THE ANATOMICAL RECORD 290:341–348 (2007) Light-Dependent Retinal Innervation of the Rat Superior Colliculus J. ROXANNE PRICHARD,1* HILDA S. ARMACANQUI,2 RUTH M. BENCA,3 2 AND MARY BEHAN 1 Behavioral Neuroscience Program, University of St. Thomas, St. Paul, Minnesota 2 Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin 3 Department of Psychiatry, University of Wisconsin, Madison, Wisconsin ABSTRACT Mammalian retinal projections are divided into two anatomically and functionally distinct systems: the primary visual system, which mediates conscious visual processing, and the subcortical visual system, which mediates nonconscious responses to light. Light deprivation during a critical period in development alters the anatomy, physiology, and function of the primary visual system in many mammalian species. However, little is known about the inﬂuence of dark-rearing on the development of the subcortical visual system. To evaluate whether the early lighting environment alters the anatomy of the subcortical visual system, we examined the retinas and retinofugal projections of rats reared in a 12:12 light/dark cycle or in constant dark from birth to 4 months of age. We found that dark-rearing was associated with a reduction in the distribution of retinal ﬁbers in the stratum opticum of the contralateral superior colliculus. In contrast to the plasticity of the retinocollicular projection, retinal input to sleep, circadian, and pupillary control centers in the hypothalamus, pretectum, and lateral geniculate complex was unaffected by dark-rearing. A decrease in retinal innervation of the stratum opticum and intermediate layers of the superior colliculus may account for some of the deﬁcits in multisensory integration that have been observed in dark-reared animals of several species. Anat Rec, 290:341–348, 2007. Ó 2007 Wiley-Liss, Inc. Key words: superior colliculus; dark-rearing; retinofugal projections; subcortical visual system; plasticity Mammalian retinofugal projections are divided into two anatomically and functionally distinct systems. The primary visual system, consisting of the retinorecipient dorsal lateral geniculate nucleus (dLGN) and the visual cortex, mediates conscious visual processing; the subcortical visual system, a collection of retinorecipient nuclei throughout the hypothalamus and midbrain, mediates non-conscious responses to light, including entrainment of the circadian rhythm, acute changes in behavioral state, pupil dilation and constriction, and reﬂexive movements of the eyes, neck and head (Schneider, 1969; Harrington, 1997; Morin and Blanchard, 1998). Dark-rearing during a critical period in development has a deleterious effect on the development of the primary visual system in many mammalian species. Beginning with Hubel and Wiesel’s seminal studies on darkreared and monocularly deprived kittens (Hubel and Ó 2007 WILEY-LISS, INC. Wiesel, 1963; Wiesel and Hubel, 1965), substantial research conﬁrms that a functionally signiﬁcant portion of the development of the primary visual system occurs postnatally and is light- and activity-dependent. Speciﬁcally, dark-rearing impairs the development of the dLGN by reducing the proportion of relay Y-cells (Kratz et al., 1979; Mower et al., 1981) and by increasing the convergence of on- and off-center receptive ﬁeld maps (Akerman et al., 2002). In the visual cortex, dark-rear- *Correspondence to: J. Roxanne Prichard, University of St. Thomas, Mail JRC LL56, 2115 Summit Avenue, St. Paul, MN 55105. Fax: 651-962-5051. E-mail: firstname.lastname@example.org Received 27 July 2005; Accepted 4 January 2007 DOI 10.1002/ar.20424 Published online 23 February 2007 in Wiley InterScience (www. interscience.wiley.com). 342 PRICHARD ET AL. ing impairs development by slowing the maturation of glia and pruning of neurons (reviewed in Sherman and Spear, 1982), disrupting columnar and orientation-speciﬁc organization (Chapman et al., 1999), impairing tissue vascularization (Argandona and Lafuente, 1996, 2000), and altering gamma aminobutyric acid (GABA)ergic (Benevento et al., 1995) and glutamatergic synaptic transmission (Czepita et al., 1994). Collectively, these dark-rearing-induced anatomical and physiological changes in the primary visual system signiﬁcantly impair visual processing (Sherman, 1973; Caspy and Babkoff, 1983; Fagiolini et al., 1994). By comparison with the dLGN and visual cortex, the effects of dark-rearing on the subcortical visual system have received less attention. The neonatal lighting environment has been shown to inﬂuence some physiological properties of neurons in the superior colliculus (SC), a laminated structure that receives topographically organized inputs encoding visual, auditory, and somatosensory stimuli, and that orchestrates multiple immediate motor responses to these stimuli (Huerta and Harting, 1984; Stein et al., 1993; McHafﬁe et al., 2002). For example, dark-rearing is associated with a nonreversible loss in directionality and binocular convergence in neurons of the cat superior colliculus (WickelgrenGordon, 1972; Hoffman and Sherman, 1975; Flandrin and Jeannerod, 1977). In rodents, dark-rearing has been shown to slow the developmentally regulated increase in apoptotic neurons in the mouse SC (Sasaki et al., 1999) and reduce receptive ﬁeld reﬁnement in the rat SC (Binns and Salt, 1997). However, very little is known about the inﬂuence of early light exposure on the development of retinofugal projections per se to the SC and other nuclei of the subcortical visual system. Research from our laboratory has shown that sleep responses to light and dark are inﬂuenced by dark-rearing; speciﬁcally, dark-rearing enhances dark pulse-induced REM sleep triggering (Prichard et al., 2004), a phenomenon mediated by the pretectal nuclei in albino rats (Miller et al., 1999). Similarly, dark-reared rats have a delayed acquisition of circadian rhythm, as compared to light-dark (LD)-reared rats (Cambras and DiezNoguera, 1991). Given that the anatomy and physiology of the primary visual system is disrupted by dark-rearing, and that sleep and circadian responses to light mediated by the subcortical visual system are compromised by dark-rearing, we hypothesized that darkrearing might inﬂuence the development of retinofugal projections to the subcortical visual system. To test this hypothesis, we used the highly sensitive anterograde tracer cholera toxin b (CTb) to label retinal projections in dark- and LD-reared rats. The results from this experiment provide the ﬁrst systematic investigation of the effects of dark-rearing on retinal input to the rat subcortical visual system. MATERIALS AND METHODS Albino rats (Charles River F344) were reared in either constant dark (DD; 0 lux) or in a control 12:12 LD environment (lights-on at 06:00, 65 lux; lights-off at 18:00, 0 lux) from before birth until adulthood (4 months). One week prior to giving birth, pregnant dams were individually housed in clear acrylic cages (26 cm width 3 40 cm length 3 60 cm height) in the two lighting condi- tions. Dark-reared rats were totally deprived of light; the routine care of the DD rats was done by personnel wearing infrared goggles (D-2MV; R and R International Trade, Fountain Valley, CA). At postnatal day (P) 21, dams were removed from the experiment and pups were housed in same-sex groups of three and had access to food and water ad libitum throughout the experiment. Research protocols were approved by the University of Wisconsin School of Medicine Animal Care and Use Committee and conform to National Institutes of Health guidelines. Although albino rats are known to have several anatomical anomalies in the visual system projections, such as an enhanced contralateral retinal projection to the dorsal lateral geniculate nucleus, as compared with pigmented rats (reviewed in Sefton and Dreher, 1995), they are a valuable model for examining the effects of early lighting environment on the subcortical visual system. First, albino rats have an exaggerated and observable sleep-wake response to acute light-dark shifts, as compared to pigmented rats (Benca et al., 1991, 1998; Obermeyer and Benca, 1999), and sleep and circadian locomotor activity is disrupted in albino rats by dark- and light-rearing (Canal-Corretger, 2001; Prichard et al., 2004). Second, the difference in retinofugal projections between pigmented and albino rats is relatively minor; of the 13 nuclei in the subcortical visual system, only 4 (the ventral lateral hypothalamus, the commissural pretectal area, the posterior limitans, and the intergeniculate leaﬂet) show a quantiﬁable enhancement of contralateral retinal input in albino versus pigmented rats (Fleming et al., 2006). In order to visualize retinofugal projections, nine DD and eight LD rats were injected intraocularly with the anterograde tracer CTb (0.4 ml; List Biologicals) at 4 months of age in their home lighting environment (DD or LD), as described previously (Prichard et al., 2002). CTb, which does not travel transsynaptically (Coolen et al., 1999), was chosen for its superior uptake, sensitivity, rapid transport, and frequent use in analysis of the retinohypothalamic tract (Angelucci et al., 1996). After a 5-day survival in their home lighting environment (LD or DD), rats were euthanized by overdose with sodium pentobarbital (1.0 mg/kg, i.p.), received heparin intracardially (0.07 mg/kg), and were perfused transcardially with 500 ml of cold 4% paraformaldehyde (pH 7.4). DD rats were euthanized in the dark, and their eyes were bandaged to prevent exposure to light during the perfusion. Brains and eyes were dissected and stored overnight at 48C, then cryoprotected (20% sucrose and 5% glycerin in 0.1 M phosphate buffer) for 24 hr at 48C. Eyes were embedded in parafﬁn, sectioned sagitally at 10 mm, and stained with hematoxylin and eosin. Brains were sectioned coronally at 50 mm. Sequential sections through the diencephalon and mesencephalon were reacted with antisera to CTb (goat; 1:20,000; List Biologicals; visualized with 0.04% 3,30 -diaminobenzidine). Tissues from LD and DD animals were reacted on the same day, and negative controls were run concurrently. For illustration purposes, image quality was optimized by making selected adjustments in gamma, brightness, and contrast using Photoshop version 6.0 software. To evaluate the patency of retinal projections to subcortical visual nuclei, 12–15 representative coronal sections from the level of the optic chiasm to the caudal RETINOCOLLICULAR PROJECTION IN DARK-REARING pole of the SC were examined in animals in each rearing condition. Rats were removed from the study (three LD, three DD) if sections through the SC showed patchy label in the superﬁcial layer, which would reﬂect injury to the retinal ganglion cell layer associated with intraocular injection (Redgrave et al., 1993). Under bright- and dark-ﬁeld microscopy, the distribution and density of CTb label in each of the subcortical visual nuclei was compared to published reports (Prichard et al., 2002). If a difference in CTb density or distribution between the two rearing groups was detected in any retinorecipient region, a quantitative and qualitative analysis of retinal terminal density and distribution was performed. Quantitative analysis of pixel intensity was used when the distribution of retinal terminals was dense and even, such as in the superﬁcial layers of the superior colliculus; qualitative analysis of the relative staining intensity was used when the distribution of retinal terminals was patchy and irregular, such as in the stratum opticum and intermediate layers of the superior colliculus. In areas of dense, evenly distributed label in the superior colliculus, digital photographs (16 bit; 65,536 gray levels) of brain sections were taken using a SPOT camera (Diagnostic Instruments), and quantitative image analysis was performed with ImagePro Plus software. All images were obtained under identical bright-ﬁeld illumination. Matched sections through the rostral, middle, and caudal regions of the nucleus were photographed. A circular sample area was deﬁned (27,134 mm2) and three optical density measurements (medial, middle, and lateral) were obtained from the superﬁcial layers in each section. Optical density, the relative darkness of each pixel, was determined by plotting the mean intensity level of pixels within the deﬁned area on a standard optical density curve calibrated to incident light and dark. To control for background labeling, optical density was measured in a region devoid of stain (periaqueductal gray) in each section and this value was subtracted from each measurement of density in that section. Data were analyzed using a 2 3 3 repeatedmeasures ANOVA (SigmaStat, version 2.0; Jaendel, San Raphael, CA) with rearing condition (DD or LD) and section level (medial, middle, and lateral) as the variables, and a ¼ 0.05. In areas of patchy distribution of retinal staining, two observers blinded to the identity of the animals examined six matched sections throughout the rostral, middle, and caudal region of the superior colliculus under bright-ﬁeld microscopy and ranked the animals according to overall staining density and distribution, from least to greatest. Rankings from the two observers showed high interrater reliability (r > 0.90). The mean ranking of staining intensity was evaluated for statistical signiﬁcance by a Mann-Whitney U-test, with a ¼ 0.05. To help delineate nuclear boundaries and laminae, CTb tracing of retinofugal ﬁbers was used in conjunction with immunocytochemistry for the calcium binding protein parvalbumin in two animals from each lighting condition (Paxinos et al., 1999). Free-ﬂoating 50 mm sections were incubated overnight at room temperature with primary antibody as previously described (1:60,000; Sigma) (Prichard et al., 2002). Histological examination of the retinal ganglion cell layer was performed on a subset of rats from each of the 343 rearing conditions (four LD, four DD). Sections through the eyes were examined for evidence of retinopathy (thinning of the retinal ganglion cell layer, loss of rods and cones). Cells in the retinal ganglion cell layer containing well-stained nuclei were counted in LD and DD rats by two observers blinded to the identity of the rats. Sample ﬁelds measuring 1 mm in diameter were distributed throughout the retina (two central, six peripheral). Mean cell counts from the two central and the six peripheral ﬁelds were compared across rearing groups by one-way analysis of variance (ANOVA), with a ¼ 0.05. This sampling technique produced reliable and repeatable counts of retinal ganglion cells, with high interrater reliability (r > 0.90). Observations Among the retinofugal targets of subcortical visual system, dark-rearing had the greatest impact on the superior colliculus (SC). In the contralateral SC, DD rats had a reduced retinal input to the stratum opticum layer in the SC. Additionally, retinofugal ﬁbers in DD rats did not appear to extend into the intermediate layers of the contralateral superior colliculus (Fig. 1). This reduction in retinal input was present throughout the rostrocaudal and mediolateral extent of the contralateral colliculus. Two observers blinded to the rearing condition ranked the staining intensity in the stratum opticum and intermediate gray layer of the SC for each DD rat (n ¼ 6) as lower than the staining intensity for each LD rat (n ¼ 5), with the sum of the ranks equal to 21 (S 1–6) for the DD rats and 35 (S 7–11) for the LD rats (U ¼ 0; P < 0.01). A quantitative analysis of CTb staining intensity in the superﬁcial layers of the contralateral SC showed no signiﬁcant difference between LD and DD rats in retinal input to lateral, middle, or medial sections of the stratum griseum superﬁciale layer (F1,8 ¼ 0.359; P ¼ 0.567; Table 1). There were no obvious differences in retinal input to the ipsilateral superior colliculus between the two rearing conditions. Both LD and DD rats had a sparse, patchy projection to the ipsilateral superior colliculus, with more stained ﬁbers present in the intermediate than in the superﬁcial layers, and more patches in the rostral than the caudal part of the nucleus. Both ipsilateral and contralateral retinal projections to the dLGN and all other regions of the subcortical visual system in DD rats appeared normal by comparison with LD rats and previously published reports (Fig. 2) (Prichard et al., 2002). No differences in CTb staining intensity were observed between the two rearing conditions in the hypothalamus, including the suprachiasmatic nucleus (SCN), the subparaventricular zone, and the ventral lateral preoptic area. Likewise, no differences in retinofugal projections to the ventral lateral geniculate nucleus (vLGN), the intergeniculate leaﬂet (IGL), or the pretectum were observed. Pretectal nuclei examined included the olivary pretectal nucleus (OPN), anterior pretectal nucleus, medial pretectal nucleus, nucleus of the optic tract, the commissural pretectal area, and the posterior limitans (PLi). Histological examination of the retinae revealed no retinopathy in DD rats by comparison with LD rats. There was no indication of retinal thinning or loss of rods and cones and counts of retinal ganglion cells in Fig. 1. Cytoarchitecture and retinal innervation of the superior colliculus (SC) in light-dark-reared rats (A, C, and E) and dark-reared rats (B, D, and F). A and B: As seen with parvalbumin immunoreactivity, there is no compression of the superﬁcial gray layer (SGS), the stratum opticum (SO), or the intermediate layer following dark-rearing. C– F: Cholera toxin b immunoreactive retinal ﬁbers in the contralateral SC. Note the reduction in labeled ﬁbers in the SO of dark-reared rats by comparison with light-dark-reared rats (C and D; magniﬁed in E and F). Scale bars ¼ 250 mm (A and B); 100 mm (C and D); 30 mm (E and F). RETINOCOLLICULAR PROJECTION IN DARK-REARING TABLE 1. Mean optical density measurements of CTb-labeled retinal ﬁbers in the contralateral stratum griseum superﬁciale layer of the superior colliculus. No signiﬁcant differences in retinal input to the lateral, middle or medial sections of this layer were found between light-dark-reared (LD, n 5 5) and dark-reared (DD, n 5 4) rats. Rearing condition LD LD LD LD DD DD DD DD Sample Optical density St. Dev. Lateral Middle Medial All Samples Lateral Middle Medial All samples 0.852 0.772 0.768 0.807 0.762 0.707 0.778 0.768 0.01 0.03 0.04 0.01 0.07 0.06 0.12 0.02 representative sections from each retina showed no signiﬁcant differences between rearing groups in the central or peripheral retina (P > 0.05). DISCUSSION The principal ﬁnding of this study is that dark-rearing is associated with a reduction in retinal input to the contralateral stratum opticum of the SC. In contrast, darkrearing does not appear to inﬂuence retinal ganglion cell density or the distribution of retinofugal projections to the dLGN or to other nuclei of the subcortical visual system. The rat superior colliculus, which orchestrates multiple immediate motor responses to sensory stimuli, receives dense retinal input in the stratum griseum superﬁciale layer, as well as direct input from retinal terminals in the stratum opticum (Beckstead and Frankfurter, 1983; Boka et al., 2006), and multisensory inputs from auditory, somatosensory, and cortical visual sources in the intermediate and deep layers (Schiller and Stryker, 1972; Stein and Clamann, 1981; Sahibzada et al., 1986; Dean et al., 1988; Behan and Kime, 1996). In particular, neurons in the stratum opticum and the intermediate layers of the rat SC play an important role in communication between the sensory superﬁcial layers and the deep premotor layers by gating synchronous depolarizations and activating intralaminar pathways (Saito and Isa, 1999, 2005). In awake behaving rats, chemical stimulation of the SC results in immediate defensive head movements (Redgrave et al., 1981), and electrical stimulation of the lateral intermediate layers of the SC induces horizontal head movements (King et al., 1991). Patterned ﬁring in neurons of the intermediate layers of the SC likely modulates the transfer of retinal information during such orienting movements (Lee et al., 1997; Pettit et al., 1999; Helms et al., 2005; Lee and Hall, 2006). The stratum opticum and intermediate layers of the rodent SC are particularly prone to postnatal reorganization following sensory deprivation. For example, congenitally deaf mice show an increase in retinal axons in the stratum opticum and in the intermediate layers of the SC, areas that are usually heavily innervated by neurons in the inferior colliculus (Hunt et al., 2005). Furthermore, hamsters with neonatal lesions of the superﬁcial layers of the SC ipsilateral to a monocularly 345 enucleated eye show signiﬁcantly increased retinal innervation of the intermediate layers of the SC, as well as to other nuclei of the subcortical visual system (Carman and Schneider, 1992). The reduction in retinal input that we observed in the stratum opticum of the SC following dark-rearing could be due to a lack of patterned retinal ganglion cell activity resulting in pruning of retinal ganglion cell axons, or to a developmental failure to establish synaptic connections followed by a compensatory increase in axonal input from other sensory modalities (e.g., afferent auditory ﬁbers). Whether one or both of these situations occur will require further study. The capacity of SC neurons to integrate visual, auditory, and somatosensory inputs develops postnatally (King et al., 1988; Wallace and Stein, 1997), and light deprivation during development has a negative impact on this multisensory integration. For example, darkrearing inhibits acoustic spatial map formation in the SC of ferrets and guinea pigs (Withington, 1992; King and Carlile, 1993). Similarly, early blind humans show deﬁcits in sound elevation localization, a task partially mediated by the SC (Zwiers et al., 2001). Wallace et al. (2004) reported that dark-rearing disrupts the ability of multisensory neurons in the intermediate and deep layers of the SC to integrate bimodal cues in the cat, although these neurons were still able to respond robustly to the cross-modal inputs when presented individually. A reduction in retinal input to the stratum opticum and intermediate layers of the SC during development may underlie such failures in multisensory integration. In contrast to the reduction in retinal input to the stratum opticum of the SC following light deprivation, retinofugal projections to the dLGN and to other subcortical visual nuclei appeared to be unaffected by darkrearing. The robustness of retinal projections to the hypothalamic and pretectal nuclei of the subcortical visual system is not too surprising, as these projections are preferentially preserved in the naturally dark-reared rodent, Spalax ehrenbergi. In this blind mole rat, retinal input to the SCN and the pretectum is well-developed by comparison with the dLGN and the SC and is comparable in size to that of land-dwelling rodents (Bronchti et al., 1991; Cooper et al., 1993). Thus, projections to these retinorecipient regions seem to be highly conserved, despite natural or induced dark-rearing. It is possible that the normal variability in CTb staining could have obscured any minor dark-rearing-induced changes in the intensity of retinal innervation; however, we observed no changes in the distribution of retinal input to these nuclei. Furthermore, the quantitative analysis of optical density showed no signiﬁcant differences in the superﬁcial gray layer of the SC, the largest target of retinofugal ﬁbers. Dark-rearing may have altered bouton size or the number of boutons associated with each axon, resulting in changes in synaptic efﬁcacy. Even small changes, beyond our ability to measure with light microscopic techniques, could potentially have physiological consequences. The critical period during which retinal innervation of the SC can be disrupted by dark-rearing is not yet known. Retinal ganglion cells are generated between E13 and E19, and the earliest axons reach the SC by E16 (Lund and Bunt, 1976; Dallimore et al., 2002). 346 PRICHARD ET AL. Fig. 2. Cholera toxin b immunoreactive retinal ﬁbers in retinofugal targets of the primary and subcortical visual system in light-dark-reared (A, C, and E) and dark-reared (B, D, and F) rats. The distribution of retinal ﬁbers in the suprachiasmatic nucleus (SCN), the dorsal lateral geniculate nucleus (dLGN), intergeniculate leaﬂet (IGL), ventral lateral geniculate nucleus (vLGN), olivary pretectal nucleus (OPN), posterior limitans (PLi), and commissural pretectal area (CPA) was similar in rats reared in both lighting conditions. Scale bars ¼ 200 mm (A and B); 400 mm (C and D); 300 mm (E and F). Because we observed no evidence of retinopathy or loss of retinal ganglion cells in the DD rats, we can assume that the reduction in the retinocollicular projection was not the direct result of major pathology in the retina itself. However, dark-rearing has been shown to induce more subtle changes in retinal anatomy and physiology (Guenther et al., 2004; Zhang et al., 2005), which might in turn inﬂuence the activity-dependent development of retinofugal projections, and which might underlie some of the altered sleep/wakefulness responses to light that are observed in dark-reared rats (Prichard et al., 2004). Further studies will be required to determine whether the reduction of input to the stratum opticum and intermediate gray layers of the SC in DD rats is a result of impaired axonal arborization, or aberrant remodeling of an initially diffuse projection, and whether this reduction can be reversed in adulthood by a period of normal light exposure. ACKNOWLEDGMENTS Supported by National Institutes of Health MH52226 (to R.M.B.) and GM07507 (to J.R.P.). The authors are grateful to Agnieszka Kubica for help with data analysis. LITERATURE CITED Akerman CJ, Smyth D, Thompson ID. 2002. 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