In contrast to findings in White Leghorn chicks (Li et al. 1995), high hyperopia was not a feature of constant light rearing in unoperated birds of the dark-feathered Shaver Black strain used here (Table 3). Thus, although some unoperated chicks did develop a degree of hyperopia commensurate with that seen in previous work (i.e., +8.00 to +10.00 D), the remaining chicks were either isometropic with, or more myopic than, age-matched chicks raised under diurnal lighting. Unlike unoperated chicks, birds with unilateral disruption of centrifugal neurons raised under constant light developed a transient hyperopic shift in the eye contralateral to the lesion. Thus, contralateral eyes were more hyperopic than ipsilateral eyes 7 days post-surgery, but not 21 days post-surgery. In contrast, ipsilateral eyes showed a lesion-induced hyperopia at 21 days post-surgery that was not evident at the earlier 7 days post-surgery time point (Fig. 6).
The finding of a transient, initial hyperopia in the contralateral eyes of chicks with a highly successful ION/IOTr lesion concurs with our findings in ION/IOTr-lesioned chicks raised under normal diurnal lighting (Dillingham et al. 2013). In the present constant light study, by the end of the 21-day observation period, contralateral eyes had developed the same ocular phenotype as found in unoperated chicks, i.e., anterior chamber shortening and vitreous chamber elongation. More surprisingly, the degree of centrifugal disruption was significantly correlated with relative axial shortening in ipsilateral eyes by the end of the 21-day follow-up period.
From prior work, the ocular phenotype resulting from constant light is known to be characterized by hyperopia, corneal flattening, increased intra-ocular pressure (IOP), reduced anterior chamber depth, lens thinning and vitreous chamber elongation (Bartmann et al. 1994; Li et al. 1995; Wahl et al. 2015). These effects on the eyes are principally due to the disruption of circadian rhythms (Weiss and Schaeffel 1993) and the concomitant absence of the normal diurnal fluctuations in dopamine and melatonin levels (Parkinson and Rando 1983). In the present study, chicks raised in constant light conditions developed some but not all of these features: reduced anterior chamber depth, lens thinning and vitreous chamber elongation were present, while hyperopia was not. This lack of hyperopia appeared to be because the constant light-induced anterior chamber shortening was matched by commensurate VCD elongation, suggesting an emmetropization response to the reduced power of a flatter cornea. Presumably, our results for constant light rearing may differ from those of other investigators either because of inter-strain variability (e.g., heavy versus light pigmentation) and/or differences in experimental conditions, e.g., light intensity.
Interestingly, primates raised under constant light do not exhibit an ocular phenotype comparable to that in chicks (Smith et al. 2001). Instead, ocular development is normal albeit with the suggestion of increased incidence of refractive anomalies, including myopia and slight axial anisometropias, implying that the emmetropization process may be affected, but to a far lesser degree than observed in the chicken. In other ways, ocular development in both chick and primate models has shown a great degree of commonality under a number of other experimental conditions (Yinon 1984; Smith et al. 1999, 2009). Nevertheless, in the case of constant light rearing, it does not appear that findings in the chick can be extrapolated to the epidemiology of human ametropias (Smith et al. 2001). Thus, while our findings are, perhaps, only relevant to mechanisms of emmetropization in the chick model, the significance of understanding the limitations of the relevance of results obtained using lower vertebrate animal models to higher vertebrates cannot be overstated. Primates have a far less well-established centrifugal projection (Gastinger et al. 2006) than the chick, with distinct anatomical and neurochemical and characteristics that are, if at all, more comparable to the ectopic centrifugal projection in birds. One possibility is that the functionality achieved through tectal feedback to the retina in birds is achieved, in primates, through cortical feedback to visual thalamic nuclei. Indeed, it is interesting to note that those bird species with a more diffuse CVS organization, i.e., without an isthmo-optic nucleus, tend to have a residual population of ectopic neurons (Gutierrez-Ibanez et al. 2012). Such variations in the size and/or degree of organization in the CVS, both within and between orders, are likely to be critical to establishing its function.
The refractive state of contralateral eyes (REFcontra
) of IOTr lesioned chicks, measured at 7 days post-surgery, showed a significant positive relationship with SuccessION+EA, a feature analogous to that observed in lesioned chicks raised under diurnal light conditions, in which disruption of centrifugal neurons caused an initial transient hyperopic shift in contralateral eyes at 7 days post-surgery that had resolved by 21 days post-surgery (Dillingham et al. 2013). In chicks raised under constant light conditions, the VCD of ipsilateral eyes, i.e., ipsilateral to the IOTr lesion, exhibited a significant negative relationship with SuccessION+EA, i.e., high lesion success was correlated with reduced VCD. In a previous study (Dillingham et al. 2013), we have discussed possible explanations for this effect. Briefly, we proposed that the initial intra-retinal effect of the lesion was one of transient hyperactivity of the nitric oxide producing target cells of ION neuron projections (IOTCs), resulting from their partial deafferentation (i.e., diaschisis; Stavraky 1961; Sharpless 1964; von Monakow 1969). We proposed that this may have caused a short-term increase in the rate of nitric oxide release. Such an effect would result in the inhibition of normal vitreous chamber elongation and, thus, explain the observed vitreous chamber-dependent hyperopia. Whatever the mechanism for this transient hyperopia, the focus of this discussion will be on the unexpected finding that, by 3 weeks post-surgery, the ipsilateral eyes of chicks showed a significant trend towards becoming shorter with increased IOTr lesion success SuccessION+EA (Fig. 7).
Our findings are best considered in the context of optic nerve section (ONS) emmetropization studies (Table 3), as severance of the optic nerve not only transects retinal efferent axons, but also centrifugal neurons to the retina. Under diurnal light conditions, chicks with ONS develop moderate hyperopia in the affected eye (Troilo et al. 1987). To our knowledge, only one study has investigated the combined effects of constant light treatment and ONS surgery on emmetropization (Li and Howland 2000). In that study, chicks were raised under constant light for 7 days before undergoing unilateral ONS surgery. Four weeks following surgery, both contralateral and ipsilateral eyes showed no differences from previously reported constant light effects in chicks with intact optic nerves (Li et al. 1995). Specifically, no significant refractive or ocular component dimension asymmetry was observed. In addition to the differences in experimental time points, the constant light + ONS study of Li and Howland and the present study differ in two important ways. Firstly, our experimental paradigm did not disrupt retinal efferent axons in the optic nerve. By lesioning the IOTr, only ~10,000 centrifugal efferent axons (~0.5 % of the total number of optic nerve fibers) were targeted, deafferenting only the small proportion of retinal neurons that receive projections from/are innervated by the CVS. Secondly, while ONS surgery effects are confined to the contralateral eye, IOTr or ION lesions are likely to disrupt pathways to both eyes as a result of the ipsilateral EA pathway. Thus, the complex pattern of lesion-associated changes reported here might be a consequence of both of these pathways being disconnected, raising the possibility that the bilateral centrifugal projections are a requirement for consensual, i.e., symmetric, refractive development. It is worthy of note that direct retino-retinal connections have been shown to exist, e.g., Avellaneda-Chevrier et al. (2015) and Nadal-Nicolás et al. (2015). While such connections could, in theory, underlie the effects described here, their significance is unclear and they are, in all likelihood, too few in number to sustain any physiological influence.
An ipsilateral eye effect was evident from the significant negative association that was observed between VCDipsi and SuccessION+EA for individual subjects (F
1,15 = 12.720, p = 0.003), while only a much weaker, non-significant negative correlation existed between VCDcontra and percentage lesion success (F
1,15 = 2.203, p = 0.158), again consistent with an ipsilateral eye effect. These data raise the possibility that both contralateral and ipsilateral centrifugal pathways may act to regulate emmetropization at some level, perhaps via independent pathways. It is, therefore, clear that, with hindsight, a limitation of this study was the inability to quantify the degree to which the ipsilateral EA projection was disrupted by the lesion of the IOTr. A number of emmetropization studies have reported fellow untreated eye effects following unilateral experimental paradigms. For example, changes in ZENK expression were found predominantly in ipsilateral eyes following unilateral plus or minus lens wear (Bitzer and Schaeffel 2002). Furthermore, as mentioned above, when chicks were form deprived unilaterally, the normal oscillatory diurnal growth (i.e., growth during the day and slight shrinkage at night) of the ipsilateral eye was also disrupted, and although the eye remained emmetropic, growth was consistently slow and did not exhibit a diurnal pattern (Weiss and Schaeffel 1993). Such experiments, like the present study, provide evidence that a reciprocal connection between the eye and the brain is necessary for normal ocular growth patterns associated with circadian rhythms. Moreover, our results offer additional support for the hypothesis that symmetrical growth of fellow eyes is dependent on inter-ocular communication, presumably either through systemic, i.e., endocrine, responses to visual stimuli, or through cross-talk between higher visual centers, such as the CVS, given its bilateral projection to the two eyes.
The neurochemical properties of IOTCs have been well established as exhibiting nitric oxide synthase and NADPH-diaphorase immunoreactivity (Morgan et al. 1994; Fischer and Stell 1999; Wilson and Lindstrom 2011). Indeed, these findings, in part, formed the basis for our proposed interpretation of previous findings in CVS-lesioned chicks raised under diurnal light (Dillingham et al. 2013), given that nitric oxide has been implicated in ocular growth mechanisms (Fujikado et al. 1997; Nickla et al. 2009). On the other hand, equivalent neurochemical data are not available for the target cells of ectopic centrifugal neurons, including the subpopulation that projects to the ipsilateral retina. Under normal diurnal light conditions, disruption of centrifugal neurons in the same strain of chicks implicated isthmo-optic, rather than ectopic, centrifugal neurons in the observed anisometropia (characterized by transient, contralateral eye hyperopia; Dillingham et al. 2013). In the present study, although no difference in the strength of association between ION or EA lesion success and anisometropia was evident, ION lesion success explained a greater amount of the variance in ΔVCD than EA lesion success. In contrast, and of particular interest, while both were significant, the absolute measurements for VCD in ipsilateral eyes (at 21 days post-surgery) showed a stronger, negative association with EA, rather than ION, lesion success (Table 2). The general consensus that ipsilaterally projecting centrifugal neurons to the retina are derived from the ectopic neuronal population, combined with their implication in the ipsilateral eye effect described here, raises the possibility that ectopic centrifugal neurons play a role in circadian rhythm-driven modulation of emmetropization mechanisms.
As the electrophysiological and neurochemical mechanisms of the ectopic centrifugal projection have not been elucidated, any mechanistic explanation for the observed ipsilateral eye hyperopia in the context of the ipsilateral centrifugal pathway would only be speculative. Instead, we will consider two biogenic amines, melatonin and dopamine, in the broader context of our results. First, on the one hand, melatonin is likely to be a critical factor in the development of the ocular phenotype associated with constant light rearing. Endocrine melatonin synthesis by the pineal gland is light dependent. Chicks reared under constant light conditions, but wearing hoods that shield the pineal gland from the surrounding illumination, do not develop the same anterior chamber characteristics (Li and Howland 2006). Similarly, chemical destruction of the retina does not disrupt corneal growth rates (Oishi et al. 1996). Thus, it is likely that systemic, rather than local, melatonin mechanisms are responsible for the observed anterior chamber effects.
In the present study, disruption of centrifugal neurons did not result in an altered anterior chamber phenotype, suggesting that the observed CVS lesion-associated posterior chamber effects were presumably the consequence of disrupted local retinal mechanisms (i.e., the absence of CVS input), rather than a compensation for corneal or lenticular changes. On the other hand, dopamine has similarly been implicated in the mechanisms responsible for the axial elongation associated with form deprivation (Bartmann et al. 1994). Unilateral form deprivation results in the bilateral disruption of diurnal patterns of ocular growth (Weiss and Schaeffel 1993), i.e., in both contralateral and ipsilateral eyes. In addition, dopamine and DOPAC (a metabolite of dopamine) levels are reduced following both form deprivation (Bartmann et al. 1994) and constant light rearing. In both cases, the reduction was between 30 and 40 % and peaked following 8–14 days of either treatment. In the context of our findings, the delay between the onset of a constant light treatment and the point at which dopamine levels are significantly reduced is suggestive, given that the major changes in eye growth rates reported here took place during this time. In addition, dopamine may be an important explanatory factor as a result of its reported interactions with retinal nitric oxide in the bovine retina. Nitric oxide antagonists were shown to increase endogenous dopamine levels, while nitric oxide generators reduced retinal dopamine levels (Bugnon et al. 1994).
A recent study suggests dopamine and nitric oxide mechanisms are interdependent in the chick retina, with dopamine seemingly acting upstream of nitric oxide (Nickla et al. 2013). In that study, inhibition of axial elongation in response to hyperopic defocus or form deprivation, through treatment with dopamine agonists, was prevented by intravitreal injection of nitric oxide antagonists, i.e., inhibition of nitric oxide led to disinhibition of vitreous chamber elongation. Thus, in the present study, a potential mechanistic explanation for the additional influence of constant light on emmetropization following disruption of the CVS is that constant light-induced increased dopamine levels occur simultaneously with lesion-induced reductions in nitric oxide release, resulting in the observed inhibition of vitreous chamber elongation. However, this does not provide an explanation for the temporal pattern of changes observed first in the contralateral eye and then in the ipsilateral eye.
In summary, under constant light conditions, unilateral disruption of centrifugal ION and EA neurons (and/or their axons) projecting to the retina of the contralateral eye induced an initial, transient hyperopia in the contralateral eye, which had resolved 2 weeks later, in a manner similar to the lesion-induced changes that have previously been reported under normal diurnal conditions. However, under constant light conditions, centrifugal disruption induced relative axial hyperopia in the eye ipsilateral to the lesion, which persisted for at least the 21-day duration of the experiment. This finding implicates ipsilaterally projecting centrifugal EA neurons in the regulation of emmetropization mechanisms, perhaps via nitric oxide/dopaminergic pathways. The complexity of ametropias associated with CVS lesions in chicks under different lighting conditions suggests that the CVS may play an important role in regulating a light-dependent lateralization of development. Indeed, the rudimentary nature of the CVS in mammals might help explain the fact that constant light rearing in primate models only produces mild ametropias, if any.