Intravitreal Interleukin-2 modifies retinal excitatory circuits and retinocollicular innervation.

Interleukin-2 is a classical immune cytokine whose neural functions have received little attention. Its levels have been found to be increased in some neuropathologies, such as Alzheimer's disease, multiple sclerosis and uveitis. Mechanistically, it has been demonstrated the role of IL-2 in regulating glutamate and acetylcholine transmission, thus being relevant for CNS physiology. In fact, our previous work showed that an acute intravitreal IL-2 injection during retinotectal development promoted contralateral eye axonal plasticity in the superior colliculus, but the involved mechanisms were not explored. So, our present study aimed to investigate the effect of increased intravitreal IL-2 levels on the retinal glutamatergic and cholinergic signalling required for retinotectal normal development. We showed through HRP neuronal tracing that intravitreal IL-2 also induces ipsilateral eye axonal sprouting. Protein level and/or immunolocalization analysis in the retina confirmed IL-2 pathway activation by increased expression of phospho-STAT-3, coupled to transient (24h) reduced levels of Egr1, PSD-95 and nicotinic acetylcholine receptor ß2 subunit, suggesting reduced neural activity and synaptic sites. Also, AChE activity and GluN2B and GluA2 contents were reduced within 96h after IL-2 treatment. Therefore, IL-2-induced retinotectal plasticity might be driven by changes in cholinergic and glutamatergic pathways of the retina.

Nicotine-induced plasticity in the retinocollicular pathway: Evidence for involvement of amyloid precursor protein.

During early postnatal development retinocollicular projections undergo activity-dependent synaptic refinement that results in the formation of precise topographical maps in the visual layers of the superior colliculus (SC). Amyloid Precursor Protein (APP) is a widely expressed transmembrane glycoprotein involved in the regulation of several aspects of neural development, such as neurite outgrowth, synapse formation and plasticity. Stimulation of cholinergic system has been found to alter the expression and processing of APP in different cell lines. Herein, we investigated the effect of nicotine on the development of retinocollicular pathway and on APP metabolism in the SC of pigmented rats. Animals were submitted to intracranial Elvax implants loaded with nicotine or phosphate-buffered saline (vehicle) at postnatal day (PND) 7. The ipsilateral retinocollicular pathway of control and experimental groups was anterogradely labeled either 1 or 3 weeks after surgery (PND 14 or PND 28). Local nicotine exposure produces a transitory sprouting of uncrossed retinal axons outside their main terminal zones. Nicotine also increases APP content and its soluble neurotrophic fragment sAPPα. Furthermore, nicotine treatment upregulates nicotinic acetylcholine receptor α7 and ß2 subunits. Taken together, these data indicate that nicotine disrupts the ordering and topographic mapping of axons in the retinocollicular pathway and facilitates APP processing through the nonamyloidogenic pathway, suggesting that sAPPα may act as a trophic agent that mediates nicotine-induced morphological plasticity.

A critical period for omega-3 nutritional supplementation in the development of the rodent visual system.

Retinocollicular connections form precise topographical maps that are normally completed through the selective elimination of misplaced axons and the stabilization of topographically ordered axon terminals during early development. Omega-3 fatty acids, acquired exclusively through the diet, and its main metabolite, docosahexaenoic acid (DHA), are involved in brain development and synaptic maturation. We have previously shown that the nutritional restriction of omega-3/DHA results in abnormal retinocollicular topographical fine-tuning. Therefore, we studied the role of omega-3 fatty acids nutritional supplementation and the developmental time windows during which this postnatal supplementation would restore normal topographical maps in the visual system. Female rats and their litters were chronically fed with either control (soy oil) or restricted omega-3 (coconut oil) diets. Fish oil supplementation was introduced between either postnatal day (PND) 7-13, PND7-28 or PND21-42. At PND13, PND28 or PND42, animals received an anterograde eye injection of a neuronal tracer to visualize retinocollicular axons. Confirming previous observations we found that an omega-3/DHA deficiency resulted in an abnormally high innervation density of retinal axons at the visual layers of the superior colliculus (SC). Although a short-term fish oil supplementation between PND7-13 could not restore normal retinocollicular topography, an extended treatment between PND7-28 completely recovered normal innervation densities of retinotectal axons. However, a late onset supplementation protocol, between PND28-42, was no longer effective in the restoration of the abnormal topographical pattern induced by an early omega-3 nutritional malnutrition. The results suggest a critical period for omega3/DHA dietary intake for the proper development of visual topographical maps.

Intravitreous interleukin-2 treatment and inflammation modulates glial cells activation and uncrossed retinotectal development.

Interleukin-2 (IL-2) plays regulatory functions both in immune and nervous system. However, in the visual system, little is known about the cellular types which respond to IL-2 and its effects. Herein, we investigated the influence of IL-2 in the development of central visual pathways. Lister Hooded rats were submitted to multiple (at postnatal days [PND]7/10/13) or single (at PND10) intravitreous injections of phosphate-buffered saline (PBS) (vehicle), zymosan, or IL-2. IL-2 receptor α subunit was detected in the whole postnatal retina. Chronic treatment with either PBS or IL-2 increases retinal glial fibrillary acidic protein (GFAP) expression, induces intravitreous inflammation revealed by the presence of macrophages, and results in a slight rearrangement of retinotectal axons. Acute zymosan treatment disrupts retinotectal axons distribution, confirming the influence of inflammation on retinotectal pathway reordering. Furthermore, acute IL-2 treatment increases GFAP expression in the retina without inflammation and produces a robust sprouting of the intact uncrossed retinotectal pathway. No difference was observed in glial cells activity in superior colliculus. Taken together, these data suggest that inflammation and interleukin-2 modulate retinal ganglion cells development and the distribution of their axons within central targets.

A time-dependent effect of caffeine upon lesion-induced plasticity.

During a critical period, unilateral retinal lesions induce rapid axonal sprouting of intact axons into denervated territories within the collicular visual layers. We investigated the effect of caffeine, a non-selective A(1) and A(2a) antagonist, upon the lesion-induced plasticity of retinotectal axons. Pigmented rats submitted to a temporal retinal lesion received either caffeine (30mg/kg, ip) or saline treatment. The anterograde tracing revealed that caffeine treatment during the critical period resulted in a clear reduction on the sprouting of ipsilateral fibers but to an amplification of the plasticity after PND21, thus revealing opposite effects depending on the developmental time window.

Evidence for a role of calcineurin in the development of retinocollicular fine topography.

The fine-tuning of topographically organized projections in sensory systems is strongly influenced by electrical activity and use-dependent modifications in synaptic strength. Since calcineurin (CaN), a Ca(2+)-calmodulin dependent serine/threonine phosphatase has been associated with activity-dependent modifications in synaptic efficacy we studied the effects of systemic and local administration of CaN inhibitors during the critical period of development of the uncrossed retinocollicular projection in pigmented rats. We found that the expression of the catalytic subunit of calcineurin (CaNA) occurs throughout early development in the visual layers of the superior colliculus and peaks at PND14 when eye opening is complete. The functional blockade of CaN activity by means of a systemic treatment with cyclosporine A (CsA) during the second postnatal week, induces sprouting of uncrossed retinal axons outside their main terminal zones. Additionally, the local treatment with intracranial implants of Elvax loaded with either CsA or a cell-permeable CaN inhibitory peptide (CIP) resulted in a similar expansion of retinocollicular terminal fields. Taken together, these results suggest CaN as a key element for the development of fine tuning of retinocollicular topography.

Purinergic modulation in the development of the rat uncrossed retinotectal pathway.

Adenosine is a neuromodulator implicated in nervous system development and plasticity and its effects are mediated by inhibitory (A(1), A(3)) and excitatory (A(2a), A(2b)) receptors. The role of adenosine in the synaptic activity depends mainly on a balanced activation of A(1) and A(2a) receptors which are activated by various ranges of adenosine concentrations. Herein, we investigated the expression of A(1) and A(2a) receptors and also the accumulation of cAMP in the superior colliculus at different stages of development. Furthermore, we examined the effects of an acute in vivo blockade of adenosine deaminase during the critical period when the elimination of misplaced axons/terminals takes place with a simultaneous fine tuning of terminal arbors into appropriate terminal zones. Lister Hooded rats ranging from postnatal days (PND) 0-70 were used for ontogeny studies. Our results indicate that A(1) expression in the visual layers of the superior colliculus is higher until PND 28, while A(2a) expression increases after PND 28 in a complementary developmental pattern. Accordingly, the incubation of collicular slices with 5'-N-ethylcarboxamido-adenosine, a non-specific adenosine receptor agonist, showed a significant reduction in cAMP accumulation at PND 14 and an increase in adults. For the anatomical studies, the uncrossed retinotectal projections were traced after the intraocular injection of horseradish peroxidase. One group received daily injections of an adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl adenine), 10 mg/kg i.p.) between PND 10 and 13, while control groups were treated with vehicle injections (NaCl 0.9%, i.p.). We found that a short-term blockade of adenosine deaminase during the second postnatal week induced an expansion of retinotectal terminal fields in the rostrocaudal axis of the tectum. Taken together, the results suggest that a balance of purinergic A(1) and A(2a) receptors through cAMP signaling plays a pivotal role during the development of topographic order in the retinotectal pathway.

Blockade of arachidonic acid pathway induces sprouting in the adult but not in the neonatal uncrossed retinotectal projection.

The uncrossed retinotectal projection of rats undergoes extensive axonal elimination and subsequent growth of axonal arbors in topographically appropriate territories within the first two/three postnatal weeks. Nitric oxide has been implicated in development and stabilization of synapses in the retinotectal pathway since blockade of nitric oxide synthesis disrupts the normal pattern of retinal innervation in subcortical nuclei. The present work investigated the role of arachidonic acid pathway in the development and maintenance of ipsilateral retinotectal axons. We also investigated the role of this retrograde messenger in the modulation of plasticity that follows retinal lesions in the opposite eye. Pigmented rats received systemic treatment with quinacrine, a phospholipase A2 inhibitor, indomethacin, a cyclooxygenase inhibitor, nordihydroguaiaretic acid, a 5-lipoxygenase inhibitor or vehicle during 4-8 days at various postnatal ages. Rats given a unilateral temporal retinal lesion were treated with either quinacrine or vehicle during the same period. For anterograde tracing of ipsilateral retinal projections, animals received intraocular injections of horseradish peroxidase. Before the third postnatal week no difference was observed in the laminar or topographic organization of the ipsilateral retinotectal projection between vehicle and treated rats in either normal or lesion conditions. After the third postnatal week, however, systemic blockade of phospholipase A2 or 5-lipoxygenase, but not cyclooxygenase induced sprouting of uncrossed axons throughout the collicular visual layers in unoperated rats. In retinal lesion groups, phospholipase A2 blockade increased the sprouting of uncrossed intact axons to the collicular surface in the same period. The results suggest that arachidonic acid or lipoxygenase metabolites play a role in the maintenance of the retinotectal synapses after the critical period and that the blockade of the arachidonic acid pathway induces reactive sprouting of retinal axons late in development.

Rapid and long-term plasticity in the neonatal and adult retinotectal pathways following a retinal lesion.

The uncrossed retinotectal projection restricts its terminal fields to the ventral boundary of the visual layers at the rostral tectum during early post natal development. During this critical period, temporal retinal lesions in one eye induce laminar rearrangements in the uncrossed pathway of the intact eye toward the collicular surface previously occupied, almost exclusively, by the crossed retinal axon population. We have compared, using anterograde tracing techniques, the time course and magnitude of the axonal sprouting resulting from retinal lesions in neonates and adults. Early retinal lesions (within the first two post natal weeks) induced extensive and rapid plasticity of the ipsilateral projection 48 h after the lesions. On the third post natal week, similar retinal lesions induced a small reorganization of the intact eye's uncrossed projection within a 3-week survival time. Nevertheless, giving the animals a long-term survival, resulted in an increased plastic capability, suggesting that even after the critical period, intact retinal axons can respond efficiently to injury. The results suggest two phases of axonal reorganization within this subcortical pathway: a rapid plasticity within the critical period and a slow, but continuous plasticity in adulthood.

Acute blockade of nitric oxide synthesis induces disorganization and amplifies lesion-induced plasticity in the rat retinotectal projection.

In the rat visual system, the uncrossed retinotectal projection undergoes a topographical refinement within the first two postnatal weeks. We have studied the role of nitric oxide (NO), a retrograde messenger which couples pre- and postsynaptic activation, in the development of the uncrossed retinotectal projection and in the plasticity of this pathway as a result of a restricted retinal lesion in the opposite eye. During development, maximal nitric oxide synthase (NOS) activity was observed in homogenates of tectal tissue at postnatal day 5 (PND 5), followed by a two-step decrease at the end of the topographical fine tuning period (PND 21) and the adult stage (PND 42). We also tested the effects of an acute in vivo blockade of NOS during the development of both animals that had not been operated on, and lesioned animals. Animals ranging from PND 4 to PND 42 were treated either with the NOS inhibitor, L-nitro-arginine (Narg 50 mg/kg ip.) or vehicle (NaCl 0.9%) during 4 days (from PND 4-7 or PND 9-12) or 8 days (from PND 20-27 or PND 34-41). Reduction of NOS activity induced sprouting of the ipsilateral pathway up to the second postnatal week in the animals that had not been operated on. Rats that had been operated on, however, showed an amplification of the lesion-induced plasticity up to the fourth postnatal week under NOS blockade. The data suggest that NO plays a role in the stabilization of retinotectal synapses during the critical period of topographic refinement, and indicate that an acute blockade of retrograde signals enables plastic rearrangements in the visual system within this time window.

Fluoxetine-induced plasticity in the rodent visual system.

We studied the effect of fluoxetine, a selective serotonin reuptake inhibitor, in the development and lesion-induced plasticity of retinotectal axons in pigmented rats. Neonatal rats received a daily injection of either fluoxetine or vehicle from postnatal day 1 (PND 1) to PND 10 or from PND 14 to PND 28 (fluoxetine, 7.5 and 10.0 mg/kg, respectively). In the latter group, some animals received a single lesion at the temporal periphery of the left retina at PND 21. Unoperated animals were use as the control. At the end of the treatment, the animals received an intraocular injection of horseradish peroxidase (HRP) in the right (intact) eye to trace the uncrossed retinotectal pathway. Chronic fluoxetine treatment, induced, in unoperated rats, an expansion of the retinal terminal fields along the rostro-caudal axis of the tectum both in the PND 10 and PND 28 groups. Following a retinal lesion in the left eye at PND 21, the vehicle-treated group showed a small reorganization of the intact uncrossed projection. In this group only a few terminals were labeled invading the denervated tectal surface one-week after the lesion. Fluoxetine-treated animals on the other hand, showed a great amplification of plasticity with a conspicuous sprouting of the uncrossed retinal axons into denervated areas. The data suggest that fluoxetine induces extensive axonal rearrangements in neonatal and juvenile central nervous system and amplifies neuroplasticity following retinal lesions late in development.

Development of abnormal lamination and binocular segregation in the retinotectal pathways of the rat.

The uncrossed retinotectal pathway of pigmented rats originates from a small fraction of the retinal ganglion cell population. This projection terminates deeply in discrete patches within the upper grey layers where crossed and uncrossed inputs overlap. However, after the experimental enlargement of the uncrossed pathway, the ipsilateral fibers are also found in a superficial tier of the upper grey layers where binocular inputs segregate [36]. We studied the development of the retinotectal projections in rats after the enlargement of the uncrossed pathway as a result of a contralateral (left) optic tract lesion (OTL) made at birth. Horseradish peroxidase (HRP) was used as an anterograde tracer. An abnormal uncrossed projection from the right eye to the collicular surface appeared at postnatal day 3 (P3). Between P5 and P10, this projection developed the bilaminar pattern seen in similar operated adults. The laminar arrangement of the aberrant terminal fields did not change significantly after an ipsilateral visual cortex ablation on the day of birth. Despite the early development of the aberrant uncrossed pathway, binocular segregation was incipient at P10. At P14, 46% of the operated rats presented gaps in the terminal labeling at the tectal surface. This figure increased to 55.5% at 6 weeks, a proportion still smaller than in adult animals of the same group (69%). Eyelid suture had no effect on segregation. This projection remains plastic for at least 3 weeks, since the removal of the ipsilateral input at either P14 or P21 resulted in the absence of gaps in the contralateral projection. We conclude that the laminar selection of retinotectal projections depends on binocular interactions and that the abnormal segregation of retinal inputs to the superior colliculus has an unusually protracted development which can be reversed long after the previously defined critical period in this system.

Evidence that the relative densities of afferents from both eyes control laminar distribution and binocular segregation of retinotectal projections in rats.

In the superior colliculus of normal rodents the crossed retinal projection overlaps the uncrossed projection. The present study describes an abnormal laminar distribution and binocular segregation of the retinotectal afferents induced after the experimental enlargement of the uncrossed retinotectal pathway in pigmented rats. Intraocular injections of anterograde tracers were used to investigate the topographic and laminar organization of retinotectal projections in adult rats given unilateral optic tract lesions at birth. These lesions are known to increase the number of ipsilaterally projecting ganglion cells in the opposite retina. The uncrossed retinal projection to the remaining superior colliculus forms an abnormal band of terminal labeling at the superficial half of the stratum griseum superficiale, markedly different from the laminar distribution of this pathway in unoperated controls. This abnormal uncrossed projection has its maximum density at the rostrolateral quadrant of the tectum. Within this region, the crossed retinotectal projection retracts from the surface of the superior colliculus, leading to partial binocular segregation. The results suggest that both the laminar distribution and the experimental binocular segregation of retinotectal afferents depend on the balance of the densities of the converging pathways from both eyes in the superior colliculus.

Cell death and interocular interactions among retinofugal axons: lack of binocularly matched specificity.

Naturally occurring ganglion cell death has been attributed to competitive interactions among axons at their targets during development of the retinofugal pathways. The present study is concerned with the hypothesis that interocular interactions leading to ganglion cell death are restricted to binocularly conjugate terminals in the optic nuclei. We tested this hypothesis in newborn rats by making localized retinal lesions, which denervate a restricted portion of the contralateral optic targets. When these rats reached adulthood, the ipsilaterally projecting ganglion cells of the intact eye were then studied following retrograde labeling with horseradish peroxidase. Results were compared with those from a normal, control group and from rats that had one eye removed on the day of birth. In those retinal loci binocularly conjugate to the lesion in the opposite eye, no localized cell rescue could be found among the ipsilaterally projecting ganglion cells. The same retinal loci, however, showed clear cell rescue after contralateral enucleation. Independent, anterograde, studies of the ipsilateral retino-collicular projection verified that lesions of equivalent size to those used in the retrograde study reliably create aberrant expanded uncrossed terminal fields. The present data suggest that the interocular interactions involved in the diminished ganglion cell loss which follows monocular enucleation are not dependent on topographically specific binocular matching. The phenomena of naturally occurring cell loss and of retinotopically specific interocular interactions may therefore be independent during normal development.

Development of abnormal lamination and binocular segregation of retinal afferents onto the rat superior colliculus.

Unilateral optic tract lesions made in newborn rats produce abnormal retinotectal pathways on the opposite side. The present investigation was designed to study the development of the abnormal retinal projections in the superior colliculus using anatomical tracing methods. The aberrant uncrossed retinotectal pathway develops within the first postnatal week. In spite of this, the retraction of the crossed projection, which indicates binocular segregation, is of late onset. This indicates that the induced segregation of retinal inputs is not dependent on regressive events such as ganglion cell death and terminal field retraction. These data and the results of lid-suture experiments are consistent with a role for spontaneous retinal activity in the regulation of the plasticity of retinal projections to the rat superior colliculus.

Development of abnormal lamination and binocular segregation of retinal afferents onto the rat superior colliculus

Unilateral optic tract lesions made in newborn rats abnormal retinotectal pathways on the opposite side. The present in investigation was designed to study the development of the abnormal retinal projections in the superior colliculus using antomical tracing methods. The aberrant uncrossed retinotectal pathway develops within the first postnatal week. In spite of this, the retration of the crossed projection, wich indicates binocular segregation, is of late onset. This indicates that the induced segregation of retinal imputs is not dependent on regressive events such as ganglion cell death and terminal field retraction. These data and the results of lid-suture experiments are consistent with a role for spontaneous retinal activity in the regulation of the plasticity of retinal projections to the rat superior colliculus

Evidence for differential effects of terminal and dendritic competition upon developmental neuronal death in the retina.

Retinal ganglion cells with ipsilaterally projecting axons were labelled with horseradish peroxidase injected unilaterally along the optic pathway in adult rats. Unoperated controls were compared with three groups of animals operated at birth, given (a) contralateral enucleation, (b) contralateral lesion to the optic tract or (c) both lesions simultaneously. The numbers of ipsilaterally projecting cells were increased in all three operated groups, presumably because of a reduction in natural neuronal death following diminished terminal and dendritic competition. The pattern of increase of labelled cell density varied with the type of lesion: enucleation led to a major increase within lower temporal retina; optic tract lesion caused its major increase in upper temporal retina, centred at the location of the area centralis; and the double lesion combined both effects above. The distribution of cell-body sizes was differentially affected by the lesions: enucleation led to a shift in the distribution towards the small cell side of the spectrum, when compared with the controls; optic tract lesion shifted the distribution towards the large cell side of the spectrum, but only outside the temporal crescent; and the double lesion led to a shift towards small cells within the temporal crescent and towards large cells outside the crescent, again combining the effects of the single lesions. Large alpha-like neurones with ipsilateral axons were common in the nasal retina of both groups given optic tract lesions but they were rare in the nasal retina of unoperated and, especially, of enucleated rats. The limits of the temporal crescent were unchanged, notwithstanding the large numbers of cells outside the crescent in operated rats. It is suggested that postnatal competitive interactions at the level of terminals and of dendrites control natural neuronal death in the rat retina with different requirements regarding retinal topography and ganglion cell types. The postnatal regulation of neuronal numbers is not responsible for the generation of the nasotemporal division but may be involved in the development of differential distributions of specific ganglion cell types across the retina.