Cone dystrophy and ectopic synaptogenesis in a Cacna1f loss of function model of congenital stationary night blindness (CSNB2A).

Congenital stationary night blindness 2A (CSNB2A) is an X-linked retinal disorder, characterized by phenotypically variable signs and symptoms of impaired vision. CSNB2A is due to mutations in CACNA1F, which codes for the pore-forming α1F subunit of a L-type voltage-gated calcium channel, Cav1.4. Mouse models of CSNB2A, used for characterizing the effects of various Cacna1f mutations, have revealed greater severity of defects than in human CSNB2A. Specifically, Cacna1f-knockout mice show an apparent lack of visual function, gradual retinal degeneration, and disruption of photoreceptor synaptic terminals. Several reports have also noted cone-specific disruptions, including axonal abnormalities, dystrophy, and cell death. We have explored further the involvement of cones in our 'G305X' mouse model of CSNB2A, which has a premature truncation, loss-of-function mutation in Cacna1f. We show that the expression of genes for several phototransduction-related cone markers is down-regulated, while that of several cellular stress- and damage-related markers is up-regulated; and that cone photoreceptor structure and photopic visual function - measured by immunohistochemistry, optokinetic response and electroretinography - deteriorate progressively with age. We also find that dystrophic cone axons establish synapse-like contacts with rod bipolar cell dendrites, which they normally do not contact in wild-type retinas - ectopically, among rod cell bodies in the outer nuclear layer. These data support a role for Cav1.4 in cone synaptic development, cell viability, and synaptic transmission of cone-dependent visual signals. Although our novel finding of cone-to-rod-bipolar cell contacts in this mouse model of a retinal channelopathy may challenge current views of the role of Cav1.4 in photopic vision, it also suggests a potential new target for restorative therapy.

Congenital stationary night blindness in mice - a tale of two Cacna1f mutants.

BACKGROUND: Mutations in CACNA1F, which encodes the Ca(v)1.4 subunit of a voltage-gated L-type calcium channel, cause X-linked incomplete congenital stationary night blindness (CSNB2), a condition of defective retinal neurotransmission which results in night blindness, reduced visual acuity, and diminished ERG b-wave. We have characterized two putative murine CSNB2 models: an engineered null-mutant, with a stop codon (G305X); and a spontaneous mutant with an ETn insertion in intron 2 of Cacna1f (nob2). METHODS: Cacna1f ( G305X ): Adults were characterized by visual function (photopic optokinetic response, OKR); gene expression (microarray) and by cell death (TUNEL) and synaptic development (TEM). Cacna1f ( nob2 ): Adults were characterized by properties of Cacna1f mRNA (cloning and sequencing) and expressed protein (immunoblotting, electrophysiology, filamin [cytoskeletal protein] binding), and OKR. RESULTS: The null mutation in Cacna1f ( G305X ) mice caused loss of cone cell ribbons, failure of OPL synaptogenesis, ERG b-wave and absence of OKR. In Cacna1f ( nob2 ) mice alternative ETn splicing produced ~90% Cacna1f mRNA having a stop codon, but ~10% mRNA encoding a complete polypeptide. Cacna1f ( nob2 ) mice had normal OKR, and alternatively-spliced complete protein had WT channel properties, but alternative ETn splicing abolished N-terminal protein binding to filamin. CONCLUSIONS: Ca(v)1.4 plays a key role in photoreceptor synaptogenesis and synaptic function in mouse retina. Cacna1f ( G305X ) is a true knockout model for human CSNB2, with prominent defects in cone and rod function. Cacna1f ( nob2 ) is an incomplete knockout model for CSNB2, because alternative splicing in an ETn element leads to some full-length Ca(v)1.4 protein, and some cones surviving to drive photopic visual responses.

Intraretinal processing following photoreceptor rescue by non-retinal cells.

Royal College of Surgeon (RCS) rats undergo retinal degeneration due to the inability of retinal pigment epithelial (RPE) cells to phagocytose shed outer segments. We explored the effect of introducing Schwann cells to the subretinal space of RCS rats (before the onset of retinal degeneration), by relying on electroretinogram (ERG) recordings and correlative retinal morphology. Scotopic ERGs recorded from cell-injected eyes showed preserved amplitudes of mixed a-wave b-wave, rod b-waves, and cone b-waves over controls (sham-injected eyes); photopic b-wave amplitudes and critical flicker fusion were also improved. Normal retinal morphology was found in areas of retinas that had received cell injections. Since Schwann cells have no phagocytic properties, their therapeutic effect is best explained through a paracrine mechanism (secretion of factors that ensure photoreceptor survival).

EphA5 and ephrin-A2 expression during optic nerve regeneration: a 'two-edged sword'.

During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.

Partial preservation of rod and cone ERG function following subretinal injection of ARPE-19 cells in RCS rats.

We quantified rod- and cone-related electroretinogram (ERG) responses following subretinal injections of the human-derived retinal pigment epithelial (hRPE) cell line ARPE-19 at age P23 to prevent progressive photoreceptor loss in the Royal College of Surgeons (RCS) rat. Culture medium-injected eyes served as sham controls. At P60, in comparison with sham-injected eyes, all recordings from hRPE-injected eyes showed preserved scotopic a- and b-waves, oscillatory potentials, double-flash-derived rod b-waves and photopic cone b-waves, and flicker critical fusion frequencies and amplitudes. Although the actual preservation did not exceed 10% of a-wave and 20% of b-wave amplitude values in non-dystrophic RCS and deteriorated rapidly by P90, rod- and cone-related ERG parameters were still recordable up to P120 unlike the virtually unresponsive sham-injected eyes.

Recruitment of local excitatory circuits in the superior colliculus following deafferentation and the regeneration of retinocollicular inputs.

The local synaptic connectivity in the superficial gray layer of the superior colliculus (SC) was assessed following retinal ganglion cell axonal regeneration through a peripheral nerve graft into the SC of Lister Hooded rats, using in vitro brain slice techniques. Repair was effected between the ipsilateral eye and SC, following bilateral lesion of optic nerves and ablation of ipsilateral occipital cortex. Deafferentation surgery alone resulted in a complete loss of synaptic potentials of extrinsic origin, once both retinal and cortical inputs were removed. Stimulation of graft insertion sites elicited synaptic responses comprising monosynaptic and network-mediated depolarising events. This activity, together with similar spontaneous bursts of depolarising events and action potential firing, was generated by the activation of non-N-methyl-D-aspartate glutamate receptors. This behaviour may reflect the development of a local recurrent synaptic connectivity following the repair surgery, as both evoked and spontaneous responses developed into large long-lasting bursts of excitatory activity when inhibition mediated by GABA receptors was blocked. These results suggest that the ultrastructural changes in the superficial layers of the SC resulting from deafferentation are reflected functionally at the synaptic level in the target structure even after repair. Such changes are likely to compromise the ability of the target structure to function normally during information processing. Therefore, although axons regenerating along peripheral nerve grafts can make functional synaptic connections, their efficacy in activating the target structure will probably be compromised by local changes in synaptic connectivity.

Enhanced cone dysfunction in rats homozygous for the P23H rhodopsin mutation.

The heterozygous P23H transgenic rat is a model of autosomal dominant retinitis pigmentosa, in which a mutation in the rhodopsin gene leads to a rapid loss of rods and a more protracted loss of cones. It has been suggested that rods play an essential role in preserving cones. We tested this hypothesis by examining whether higher levels of dysfunctional rhodopsin in rats homozygous for the P23H mutation would result in exacerbated cone dysfunction when compared with heterozygous P23H rats. Electroretinogram (ERG) responses were recorded from P21 to P250 in Sprague-Dawley (SD) and homozygous P23H rats. Both scotopic and photopic intensity response ERGs were severely depressed already at P21 when compared with age-matched SD rats. Furthermore, flicker amplitudes and critical fusion frequencies were also lower in P23H compared with SD rats at P21. Scotopic and photopic intensity responses as well as flicker amplitude and critical fusion frequencies declined rapidly up to P60, reaching a steady state that was maintained up to P200. We conclude that in rats homozygous for P23H rhodopsin mutations, the severe loss of rod function already seen by P21 is accompanied by substantial cone functional loss at that age. While rod-related responses are more severely affected than cone-related responses at all ages, their actual rate of decline with age is surprisingly similar. Both undergo a biphasic temporal pattern of decline: very rapid (P21-P60) followed by very slow (P60-P200) deterioration in response parameters, implying a tight link between rod and cone functional deterioration.

Cone function studied with flicker electroretinogram during progressive retinal degeneration in RCS rats.

The Royal College of Surgeons (RCS) rat has a primary defect in retinal pigment epithelial cells that leads to the progressive loss of photoreceptors and central visual responsiveness. While most rods are lost by 90 days of age (P90), cones degenerate more slowly, and can be detected anatomically up to 2 years of age, despite massive neuronal death and retinal remodelling. To examine how this progressive degenerative process impacts on cone function, we recorded the electroretingram to white light flashes (1.37 log cd s m(-2)) presented at frequencies ranging from 3 to 50 Hz, under light adapted conditions (29.8 cd m(-2)). Pigmented dystrophic and congenic non-dystrophic RCS rats aged from 18 to 300 days were studied. In all responsive animals at all ages, maximal amplitudes were obtained at 3 Hz. In both non-dystrophic and dystrophic rats, there was an increase from P18 to P21 in response amplitude and critical fusion frequency. After P21, these two parameters declined progressively with age in dystrophic rats. Other changes included prolongation in latency, which was first detected prior to the initiation of amplitude reduction. While phase shifts were also detected in dystrophic RCS rats, they appeared at later degenerative stages. The latest age at which responses could be elicited in dystrophic rats was at P200, with positive waves being replaced by negative deflections. The effect of increments in the intensity of background illumination was tested at P50 in both groups. This caused a diminution in flicker response amplitude and critical fusion frequencies in non-dystrophics, while in dystrophic animals, response amplitudes were reduced only at low frequencies and critical fusion frequencies were unaltered. In conclusion, although dystrophic RCS rats undergo a progressive decline in cone function with age, the flicker responsiveness at P21 is comparable to that of non-dystrophic congenic rats, suggesting normal developmental maturation of the cone system in this animal model of retinal degeneration. Flicker responses can be recorded up to P200, at which point the retina has undergone severe regressive and reactive changes in its connectivity patterns. The fact that responses at this age consist of solely negative deflections might be a reflection of the highly pathological state of the retina.

Measuring the cone contribution to the ERG b-wave to assess function and predict anatomical rescue in RCS rats.

Subretinal injections of human retinal pigment epithelial (RPE) cells early in the course of retinal degeneration in Royal College of Surgeons (RCS) rats can rescue photoreceptors. Fourteen injected animals were studied using a double flash electroretinogram (ERG): 10 were examined longitudinally and four terminally with immunohistochemistry. The proportion of cone contribution to the ERG b-wave rather than the absolute size of isolated cone response proved to be a reliable indicator of function over time and a predictor of the proportion of cones identified anatomically in the area of optimal photoreceptor rescue.

Contribution of rod and cone pathways to the dark-adapted electroretinogram (ERG) b-wave following retinal degeneration in RCS rats.

Although the RCS rat is widely used as a model of progressive photoreceptor loss, it is unclear how the relative rod and cone functions change with age. Rod and cone b-waves were isolated using a double flash ERG paradigm. In contrast to cones, rods never reached normal functional maturity levels, and the ERG b-wave changed from being predominantly rod-driven to being purely cone-driven by age 74 days, at which point, b-waves were progressively replaced by negative STR-like (scotopic threshold response) waves that persisted up to age 180 days. A double flash commonly abolished this wave and unveiled a b-wave.

Regressive and reactive changes in the connectivity patterns of rod and cone pathways of P23H transgenic rat retina.

We have used the P23H line 1 homozygous albino rat to study how progressive photoreceptor degeneration affects rod and cone relay pathways. We examined P23H retinas at different stages of degeneration by confocal microscopy of immunostained sections and electroretinogram (ERG) recordings. By 21 days of age in the P23H rat retina, there is already substantial loss of rods and reduction in rod bipolar dendrites along with reduction of metabotropic glutamate receptor 6 (mGluR6) and rod-associated bassoon staining. The cone pathway is relatively unaffected. By 150 days, when rods are absent from much of the retina, some rod bipolars remain and dendrites of rod and cone bipolar cells form synaptic complexes associated with cones and horizontal cell processes. These complexes include foci of mGluR6 and bassoon staining; they develop further by 270 days of age. Over the course of degeneration, beginning at 21 days, bipolar axon terminals atrophy and the inner retina undergoes further changes including a reduced and disorganized AII amacrine cell population and thinning of the inner plexiform layer. Electroretinogram (ERG) results at 23 days show reductions in a-wave amplitude, in rod and cone-associated b-waves (using a double flash paradigm) and in the amplitude of oscillatory potentials (OPs). By 38 days, rod scotopic a-wave responses and OPs are lost. B-wave amplitudes decline until 150 days, at which point they are purely cone-driven and remain stable up to 250 days. The results show that during the course of photoreceptor loss in the P23H rat, there are progressive degenerative changes, particularly in the rod relay pathway, and these are reflected in the changing ERG response patterns. Later reactive changes involving condensation of cone terminals and neurotransmitter receptors associated with rod and cone bipolar dendrites and with horizontal cell processes suggest that at this stage, there are likely to be complex changes in the relay of sensory information through the retina.

The relationship between full field electroretinogram and perimetry-like visual thresholds in RCS rats during photoreceptor degeneration and rescue by cell transplants.

Dark-adapted full field electroretinogram (ERG) and visual receptive field thresholds (recorded from the superior colliculus) were correlated in a model of retinal degeneration, the Royal College of Surgeons rat. In both untreated and retinal pigment epithelium cell transplanted rats, optimal correlation was between b-wave amplitude and preserved visual field area with thresholds under a defined level. The work shows that the magnitude of the b-wave can be used to predict the computed area and degree of visual field preservation recorded in the central nervous system. These observations validate using ERG to assess residual visual function and the effect of transplantation.

Preservation of visual responsiveness in the superior colliculus of RCS rats after retinal pigment epithelium cell transplantation.

The dystrophic RCS rat undergoes progressive photoreceptor degeneration due to a primary defect in retinal pigment epithelial (RPE) cells. This has a major impact on central visual responsiveness. Here we have examined how functional deterioration is contained by subretinal transplantation of immortalized human RPE cells. Transplantation was done at three to four weeks of age prior to significant photoreceptor loss and recipients were kept on cyclosporin. At six months of age, sensitivity maps and multi-unit response properties were obtained across the visual field by recording at 76 equidistant sites encompassing the whole superior colliculus.A significant degree of functional protection, both in terms of area of responsive retina and response characteristics was observed following RPE transplantation. At best, the sensitivity, latency of onset, and response rise time were all maintained within normal ranges and this was achieved with no more than half of the normal complement of photoreceptors. Although partial, the degree of anatomical preservation (both in terms of outer nuclear layer thickness and area of rescue) correlated well with the level of preserved visual sensitivities. Sham injections also resulted in rescue, though the area of preservation was strictly confined to the needle injury site and the response properties were significantly worse than with RPE injections. This study shows that central physiological responsiveness and correlated retinal morphology can be preserved in an animal model of retinal disease by implantation of an immortalized cell line. The use of retinal sensitivity measurements provides a background for assessing higher visual functions in these animals and a direct comparison for human perimetry measures.

Microglial changes accompanying the promotion of retinal ganglion cell axonal regeneration into peripheral nerve grafts.

Intravitreal injection of the microglia inhibitor tuftsin 1-3 leads to an increase in retinal ganglion cell axonal regeneration into peripheral nerve grafts and a decrease in phagocytic cells in the retina. However, the relation of phagocytic cells and particularly microglia towards axonal regeneration remains unclear. Initially, to assess this, tuftsin 1-3's effect on axonal regeneration was reexamined by doing a dose-response study. Optimal doses were found to be 2.5 microg/ml and 250 microg/ml in rats and hamsters respectively. We then studied retinal phagocytic cells in rats. Microglial cells were classified as resting or activated based on their morphology following OX42 immunolabelling. In controls, most microglial cells were in the resting state. Optic nerve cut led to an increase in the total number of microglia and a ten-fold elevation in the proportion of activated cells; changes were more pronounced at the optic nerve stump. Anastomosis of an autologous segment of sciatic nerve to the stump of the freshly cut optic nerve minimized the overall increase in microglia, and combined with 2.5 microg/ml tuftsin 1-3, lead to a marked blunting of activation. Preservation within the retina of a higher proportion of resting over active form of microglia, and not the prevention of microglial proliferation per se, may be a crucial factor in allowing additional retinal ganglion cell axons to regenerate into peripheral nerve grafts.

Subretinal transplantation of genetically modified human cell lines attenuates loss of visual function in dystrophic rats.

Royal College of Surgeons rats are genetically predisposed to undergo significant visual loss caused by a primary dysfunction of retinal pigment epithelial (RPE) cells. By using this model, we have examined the efficacy of subretinal transplantation of two independent human RPE cell lines each exhibiting genetic modifications that confer long-term stability in vitro. The two cell lines, a spontaneously derived cell line (ARPE19) and an extensively characterized genetically engineered human RPE cell line (h1RPE7), which expresses SV40 large T (tumor) antigen, were evaluated separately. Both lines result in a significant preservation of visual function as assessed by either behavioral or physiological techniques. This attenuation of visual loss correlates with photoreceptor survival and the presence of donor cells in the areas of rescued photoreceptors at 5 months postgrafting (6 months of age). These results demonstrate the potential of genetically modified human RPE cells for ultimate application in therapeutic transplantation strategies for retinal degenerative diseases caused by RPE dysfunction.

Cell transplantation as a treatment for retinal disease.

It has been shown that photoreceptor degeneration can be limited in experimental animals by transplantation of fresh RPE to the subretinal space. There is also evidence that retinal cell transplants can be used to reconstruct retinal circuitry in dystrophic animals. Here we describe and review recent developments that highlight the necessary steps that should be taken prior to embarking on clinical trials in humans.

Progressive visual sensitivity loss in the Royal College of Surgeons rat: perimetric study in the superior colliculus.

The Royal College of Surgeons rat has a retinal pigment epithelial cell defect which causes a progressive loss of rods occurring primarily over the first few months of life. We have studied the consequences of this degenerative process on visual sensitivity across the visual field. Sensitivities were determined in the superior colliculus for unit responses recorded from 22 days up to one year of age from sites encompassing the whole visual field representation. Following visual sensitivity assessment, retinae were examined anatomically at the light and electron microscopic level. At 22 days of age, sensitivities in dystrophic rats were comparable to those of non-dystrophics at any age (40+/-1 and 41+/-1dB, respectively), despite the fact that signs of degenerative events were clear at the electron microscopic level, including presence of pyknotic photoreceptor nuclei, disorganised outer segments and accumulation of debris. However, loss in sensitivity was first detected only at 28-36 days of age (27+/-4dB). From then on, sensitivities progressively decreased to reach a plateau by 180-240 days (4+/-2dB). Starting around 90 days and onward, there was a positive gradient of sensitivities from temporal to nasal field. Drops in visual sensitivity were parallelled by several changes in visual response properties, including prolonged latency, inconsistent responsiveness, appearance of bursting spontaneous activity and activation of units by stimuli presented outside their classical receptive fields. The measure of visual sensitivities by recording visual responses at specific sites in the superior colliculus provides a reliable point-to-point assessment of retinal function comparable to visual perimetry testing in humans. This experimental approach provides the background for answering questions arising during the development of potential experimental therapies for retinal degeneration using animal models like the Royal College of Surgeons rat.

Topological specificity in reinnervation of the superior colliculus by regenerated retinal ganglion cell axons in adult hamsters.

In normal rodents there is a precise topology of the retinocollicular projection, the nasotemporal and ventrodorsal axes of the retina being respectively projected onto the caudorostral and mediolateral axes of the contralateral superior colliculus (SC). We evaluated the distribution of regenerated retinal ganglion cell (RGC) axon terminals in the SC of adult hamsters in which an unbranched peripheral nerve graft was directed from the retina to the contralateral SC. Responses to visual stimulation of individual RGCs were recorded from terminal arbors of their regenerated axons in the reinnervated SC. Retinal positions of these RGCs were inferred from the locations of their visual receptive fields. At some sites in the reinnervated SC, axon terminal arbors converged from widely separated RGCs. Conversely, axon terminal arbors at widely separated sites in the SC could emanate from contiguous RGCs. To assess whether any tendency for order was superimposed on the apparent disorganization of the regenerated projection, we evaluated the relative positions of pairs of RGC terminals in the SC in relation to the relative retinal locations of the corresponding pairs of RGCs. Among the 983 pairs of RGCs able to be evaluated from nine animals studied 30-60 weeks after grafting, there was a statistically significant 3/2 tendency for the more nasally situated of two RGCs to project its terminal more caudally in the SC than that of the more temporally situated RGC. A similar tendency toward appropriate organization was not found with respect to the ventrodorsal axis of the retina and the mediolateral axis of the SC.

Schwann cell grafting into the retina of the dystrophic RCS rat limits functional deterioration. Royal College of Surgeons.

PURPOSE: To examine whether congenic Schwann cells grafted into the subretinal space of dystrophic Royal College of Surgeons (RCS) rats can prevent photoreceptor loss and maintain visual function. METHODS: Purified neonatal Schwann cells derived from congenic rats were grafted into the subretinal space of 3- to 4-week-old dystrophic RCS rats. Graft placement was confirmed using Schwann cells labeled in vitro with the fluorescent dye Hoechst 33342 or in grafted eyes processed for electron microscopy (48-hour to 1-month survival). At longer intervals, up to 9 months after surgery, animals were examined for photoreceptor survival; preservation of a visual reflex, head-tracking to moving stripes; and preservation of visual receptive fields associated with the region of graft placement. RESULTS: One week after the graft was performed, Schwann cells had integrated into the subretinal space with little evidence of a reactive response. When screened for head-tracking to moving stripes, Schwann cell-grafted animals performed better than sham-treated or control dystrophic animals. Threshold sensitivity measurements and visual field assessment made by recording from the superior colliculus also showed a significant level of preserved function compared with control animals. Functional rescue was correlated with photoreceptor survival and could be observed for at least 9 months after grafting. CONCLUSIONS: Schwann cells injected into the subretinal space limit functional deterioration and prolong photoreceptor survival. It is suggested that they act by local release of growth factors that either support photoreceptors directly and/or stimulate phagocytosis in RPE cells.

Selective innervation of retinorecipient brainstem nuclei by retinal ganglion cell axons regenerating through peripheral nerve grafts in adult rats.

The pattern of axonal regeneration, specificity of reinnervation, and terminal arborization in the brainstem by axotomized retinal ganglion cell axons was studied in rats with peripheral nerve grafts linking the retina with ipsilateral regions of the brainstem, including dorsal and lateral aspects of the diencephalon and lateral aspect of the superior colliculus. Four to 13 months later, regenerated retinal projections were traced using intraocular injection of cholera toxin B subunit. In approximately one-third of the animals, regenerated retinal axons extended into the brainstem for distances of up to 6 mm. Although axons followed different patterns of ingrowth depending on their site of entry to the brainstem, within the pretectum, they innervated preferentially the nucleus of the optic tract and the olivary pretectal nucleus in which they formed two types of terminal arbors. Within the superior colliculus, axons extended laterally and formed a different terminal arbor type within the stratum griseum superficiale. In the remaining two-thirds of the animals, retinal fibers formed a neuroma-like structure at the site of entry into the brainstem, or a few fibers extended for very short distances within the neighboring neuropil. These experiments suggest that regenerated retinal axons are capable of a highly selective reinnervation pattern within adult denervated retinorecipient nuclei in which they form well defined terminal arbors that may persist for long periods of time. In addition, these studies provide the anatomical correlate for our previous functional study on the re-establishment of the pupillary light reflex in this experimental paradigm.