyopia.

Recovery from the anatomical effects of long-term monocular deprivation in cat lateral geniculate nucleus.

Monocular deprivation (MD) imposed early in postnatal life elicits profound structural and functional abnormalities throughout the primary visual pathway. The ability of MD to modify neurons within the visual system is restricted to a so-called critical period that, for cats, peaks at about one postnatal month and declines thereafter so that by about 3 months of age MD has little effect. Recovery from the consequences of MD likewise adheres to a critical period that ends by about 3 months of age, after which the effects of deprivation are thought to be permanent and without capacity for reversal. The attenuation of plasticity beyond early development is a formidable obstacle for conventional therapies to stimulate recovery from protracted visual deprivation. In the current study we examined the efficacy of dark exposure and retinal inactivation with tetrodotoxin to promote anatomical recovery in the dorsal lateral geniculate nuclues (dLGN) from long-term MD started at the peak of the critical period. Whereas 10 days of dark exposure or binocular retinal inactivation were not better at promoting recovery than conventional treatment with reverse occlusion, inactivation of only the non-deprived (fellow) eye for 10 days produced a complete restoration of neuron soma size, and also reversed the significant loss of neurofilament protein within originally deprived dLGN layers. These results reveal a capacity for neural plasticity and recovery that is larger than anything previously observed following protracted MD in cat, and they highlight a possibility for alternative therapies applied at ages thought to be recalcitrant to recovery.

Brief daily periods of binocular vision prevent deprivation-induced acuity loss.

The role of experience in the development of the central visual pathways has been explored in the past through examination of the consequences of imposed periods of continuously abnormal or biased visual input. The massive changes in the visual cortex (area 17) induced by selected early visual experience, especially monocular deprivation (MD) or experience (ME) where patterned visual input is provided to just one eye, are accompanied by profound and long-standing visual deficits. Although the use of exclusively abnormal experience permits identification of those aspects of the visual cortex and of visual function that can be influenced by visual experience during development, this approach may provide a distorted view of the nature of the role of visual experience because of the absence of any normal visual input. In this study a different approach was used whereby animals were provided daily with separate periods of normal (i.e., binocular exposure) and abnormal (monocular exposure) visual experience. We show that 2 hr of daily normal concordant binocular experience (BE) can outweigh or protect against much longer periods of monocular deprivation (MD) and permit the development of normal visual acuities in the two eyes. This result is not what would be expected if all visual input had equal influence on visual development.

Monocular deprivation reduces reliability of visual cortical responses to binocular disparity stimuli.

While continuous monocular deprivation (MD) of patterned vision causes severe loss of visual cortical responses and visual acuity in the affected eye, these effects can be avoided by providing brief daily periods of binocular exposure [BE; D.E. Mitchell et al. (2003) Curr. Biol., 8, 1179-1182; D.E. Mitchell et al. (2006) Eur. J. Neurosci., 23, 2458-2466; D.S. Schwarzkopf et al. (2007) Eur. J. Neurosci., 25, 270-280]. In order to analyse binocular mechanisms involved in this phenomenon, we studied neuronal responses in primary visual cortex to binocular disparity stimuli in cats that had experienced mixed daily visual exposure (i.e. different amounts of daily binocular and monocular exposure). To examine whether binocular responses are as reliable in MD as in normal animals, we analysed single-trial responses to spatial phase disparity stimuli. In cats with various amounts of daily binocular experience (3.5 h, 7 h or 12 h) alone, about half of neurons (47.9%) showed reliable phase-specific binocular responses in two consecutive trials. The percentage of phase-selective cells was reduced in cats with mixed visual exposure with a decrease in the duration of daily BE. Within these neurons, a 'stable' cell population, i.e. with identical relative phases eliciting the strongest and weakest responses in two trials, was also reduced. In other words, the responses of neurons recorded from deprived animals were more likely to show different preferred phases on successive trials, although their amplitude ratios in both trials were about equal. We suggest that the detrimental effect of MD on binocular vision may begin, at least in part, with a subtle disruption of the mechanism involved in discrimination of binocular disparity signals.

Susceptibility to monocular deprivation following immersion in darkness either late into or beyond the critical period.

An extended duration of darkness starting near the time of birth preserves immature neuronal characteristics and prolongs the accentuated plasticity observed in young animals. Brief periods of complete darkness have emerged as an effective means of restoring a high capacity for neural plasticity and of promoting recovery from the effects of monocular deprivation (MD). We examined whether 10 days of darkness imposed in adulthood or beyond the peak of the critical period could rejuvenate the ability of MD to reduce the size of neuron somata within deprived layers of the cat dorsal lateral geniculate nucleus (dLGN). For adult cats subjected to 10 days of darkness before 7 days of MD, we observed no alteration in neuron size or neurofilament labeling within the dLGN. At 12 weeks of age, MD that followed immediately after 10 days of darkness produced an enhanced reduction of neuron soma size within deprived dLGN layers. For this age we observed that 10 days of darkness also enhanced the loss of neurofilament protein within deprived dLGN layers. These results indicate that, although 10 days of darkness in adulthood does not enhance the susceptibility to 7 days of MD, darkness imposed near the trailing edge of the critical period can restore a heightened susceptibility to MD more typical of an earlier developmental stage. The loss of neurofilament in juveniles exposed to darkness prior to MD suggests that the enhanced capacity for structural plasticity is partially rooted in the ability of darkness to modulate molecules that inhibit plasticity. J. Comp. Neurol. 524:2643-2653, 2016. © 2016 Wiley Periodicals, Inc.

The spatial localization deficit in visually deprived kittens.

We measured the spatial localization abilities (alignment accuracy) of visually deprived kittens by use of similar spatially bandpass stimuli (Gaussian blobs) to those employed for the assessment of human amblyopes. The tests of vision were conducted on kittens reared with either strabismus or following different periods of monocular deprivation. As with amblyopic humans, the deficits in alignment accuracy were scaled in proportion to blob size and were not only considerably larger than those of grating acuity but also were not correlated with either the acuity or contrast sensitivity losses. Tests with stimuli of various contrast revealed that the deficits could not be explained in terms of the contrast sensitivity loss in this eye. The positional deficits that arise from anomalous visual development are independent of the contrast sensitivity loss and profound.