A shot in the dark: the use of darkness to investigate visual development and as a therapy for amblyopia

Authors


Abstract

Extended periods of complete darkness have long been used among other early experiential manipulations to explore the role of visual experience in the development of the visual pathways. In the last decade, short periods of darkness have been used to facilitate the imposition of different or conflicting visual input each day to explore the manner by which processes of perinatal development controlled by gene action are refined subsequently by visual experience. Very recently, periods of complete darkness of intermediate length (10 days) have been shown to promote very fast recovery from amblyopia induced by prior monocular deprivation (MD). When imposed immediately after a period of MD, in certain circumstances, darkness appears to insulate against the development of amblyopia. It is proposed that complete darkness may reverse maturation of many of the so-called braking molecules in the visual cortex, so that it reverts to a more juvenile state.

Vision is the only sense for which its natural physical input can be eliminated completely for extended periods by immersion in a light-free external environment, for which rigorous safeguards are in place to avoid accidental stray light. Although the external environment for other senses can be made stimulus-free, unintended sensory stimulation can occur from internal sources. Consider audition for example; even with a noise-free environment, unavoidable auditory stimulation can still arise through air-borne sound or by bone conduction, as a consequence of self-induced or regular autonomic motor responses associated with respiration or cardiac activity.

The ability to eliminate light from the environment permits study of how development of the visual pathway proceeds after birth in the complete absence of sensory- (visually) driven neural activity. Arguably, this is a similar situation to that prior to birth, when development proceeds on the basis of molecular cues that may be modulated by neural activity of intrinsic origin, such as intrinsic waves of activity in the retina[1, 2] but not on the basis of neural activity that is visually driven. The initial developmental papers of Hubel and Wiesel[3] in the 1960s provided the inspiration for many studies directed toward an understanding of the role played by visual experience in postnatal development. In addition to documentation of the functional state of the neonatal cortex, these studies included the many newsworthy demonstrations of the consequences of different forms of selected or biased early visual exposure (where the visual input represented an extreme of the normal continuum as with input to just one eye or with input restricted to a single contour orientation, such as horizontal) that were imposed exclusively (that is, provided the only visual input) for extended periods in early life.[4-7] As an aid to the interpretation of studies of the effects of biased early visual exposure, it was necessary to explore the effects of complete visual deprivation. All of these experiments were designed in the context of contemporary views of the state of the neonatal cortex and by the need to investigate potential roles that subsequent visual experience could exert. As a consequence, the periods of selective visual exposure (or deprivation) were exclusive and episodes of normal visual input were specifically avoided. With the advent of new technologies, such as optical imaging of intrinsic signals,[8, 9] a consensus has begun to amass on the development of the three major response properties of cells in the primary visual cortex (their specificity for ocular dominance, orientation and direction of motion) and the emergence of the separate columnar maps for these properties onto the cortex.[10] For ocular dominance and orientation selectivity, development appears to occur in two phases, an initial experience-independent stage that is completed at or near birth, followed by a period of plasticity during which neural circuits and cortical maps are refined and subject to alteration by visual experience;[10-13] however, at least in ferrets,[14] directional selectivity may prove an exception to the two-stage viewpoints as this property emerges after birth without an experience-independent stage.

The initial studies of visual cortical development imposed periods of darkness or exclusively abnormal visual input that extended for many weeks or months; however, the recognition of a substantial anatomical and physiological scaffold at or near birth suggests that subsequent visual experience may serve more to refine a pre-existing cortical architecture than to sculpt it de novo. Examination of the former role for experience invites different approaches that include the use of mixed daily visual input, in which normal and abnormal visual inputs are pitted against each other on a daily basis. This paper summarises the major findings of studies from my laboratory of the effects of mixed daily visual experience in early postnatal life. Implementation of these studies required the use of a secure darkroom facility at Dalhousie University to allow strict supervision of the daily episodes of mixed visual exposure. The presence of the darkroom also permitted very recent and exciting research that points to previously unexpected beneficial effects of short periods of complete darkness on the developing visual system. These new results, as well as their implications for treatment of amblyopia, are also summarised. With respect to the latter studies, it is important to recognise the differences in outcome between complete darkness and binocular deprivation by other means. Detailed descriptions are provided of the darkroom facility as well as updates on the techniques employed to measure the visual capabilities of kittens as young as four weeks.

The Different Forms of Binocular Deprivation

Although a completely dark environment provides the purest form of visual deprivation, various forms of diffusers that eliminate or severely limit the transmission of patterned light have also been touted and used as alternate means of implementation. The most widely used form of such deprivation is binocular eyelid suture[15] that from an anthropomorphic perspective may even appear to be an equivalent form of deprivation to darkness. For the major animal species employed in developmental investigations, the two forms of deprivation are not comparable because the composition and thickness of the eyelids of animals are such that they are both far more transparent and less effective diffusers than those of humans. Photometric measurements of light transmission through kitten eyelids of different ages reveal a reduction of three to four log units for an adult cat but only about two log units for four- to five-week-old kittens.[16-18] Possibly as a consequence of skin pigmentation, there is considerable variation between animals, but even in a single animal the two eyelids may differ in transmission by one log unit.[18] The diffuse light transmitted through the eyelids permits cats to make luminance discriminations and allow the measurement of absolute light thresholds.[16] In addition, it is possible to plot crude receptive fields for about 30 per cent of cortical cells in kittens at four to five weeks of age[18] through closed eyelids.

Because some pattern information is transmitted through the eyelids, the visual cortex can receive limited information about the shape and orientation of large objects (low spatial frequency information) but be deprived of fine detail or high spatial frequencies. On the other hand, deprivation in the form of darkness completely removes all visual input. The two forms of binocular deprivation produce very different results. Although some outcomes may be similar if the deprivation is brief,[11] when the deprivation lasts two or more months, the consequences for the visual cortex and for vision are quite different.[19-22] Binocular eyelid suture appears to allow the cortex to be crudely specified by the impoverished visual input, while complete darkness leaves the visual cortex in a highly plastic state, so that it can be specified by visual input, even when it is imposed well beyond the end of the critical period.[19, 23, 24]

The Construction of a Darkroom Facility

The darkroom I employ was constructed over 40 years ago for Dr Max Cynader, now Director of the Brain Research Centre at the University of British Columbia. It complies with the standard requirements for all animal holding areas, including washable floors and walls, as well as the ability to change and monitor air humidity levels. The facility incorporates in its design (Figure 1) features that make it possible to clean and change cages regularly and even monitor the weight of the kittens. The room in which the animals are housed (C1) can be entered only through two small dark anterooms (A1 and A2) that serve as safety features to ensure that the housing room remains light-tight at all times. Adjacent to the housing room is a slightly smaller darkroom (C2), for which external entry can be made only through a third dark anteroom (A3). The second darkroom allows the large cages, in which the animals are housed, to be changed for regular cleaning. An unclean cage is wheeled into the second darkroom, the doors between the two rooms closed and then the cage removed via the anteroom to be cleaned. A new clean cage can then be moved into the housing room by reversal of this process. Most kittens are placed in the darkroom facility well before weaning and are housed with their mother and their littermates in a large communal cage (1.5 m long × 0.7 m wide × 0.9 m high) that has a ledge (0.2 m wide) at both ends located 0.4 m from the cage floor.

Figure 1.

A scale drawing of the floor plan of the darkroom facility. The darkroom holding areas C1 and C2, as well as the three anterooms (A1, A2, A3) that allow access to these rooms, are shown shaded in grey. Kittens enter the main holding room, C1, in carriers through the anterooms A1 and A2, at which time there are three closed doors between them and the outside illuminated area. The kittens are placed with their mother in large rectangular cages (floor dimensions 1.5 m × 0.7 m) with a maximum of two such cages in the darkroom at any time. To clean a cage, the kittens are first removed to a carrier cage while the main cage is wheeled into the second darkroom, C2, and the two intervening doors between C1 and C2 are closed. The cage can then be removed through the anteroom, A3, to be cleaned and replaced by a fresh cage.

The darkroom facility incorporates a television camera that is trained on the cage in which animals are housed. An array of eight small light-emitting diodes that emit infrared radiation (longer than 820 nm) located adjacent to the TV camera can be turned on briefly to permit illumination of the kittens so that they can be viewed and checked for possible changes in health on a television monitor located outside of the darkroom facility. The kittens can be placed individually in a small basket that can be weighed by use of a weight-scale having a circular clock-face dial. The position of the pointer on the clock-face scale can be estimated quite accurately, even if the numbers are indistinct. Despite expressions of concern that kittens reared in darkness may lose weight, this occurs vary rarely. In fact, it has been our experience that kittens reared in the darkroom tend to gain weight faster than those reared in the light possibly because of their lower motor activity. To entrain a circadian activity cycle for the animals, a radio is automatically turned on and off at times that correspond to the lighting cycle for the regular colony rooms.

Behavioural Testing of the Acuity of Kittens

It would be convenient if the acuity of a cat could be determined by means of an eye chart, as shown by the cartoon (Figure 2). Although impractical in the form shown, the technique that we use borrows on two features of contemporary logMAR eye charts. As with such charts, we employ a descending method of limits beginning with large suprathreshold stimuli (the letters at the top of the chart) and reduce their size gradually to threshold in small logarithmic steps. Unlike an eye chart, where there are three lines per octave (a factor of two), the step sizes we employ are much smaller with 12 logarithmic steps per octave. Our impression over the years has been that such small changes, that are barely perceptible to a human, are necessary so that kittens continue to respond solely to the visual cues as the stimuli become smaller. With larger steps in stimulus size in a two-alternative choice discrimination, kittens may no longer continue to respond solely to the visual cue and instead switch to non-visual strategies, such as position preferences, alternation patterns or jumps to the side of last reward.

Figure 2.

A purely hypothetical way to test the visual acuity of a cat. Note that the acuity chart shares some of the characteristics of a human logMAR chart, such as a logarithmic progression of stimulus size from top to bottom.

To measure a visual threshold and enable documentation of fast changes in this threshold with time, it is necessary to adopt a procedure that allows both the visual discrimination and the way in which the animal indicates its choice to be learned fast. For the latter, the choice should be made by means of a motor action that is spontaneously expressed by a young animal rather than a complex motor pattern, such as a nose press or paw motion that itself may take considerable time to learn. The task we employ exploits normal play behaviour; when placed on a small, elevated surface, kittens jump quickly to the floor. Effectively, we then have only to train the kitten to jump to one of two adjacent stimuli beneath it, for a food reward and petting. Two versions of the jumping stand apparatus[25] are illustrated diagrammatically in Figure 3. On the left is the original version that employs printed square-wave gratings as stimuli, while on the right is the version employed for more complex stimuli, such as coherent motion displays (as illustrated), that are shown on a computer monitor beneath a glass plate onto which the kitten jumps. The simple jumping stand on the left is used for training all animals, measurements of grating acuity and for testing for the presence of rudimentary vision after deprivation. The two stimuli are placed on separate hinged surfaces (doors) that allow the two doors independently to swing open. For training, one of the doors is open so that the kitten is confronted with just one stimulus, a vertical grating having a large period (32 mm), adjacent to a hole as illustrated in Figure 4. The jumping platform is set at a low height (three to five centimetres) by manipulation of the two yoked laboratory jacks with one of the two stimulus doors open. The kitten is coaxed from the platform to step onto a vertical grating placed on the closed door for food (wet cat food mixed with flakes of chicken liver) and petting. About 10 repetitions of this routine are provided with the position of the grating switched from side to side in a quasi-random order. The jumping platform is raised gently between trials to establish the maximum height for which the kitten can step or jump with ease. After 10 repetitions, both stimulus doors are closed, so that the kitten is now confronted with a choice between a vertical and a horizontal grating with the same period (spatial frequency). The majority of kittens continue to jump or step onto the vertical grating. If the kitten jumps onto the horizontal grating, it is neither fed nor petted and is required immediately to repeat the trial. About 10 such trials are provided on the first training day with the jumping platform raised gently after each jump to maximise the height.

Figure 3.

Sketches of two forms of the jumping stand technique. On the left is the stand employed for measurement of grating acuity and for the initial training of all kittens. The jumping platform can be raised in a continuous fashion by suitable manipulation of the two yoked laboratory jacks, on which the platform is fastened.

Figure 4.

A photograph of a kitten that had been monocularly deprived to eight weeks of age attempting to find the closed door on the jumping stand by use of its deprived eye shortly after termination of the period of deprivation. Note that the kitten appears to employ only non-visual cues, primarily touch, to find the closed door.

The next day after the kitten repeats five trials stepping onto the vertical grating with the other stimulus door open, the door is closed and 20 trials are provided with the horizontal grating in place. Once the kitten makes 10 consecutively correct jumps to the vertical grating, the period of the gratings is reduced. After five consecutively correct trials the period is reduced another step. The total number of trials is increased gradually on successive days, while all the time increasing the height of the jumping platform. Although kittens usually learn the task in only two to three days, in practice acuity measurements are not made until the kitten can jump from 35 cm or more, which many kittens can accomplish by five weeks of age but is rarely possible at four weeks. The side on which the vertical grating appears is decided on the basis of a quasi-random Gellerman series[26] that restricts the number of consecutive presentations on the same side to avoid the emergence of a side preference, one of the most common non-visual strategies that cats adopt.

To measure visual acuity, a balance has to be struck between the number of trials at any spatial frequency and the total number of trials that a kitten can receive in a session without signs of discomfort. For gratings having low spatial frequencies, where the task is easy and the animal's choices are flawless, the spatial frequency is increased after only one or two trials. As the task becomes difficult, the minimum number of trials (where performance is flawless) is increased, first to three and then to five. After an error, the kitten has to make five consecutively correct responses or else achieve either seven, eight or nine correct responses in a maximum of 10 trials. Usually performance is near-flawless until one or two (1/12 octave) steps from threshold, where the animal's performance falls to chance accompanied by clear signs of difficulty that can include a long latency to jump, vocalisations and attempts to turn around and avoid the task. The threshold is defined conservatively as the highest spatial frequency for which the animal achieved criterion performance (that is, 70 to 100 per cent correct).

Many of the experiential manipulations described in this paper were begun when the animals were only four weeks old or else too young to obtain threshold measurements. In these cases, it was necessary for training to begin or be continued during an appropriate part of their experiential history (such as during the period of monocular deprivation) that preceded an important experiential manipulation (such as termination of monocular deprivation), during which longitudinal measurements of vision had to be made.

Immediately following some of the experiential manipulations I employ, the effects on the vision of one or both eyes may be extremely profound. On the jumping stand the animal may appear blind even after receiving extensive training prior to the experiential manipulation or even while possessing normal vision in the fellow eye. The operational definition employed for blindness can be appreciated from Figure 4, which illustrates an inability of a kitten to find the closed door on the jumping stand without the use of tactile or other non-visual cues. In the situation shown, the kitten has placed one paw on the divider between the grating and the open door on the right and is searching for the grating with its head and paw on this (the wrong) side. This particular photograph displays the inability of a kitten to find the grating by vision alone by use of its deprived eye, when it is first tested after a period of monocular deprivation from natural eye opening to eight weeks of age.

There are close parallels between the behavioural method we employ on young kittens and clinical vision testing of humans, as an assessment of visual capabilities must be obtained on the occasion of each session or visit. For kittens, the small litter sizes and the extensive time invested in rearing them with special visual exposure requires that the procedure be applicable to each animal. These requirements dictate that the visual test be simple, fast and flexible, so that the procedure can be modified to obtain data every time. For humans, this may require asking questions in different ways, while for kittens the presentation sequence may need to be modified, if the animal is inattentive, reluctant or prone to non-visual solutions, such as a position habit, at the beginning of a session. As with humans, the visual abilities of kittens after certain experiential manipulations may be so poor as to defy assessment with the usual tests. In these situations, simple operational tests are used, such as counting fingers (humans) or the ability to distinguish an open from a closed door on the jumping stand (kittens).

The Use of Mixed Daily Visual Input to Study the Role of Vision in Development

The realisation that the initial development of the central visual pathways proceeds without any guidance by visually driven neural activity implies that at an age when such activity can modulate development it acts upon a visual system that has already been partly specified. As a consequence, it is possible that at the time when visual input can modulate cortical development, not all visual input is equally effective. The possibility that certain visual input may be more effective than others stands in contrast to an extreme possibility explored in the early studies of visual system development that all visual input may be treated equally. These studies employed periods of early visual exposure that were exclusively abnormal so that they specifically excluded episodes of normal or other exposure. As a consequence, the results are difficult to interpret from the perspective of varying efficacies of visual input on cortical development. To the extent that the developing visual system is plastic, that is, can change in response to its visual input, then all visual input that could be classed as biased, unusual or abnormal, could lead to abnormal outcomes but may not easily allow conclusions to be drawn as to whether some inputs were more effective than others.

An alternative approach that finds parallels in studies of the development of birdsong[27, 28] and experiential influences on emmetropisation[29] is to pit on a daily basis, different exposures against each other, such as conspecific versus foreign songs or for emmetropisation, normal visual input versus form deprivation.[29] The strategy employed in the initial experiments[30, 31] is illustrated schematically in Figure 5 in the form of a 24 hour clock. From four to eight weeks of age, the animals received only seven hours of visual experience each day, during which a period of normal binocular exposure (BE) of various lengths was pitted against periods of (abnormal) monocular exposure (ME) of differing durations. Outside of the period of daily visual exposure, the kittens were placed with their mother and littermates in the darkroom. The initial experiments specifically explored for possible effects of the temporal order of the two exposures but as the outcomes were the same no matter whether the binocular exposure preceded or followed the monocular exposure, it appeared that the exposure received last did not receive preferential weight, a possible scenario based on work[32] that suggests that the effects of visual deprivation may be consolidated by sleep.

Figure 5.

A schematic representation of the daily mixed visual experience animals received from four to eight weeks of age. As illustrated by the 24-hour clock at the top, each animal received just seven hours of mixed visual experience each day from 9 am until 4 pm (hatched region) and at all other times remained with its littermates and mother in the darkroom (black regions). The left arrow below depicts the two main episodes of mixed visual experience that separate animals received each day, in which a period of monocular exposure (ME) either preceded (top) or followed (below) a period of normal binocular exposure (BE). The arrow to the right illustrates two other exposure conditions. For the first condition, the period of binocular exposure was split into two episodes that straddled a single period of monocular exposure. The second condition compared the effects of concordant versus discordant visual exposure during the daily periods of binocular exposure by the use of prisms (held in a special mask) that had orientations that were either the same (both base down) or opposite (one base-up, the other base-down) for the two eyes.

To implement the various regimens of mixed daily visual exposure, masks made of neoprene foam and fastened by Velcro strips were used to cover one eye, while the kittens wore a light cardboard ruff around their neck to prevent them from pawing at the mask,[33] which was removed to allow for a daily period of binocular exposure. In some experiments, the animals wore a mask that allowed for prisms of either the same or opposite orientation to be placed in front of each eye to allow for either concordant or discordant binocular exposure. Some kittens attempted to remove the masks on the first day but thereafter they were tolerated. The animals were monitored regularly to ensure that the masks had not been dislodged. Because mixed visual experience began at four weeks the kittens had to be trained on the jumping stand during the daily periods when both eyes were open. At the end of four weeks of mixed daily visual experience measurements were made of the visual acuity of the eye that had been occluded for part of each day.

The outcomes following four weeks of the two extreme exposure conditions, where either the length of monocular or binocular exposure was zero, were exactly as could be predicted. In the first situation (monocular exposure = 0; binocular exposure = 7) both eyes had equal visual acuity comparable to that of normally reared eight-week-old animals, while in the second condition (binocular exposure = 0) the eye that had been occluded by the mask for all seven hours each day was blind. Two possible classes of outcomes for non-zero values of either monocular or binocular exposure could be predicted based on different hypotheses as to how development of the central visual pathways is influenced by its visual input. If all visual input exerts an effect on the developing visual system, then the outcome would depend on how the two daily visual inputs are integrated with respect to their effects on cortical development. While a variety of outcomes could be proposed, they share in common the prediction that any daily period of monocular exposure should have a deleterious effect on the vision of the eye occluded each day. In other words, only when monocular exposure is zero would the two eyes achieve equal acuity. In contrast to the predictions based on positive weighting of all visual input on visual system development, the idea of a preferred visual input predicts a very different outcome. On the basis of the highly developed system of ocular dominance that exists at birth in primates, it could be reasonably assumed that the preferred input would be binocular. If the daily period of mixed visual input includes an episode of visual exposure that is matched closely to the preferred visual input and is of sufficient duration, then the visual system could develop normally, as a result of which the visual acuities of both eyes could be normal. The results, shown in Figure 6, in which the visual acuity of the deprived eye is plotted as a function of the amount of daily visual exposure, provide strong support for a preferred visual input. Even 30 minutes of daily binocular exposure was sufficient to partially overcome a period of monocular deprivation 13 times longer to permit some vision in the deprived eye. Even more remarkable was the demonstration that only two hours of daily visual exposure was sufficient to outweigh or protect against five hours of monocular exposure to allow the vision of the deprived eye to achieve normal levels as indicated by the horizontal dashed lines that show the range of values for normal kittens at eight weeks of age. This result provides a strong support for the view that binocular visual input is preferred over other visual input, a result that fits well with the early maturation of ocular dominance columns in kittens,[34] as well as the suggestion that links the timing of critical periods in the visual cortex to the special requirements for stereoscopic vision.[35] This possibility receives further support from the experiment depicted in Figure 5, which explored whether it was sufficient only for the two eyes to receive visual input at the same time or whether it further required that simultaneous visual input be concordant. These alternative explanations were examined by the use of prisms worn in special masks during a daily two hour period of binocular exposure (accompanied by a five hour period of monocular exposure) that had either the same orientation for the two eyes (both base-down) to allow concordant visual input or else had opposite orientations (one base-up, the other base-down) to produce discordant input to the two eyes. The results were very clear, as only in the former condition did the animal develop normal visual acuity in the deprived eye, indicating that the daily binocular exposure had to be both simultaneous and concordant to be effective.[31]

Figure 6.

Evidence of a preferred visual input for the developing visual system. A replot of published data[31] that displays the outcome of four weeks of daily mixed visual experience initiated at four weeks of age. The visual acuity of the deprived eye measured immediately at the end of the period of mixed daily visual input as a function of the amount of daily binocular visual experience. The horizontal dashed lines indicate the range of acuity values observed among normal kittens at eight weeks of age.

A further result from the experiments of mixed visual exposure that deserves mention (also depicted on the right of Figure 5) is one where the two hour binocular exposure was split into two equal one hour exposures that straddled the five hour period of monocular exposure; here it was as if the second one hour binocular exposure was ineffective as the result was identical to that for a single one hour binocular exposure each day.[31] To be effective, it appears that the binocular exposure must be provided in a continuous block and not distributed in two episodes.

The results of mixed daily visual experience could be expressed as a minimum amount of binocular exposure each day (two hours), or alternatively as a proportion of the total daily visual exposure (two of seven or approximately 30 per cent). To ascertain the most appropriate expression of the result, the study was repeated on two additional cohorts of kittens that received either 3.5 or 12 hours total daily visual exposure. If the outcomes were determined by an absolute amount of daily binocular exposure, the result would be the same for these two additional cohorts as well; two hours daily binocular exposure would be sufficient for the deprived eye to achieve normal visual acuity. By contrast, if the result were determined by the proportion of total exposure that was binocular, different amounts of binocular exposure would be required for the different cohorts corresponding to the same proportion. The results,[36] displayed as a graph in Figure 7, in which the acuity of the deprived eye is plotted as a function of the daily binocular exposure expressed as a proportion of the total visual exposure, support the idea that the critical daily binocular dose is approximately 30 per cent of the total visual exposure. Virtually concurrent with the experiments on kittens, another group[37, 38] carried out comparable studies of mixed early visual experience on monkeys. Despite a different experimental design, their results also suggest that a critical daily binocular exposure representing 30 per cent of the total visual exposure was sufficient to allow both the development of normal visual resolution and cortical ocular dominance.

Figure 7.

The acuity of the deprived eye plotted relative to that of the fellow eye as a function of the amount of daily binocular exposure expressed as a fraction of the total daily visual experience. Data are shown for three cohorts of kittens that received either 3.5, 7 or 12 hours total visual experience each day. Separate logistic functions have been fitted to the data for the 3.5- (dotted line) and seven-hour (dashed) cohorts, as well as for the two cohorts combined (black). Reproduced from Mitchell and colleagues.[36]

Potential Therapeutic Effects of Complete Darkness

Although the experiments on mixed visual exposure could have clinical applications, as they suggest a possible way to insulate against the development of amblyopia, a potentially far more clinically important outcome has emerged from a very recent study[39] in collaboration with Dr Kevin Duffy on the effects of a 10-day period of complete darkness on amblyopic kittens. The investigation was motivated by prior findings[40] that suggested that a short period of darkness might effectively reset the maturation of stable cytoskeletal components in the kitten visual cortex to an earlier and more plastic stage of development. It follows that a period of darkness imposed on an amblyopic kitten might set the stage for greater recovery than would otherwise occur. The studies that support the idea that darkness might reset the cortex to a more plastic stage are bypassed for now to highlight the behavioural findings that in many respects were unexpected but in all cases remarkable. The behavioural studies examined two times of imposition of darkness with respect to a one-week period of monocular deprivation (MD) imposed at 30 days of age (P30). Although the most unexpected results were obtained in the situation where darkness was imposed immediately after the period of monocular deprivation, the ‘immediate dark’ (ID) condition, the most insightful findings from a clinical perspective occurred in animals in which darkness was delayed with respect to the period of monocular deprivation, the ‘delayed dark’ (DD) condition. For the latter, darkness was imposed five to eight weeks after termination of the period of monocular deprivation at a time when the visual acuity of the deprived eye had recovered to a stable level that indicated a substantial amblyopia. The timing of the episodes of darkness imposed on the kittens in the immediate dark and delayed dark conditions are shown in Figure 8, in relation to the profile of the sensitive period for ocular dominance plasticity in the striate cortex.[41, 42]

Figure 8.

The length and timing of the 10-day period of darkness imposed on the two groups of kittens are shown by the horizontal black bars (labelled ID and DD) at the top in relation to the profile of the critical period for ocular dominance plasticity in the striate cortex. The latter profile has been calculated on the basis of the effects of a constant period of monocular deprivation imposed on animals at different ages. Black circles and open square symbols display the deprivation effect expressed as a deprivation index calculated from, respectively, the data of Olson and Freeman[41] (10 days of monocular deprivation) and Jones, Spear and Tong[42] (one month of monocular deprivation). The deprivation index represents the proportion of cells in the visual cortex dominated by the non-deprived eye computed in relation to the proportion observed in normal animals (expressed as a percentage). The index adopts a value of zero in a normal animal. The arrow indicates the start of the one-week period of monocular deprivation. The deprivation index has been plotted as a function of postnatal age. The dark period for the ‘immediate dark’ animals was initiated close to the peak of the critical period, while for the ‘delayed dark’ animals it occurred at about three months at a time when plasticity was in steady decline.

The results for the animals when darkness was delayed (DD animals) were remarkable as the amblyopia disappeared rapidly in just five to seven days.[39] To illustrate that this result was not confined to the temporal specifics of the prior period of monocular deprivation, results are shown in Figure 9 for two animals for which the period of monocular deprivation was far more extensive beginning at one week of age and terminated at six weeks. The procedures employed on these kittens were identical to those employed for the animals in the earlier studies.[39] One of the two animals (C219) was placed in darkness seven weeks after the end of the period of monocular deprivation, while its littermate (C218) remained in the illuminated colony room. In Figure 9, the acuity of the deprived eye is shown as a function of the time since termination of monocular deprivation but to highlight the changes after the period of darkness, the recovery in the first six weeks has not been shown. At the time that C219 was placed in darkness, the acuity of the deprived eye of both animals had virtually stabilised at a level about two octaves below that of the non-deprived eye. On removal from the darkroom the acuity of neither eye of C219 had changed but during the next seven days, the acuity of the deprived eye climbed rapidly to match that of the other eye; in other words, the amblyopia had disappeared. In striking contrast, the acuity of the deprived eye of the littermate (C218) that had remained in the light and was also tested daily did not change. That the finding from C219 was so similar to the data from the animals described earlier[39] that were deprived at P30 and for only a week indicates that darkness is just as effective, when imposed on animals that had no prior binocular experience before an amblyogenic period of monocular deprivation that was considerably longer than that employed earlier. In addition to the fact that the recovery of the grating acuity of the deprived eye was complete, it occurred while both eyes were open and without the need to occlude the fellow eye.

Figure 9.

The grating acuity of the deprived eye (open circles) and non-deprived eye as assessed by either monocular measurement (filled circles) or from measurement of binocular acuity (filled squares) of two kittens plotted as a function of the time since termination of monocular deprivation at six weeks of age. Data for the first six weeks of recovery are not shown. One kitten, C219, received a 10-day period of darkness at 92 days of age, while its littermate, C218, remained in the light. Note that the fast recovery of the vision of the deprived eye of C219 following the period of darkness was not accompanied by any change of vision in the equivalent eye of its littermate that remained in the light at the same time.

The results for the animals in the immediate dark condition were surprising, as they revealed some unanticipated effects of a brief period of darkness imposed early in postnatal life. At the end of the period of monocular deprivation, kittens were only 37 days old, an age close to the peak of the critical period (Figure 8). On removal from the light, the kittens appeared blind in not just the deprived eye as expected but also in the non-deprived eye. Thereafter, the vision of the two eyes improved in a similar way for both eyes; measurements of acuity showed that the acuities of the two eyes increased at the same rate but slowly in each eye, such that amblyopia never developed. The data from the delayed dark animals revealed no effects of darkness on the acuity of the non-deprived eye when imposed at 10 to 13 weeks of age; this and subsequent work demonstrates that the critical period for the deleterious effects of darkness on the visual acuity of the non-deprived eye is very short (less than 10 weeks).

These experiments demonstrated two previously unexpected results of a period of darkness. First, when imposed immediately after an early amblyogenic event, temporarily, there may be implications for the vision of both eyes but thereafter, the vision of the two eyes improves slowly together to normal levels in about seven weeks and amblyopia does not develop. Second, when darkness is delayed after a stable amblyopia has become entrenched, the subsequent recovery is fast, five to seven days, and at least for grating acuity, is complete. These results beg many questions, including the consequences of a dark interval for other visual capacities such as stereoscopic vision and whether the effectiveness of a dark period declines with age. Prior work on rats suggests that darkness may be effective even in adults.[43] There are also many unanswered issues with respect to the possible underlying molecular mechanisms.

Although the original study was motivated by observations of the effects of darkness on neurofilament protein levels, there are many other potential molecular candidates that may also be influenced by this particular experiential manipulation. In the parlance of current formulations[44, 45] of the molecular underpinnings of developmental plasticity, darkness could influence the expression of many if not all the known proteins (braking molecules) that may serve to reduce cortical plasticity. Additionally, the effectiveness of darkness may hinge on the fact that this experiential manipulation may cause levels of the various braking molecules to change in a propitious temporal order.

Clinical Implications

Although the therapeutic effects of darkness have been demonstrated only in relation to an animal model of deprivation amblyopia, it is most likely that a similar outcome would be observed on animals reared with a unilateral refractive error to induce anisometropic amblyopia. On the other hand, a test of the applicability of darkness as a therapy for strabismic amblyopia would face a difficult practical hurdle as the strabismus would have to be eliminated immediately prior to the interval of darkness for it to be of potential benefit. If any ocular deviation were present at the time the animal was removed from darkness, the subsequent period of heightened cortical plasticity could cause the pre-existing strabismic amblyopia to be exacerbated. Because of its severity and high prevalence, it will be important to examine the potential therapeutic benefits of darkness on animal models of strabismic amblyopia.

As the underlying molecular mechanisms of developmental plasticity are likely highly conserved between mammalian species, it is possible to contemplate the potential future use of 10 days of darkness therapy as an adjunct to treatment of amblyopia. Indeed, there exists a report[46] of the use of bilateral occlusion for 17 days on a single neonate after two weeks of dense unilateral vitreous haemorrhage and hyphaema; follow-up at three years of age revealed equal visual acuities (6/9) in the two eyes as if the therapy had insulated against amblyopia. Of particular value is its potential applicability to children who benefit more than adults in terms of life and career prospects from successful treatment. The prospect that benefits follow very fast on the heels of the period of darkness, means that certain impediments to success with conventional treatments, such as compliance with patching and poor motivation, could be bypassed. Of course, there are many practical issues yet to be confronted, including the challenging but not insurmountable means by which to implement a 10-day period of complete darkness. The difficulty of implementation of the therapy will be impacted by the results of current work that examines the parameters of the dark period, including the benefits of shorter periods of darkness, as well as the impact of brief daily episodes of scotopic illumination that may alleviate a number of the obvious practical considerations associated with immersion in a completely dark environment. Even at this stage, it is possible to predict some of the requirements for potential success. Because the benefits of darkness appear to be linked to its ability to reset the cortex to an earlier stage of development, impediments to clear concordant binocular visual input should be eliminated or substantially reduced before darkness is imposed, as otherwise the amblyopia may be made worse. Potentially, darkness therapy may serve best to boost and hasten the outcomes from newly developed binocular therapies for amblyopia that could be implemented immediately after the period of darkness.[47]

Acknowledgements

I wish to thank Drs Kathryn Murphy, Frank Sengpiel, Peter Kind, Sam Schartzkopf and especially Kevin Duffy for many discussions and their invaluable contributions to this research. The research also benefitted from the many important contributions of various technicians and students, particularly Jan Kennie, Kaitlyn Holman, Matthew Smithen, Katleyn MacNeill and Jordan Boudreau. Special thanks to the cats and particularly Batman, Robin, Tipster, Joker, Bah, Humbug, Scroogina, Novak and Samantha. The research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (A7660), from the Canadian Institutes of Health Research to Kevin Duffy, N. Crowder and DEM (102653) and from the Critical Period Revisited Network of the James S MacDonnell Foundation.

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