Retinotectal plasticity induced by monocular enucleation during the critical period is dependent of A2a adenosine receptor: A possible role of astrocytes.

The retinotectal topography of rats develops within the first three postnatal weeks during the critical period. Previous studies have shown that monocular enucleation results in plasticity of the intact retinotectal pathway in a time-dependent manner. Glial fibrillary acidic protein (GFAP), an astrocyte marker, is up-regulated after central nervous system injury. Adenosine is a neuromodulator involved in the development and plasticity of the visual system acting through the inhibitory A1 and excitatory A2a receptor activities. Herein, we examined whether adenosine receptors and astrocytes are crucial for monocular enucleation (ME)-induced plasticity. We also investigate whether A2a blockade alters retinotectal plasticity in an astrocyte-dependent manner. Lister Hooded rats were submitted to monocular enucleation at postnatal day 10 (PND10) or PND21 and, after different survival times, were processed for immunohistochemistry or western blotting assays. Another group underwent subpial implantation of ELVAX containing vehicle (DMSO) or SCH58261 (1 µM - an A2a receptor antagonist), simultaneously with ME at PND10. After a 72 h survival, GFAP content and the retinotectal plasticity were evaluated. Our data show that monocular enucleation leads to an upregulation in GFAP expression in the contralateral superior colliculus. At PND10, a slight increase in GFAP labeling was observed at 72 h post-enucleation, while at PND21 GFAP increase was detected in the deafferented superior colliculus after 1 to 3 weeks of survival. The content of adenosine receptors also varies in the contralateral target after ME. A transient increase in A1 receptors is observed in the early periods of plasticity, while A2a receptors are upregulated later. Interestingly, the local blockade of A2a receptors abolished the increase in GFAP and the retinotectal reorganization induced by monocular enucleation during the critical period. Taken together these results suggest a correlation between astrocytes and A2a adenosine receptors in the subcortical visual plasticity.

Signaling pathways modulated by monocular enucleation in the superior colliculus of juvenile rats.

Monocular eye enucleation (ME) is a classical paradigm to induce neural plasticity in retinal ganglion cells (RGCs) axons from the intact eye, especially when performed within the critical period of visual system development. However, the precise mechanisms underlying the axonal sprouting and synaptogenesis seen in this model remain poorly understood. In the present work, we investigated the temporal alterations in phosphorylation of three kinases related to axonal growth and synaptogenesis-GSK3ß (an important repressor of axonal outgrowth), AKT, and ERK-in superior colliculus of rats submitted to ME during early postnatal development. Western blotting analysis showed an increase in pGSK3ß, the inactive form of this enzyme, 24 and 48 hr after ME. Accordingly, an increase in pERK levels was detected 24 hr after ME, indicating that phosphorylation of these enzymes might be related to axonal reorganization induced by ME. Interestingly, AKT phosphorylation was increased just 1 week after ME, suggesting it may be involved in the stabilization of newly formed synapses, rising from the axonal reorganization of remaining eye. A better understanding of how signaling pathways are modulated in a model of intense axonal sprouting can highlight possible therapeutic targets in RGCs injuries in adult individuals, where axonal regrowth is nearly absent.

Rapid plasticity of intact axons following a lesion to the visual pathways during early brain development is triggered by microglial activation.

Lesions in the central nervous system (CNS) can often induce structural reorganization within intact circuits of the brain. Several studies show advances in the understanding of mechanisms of brain plasticity and the role of the immune system activation. Microglia, a myeloid derived cell population colonizes the CNS during early phases of embryonic development. In the present study, we evaluated the role of microglial activation in the sprouting of intact axons following lesions of the visual pathways. We evaluated the temporal course of microglial activation in the superior colliculus following a contralateral monocular enucleation (ME) and the possible involvement of microglial cells in the plastic reorganization of the intact, uncrossed, retinotectal pathway from the remaining eye. Lister Hooded rats were enucleated at PND 10 and submitted to systemic treatment with inhibitors of microglial activation: cyclosporine A and minocycline. The use of neuroanatomical tracers allowed us to evaluate the time course of structural axonal plasticity. Immunofluorescence and western blot techniques were used to observe the expression of microglial marker, Iba-1 and the morphology of microglial cells. Following a ME, Iba-1 immunoreactivity showed a progressive increase of microglial activation in the contralateral SC at 24 h, peaking at 72 h after the lesion. Treatment with inhibitors of microglial activation blocked both the structural plasticity of intact uncrossed retinotectal axons and microglial activation as seen by the decrease of Iba-1 immunoreactivity. The local blockade of TNF-α with a neutralizing antibody was also able to block axonal plasticity of the intact eye following a ME. The data support the hypothesis that microglial activation is a necessary step for the regulation of neuroplasticity induced by lesions during early brain development.

Interleukin 4 Leads to Sustained Phosphorylation of the STAT6 and ERK Pathways in the Retina and Disrupts Subcortical Visual Circuitry in Rodents.

OBJECTIVE: Interleukin 4 (IL-4) is an anti-inflammatory cytokine related to different aspects of central nervous system development such as survival, proliferation, and differentiation, among others. Our goals were to investigate the effect of intravitreous treatment with IL-4 on the activation of downstream signaling pathways in the retina and the distribution of retinal axons within the superior colliculus (SC). MATERIAL AND METHODS: Lister hooded rats were submitted to an intravitreous injection of either IL-4 (5 U/µL) or PBS (vehicle) at postnatal day 10 (PND10). At PND11 or PND14, retinas were processed for Western blot or immunohistochemistry. At PND13, a group of animals received an intraocular injection of an anterograde tracer in the left (untreated) eye in order to label the uncrossed retinotectal axons. RESULTS: Our data revealed that intravitreous treatment with IL-4 at PND10 leads to a decrease in GFAP content and a sustained increase in the phosphorylation of STAT6 and ERK levels in the retina. IL-4 also increases retinal axonal arbors within the SC, compared to control groups. CONCLUSIONS: These data suggest that a single in vivo treatment with IL-4 during the early stages of development modulates signaling pathways in the retina, resulting in altered binocular subcortical visual connectivity.

Serotonin transporter immunoreactivity is modulated during development and after fluoxetine treatment in the rodent visual system.

The serotonin transporter (5-HTT) regulates serotonin homeostasis and has been used as a target for different drugs in depression treatment. Although the serotonergic system has received a lot of attention, little is known about the effects of these drugs over serotonin transporters. In this work, we investigated the expression pattern of 5-HTT during development of the visual system and the influence of fluoxetine on different signaling pathways. Our data showed that the expression of 5-HTT has a gradual increase from postnatal day 0 until 42 and decrease afterwards. Moreover, chronic fluoxetine treatment both in childhood and adolescence induces down regulation of 5-HTT expression and phosphorylation of ERK and AKT signaling pathways. Together these data suggest that the levels of 5-HTT protein could be important for the development of the central nervous system and suggest that the ERK and AKT are involved in the molecular pathways of antidepressants drugs, acting in concert to improve serotonergic signaling.

Monocular denervation of visual nuclei modulates APP processing and sAPPα production: A possible role on neural plasticity.

Amyloid precursor protein (APP) is essential to physiological processes such as synapse formation and neural plasticity. Sequential proteolysis of APP by beta- and gamma-secretases generates amyloid-beta peptide (Aß), the main component of senile plaques in Alzheimer Disease. Alternative APP cleavage by alpha-secretase occurs within Aß domain, releasing soluble α-APP (sAPPα), a neurotrophic fragment. Among other functions, sAPPα is important to synaptogenesis, neural survival and axonal growth. APP and sAPPα levels are increased in models of neuroplasticity, which suggests an important role for APP and its metabolites, especially sAPPα, in the rearranging brain. In this work we analyzed the effects of monocular enucleation (ME), a classical model of lesion-induced plasticity, upon APP content, processing and also in secretases levels. Besides, we addressed whether α-secretase activity is crucial for retinotectal remodeling after ME. Our results showed that ME induced a transient reduction in total APP content. We also detected an increase in α-secretase expression and in sAPP production concomitant with a reduction in Aß and ß-secretase contents. These data suggest that ME facilitates APP processing by the non-amyloidogenic pathway, increasing sAPPα levels. Indeed, the pharmacological inhibition of α-secretase activity reduced the axonal sprouting of ipsilateral retinocollicular projections from the intact eye after ME, suggesting that sAPPα is necessary for synaptic structural rearrangement. Understanding how APP processing is regulated under lesion conditions may provide new insights into APP physiological role on neural plasticity.

Nutritional restriction of omega-3 fatty acids alters topographical fine tuning and leads to a delay in the critical period in the rodent visual system.

The development and maturation of sensory systems depends on the correct pattern of connections which occurs during a critical period when axonal elimination and synaptic plasticity are involved in the formation of topographical maps. Among the mechanisms involved in synaptic stabilization, essential fatty acids (EFAs), available only through diet, appear as precursors of signaling molecules involved in modulation of gene expression and neurotransmitter release. Omega-3 fatty acids, such as docosahexaenoic acid (DHA), are considered EFAs and are accumulated in the brain during fetal period and neonatal development. In this study, we demonstrated the effect of omega-3/DHA nutritional restriction in the long-term stabilization of connections in the visual system. Female rats were fed 5 weeks before mating with either a control (soy oil) or a restricted (coconut oil) diet. Litters were fed until postnatal day 13 (PND13), PND28 or PND42 with the same diets when they received an intraocular injection of HRP. Another group received a single retinal lesion at the temporal periphery at PND21. Omega-3 restriction induced an increase in the optical density in the superficial layers of the SC, as a result of axonal sprouting outside the main terminal zones. This effect was observed throughout the SGS, including the ventral and intermediate sub-layers at PND13 and also at PND28 and PND42. The quantification of optical densities strongly suggests a delay in axonal elimination in the omega3(-) groups. The supplementation with fish oil (DHA) was able to completely reverse the abnormal expansion of the retinocollicular projection. The same pattern of expanded terminal fields was also observed in the ipsilateral retinogeniculate pathway. The critical period window was studied in lesion experiments in either control or omega-3/DHA restricted groups. DHA restriction induced an increased sprouting of intact, ipsilateral axons at the deafferented region of the superior colliculus compared to the control group, revealing an abnormal extension of the critical period. Finally, in omega-3 restricted group we observed in the collicular visual layers normal levels of GAP-43 with decreased levels of its phosphorylated form, p-GAP-43, consistent with a reduction in synaptic stabilization. The data indicate, therefore, that chronic dietary restriction of omega-3 results in a reduction in DHA levels which delays axonal elimination and critical period closure, interfering with the maintenance of terminal fields in the visual system.

Nutritional tryptophan restriction and the role of serotonin in development and plasticity of central visual connections.

Tryptophan is an essential amino acid and metabolic precursor of serotonin. Serotonin is both a classical neurotransmitter and a signaling molecule that plays crucial roles in the development of neural circuits and plasticity. The specification of neural circuits in rodents occurs during the postnatal period with conspicuous influence of environmental factors including the nutritional status. Sensory, motor and cognitive systems develop during a critical period, a time window that is crucial to the use-dependent organization of neuronal circuits. This review presents recent experimental findings that disclose some mechanism of tryptophan- and serotonin-dependent plasticity in the developing and adult brain.