The Influence of Microglia on Neuroplasticity and Long-Term Cognitive Sequelae in Long COVID: Impacts on Brain Development and Beyond.

Microglial cells, the immune cells of the central nervous system, are key elements regulating brain development and brain health. These cells are fully responsive to stressors, microenvironmental alterations and are actively involved in the construction of neural circuits in children and the ability to undergo full experience-dependent plasticity in adults. Since neuroinflammation is a known key element in the pathogenesis of COVID-19, one might expect the dysregulation of microglial function to severely impact both functional and structural plasticity, leading to the cognitive sequelae that appear in the pathogenesis of Long COVID. Therefore, understanding this complex scenario is mandatory for establishing the possible molecular mechanisms related to these symptoms. In the present review, we will discuss Long COVID and its association with reduced levels of BDNF, altered crosstalk between circulating immune cells and microglia, increased levels of inflammasomes, cytokines and chemokines, as well as the alterations in signaling pathways that impact neural synaptic remodeling and plasticity, such as fractalkines, the complement system, the expression of SIRPα and CD47 molecules and altered matrix remodeling. Together, these complex mechanisms may help us understand consequences of Long COVID for brain development and its association with altered brain plasticity, impacting learning disabilities, neurodevelopmental disorders, as well as cognitive decline in adults.

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.

Chronic nutritional restriction of omega-3 fatty acids induces a pro-inflammatory profile during the development of the rat visual system.

Modern western diets have been associated with a reduced proportion of dietary omega-3 fatty acids leading to decreased levels of DHA (docosahexaenoic acid) in the brain. Low DHA content has been associated with altered development of visual acuity in infants and also with an altered time course of synapse elimination and plasticity in subcortical visual nuclei in rodents. Microglia has an active role in normal developmental processes such as circuitry refinement and plasticity, and its activation status can be modulated by omega-3 (ω3) and omega-6 (ω6) essential fatty acids. In the present study, we investigated the impact of dietary restriction of DHA (ω3-), through the chronic administration of a coconut-based diet as the only fat source. This dietary protocol resulted in a reduction in DHA content in the retina and superior colliculus (SC) and in a neuroinflammatory outcome during the development of the rodent visual system. The ω3- group showed changes in microglial morphology in the retina and SC and a corresponding altered pattern of pro-inflammatory cytokine expression. Early and late fish oil protocols supplementation were able to restore DHA levels. The early supplementation also decreased neuroinflammatory markers in the visual system. The present study indicates that a chronic dietary restriction of omega-3 fatty acids and the resulting deficits in DHA content, commonly observed in Western diets, interferes with the microglial profile leading to an inflamed microenvironment which may underlie a disruption of synapse elimination, altered topographical organization, abnormal plasticity, and duration of critical periods during brain development.

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.

In vivo effect of acute exposure to interleukin-6 on the developing visual system.

Interleukin-6 (IL-6) is involved in different processes of the central nervous system. Our aims were to investigate the effect of IL-6 on retinotectal topography and on different signaling pathways. Rats were submitted to an intravitreous injection of either IL-6 (50 ng/ml) or PBS (vehicle) at postnatal day 10 (PND10). At PND11 or PND14, different groups were processed for western blot, histochemistry or immunofluorescence analysis. IL-6 treatment leads to an increase in pSTAT-3 levels in the retina and a disruption in the retinotectal topographic map, suggesting that a transient increase in interleukin-6 levels may impact neural circuitry development.

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.

The fine tuning of retinocollicular topography depends on reelin signaling during early postnatal development of the rat visual system.

During postnatal development, neural circuits are extremely dynamic and develop precise connection patterns that emerge as a result of the elimination of synaptic terminals, a process instructed by molecular cues and patterns of electrical activity. In the rodent visual system, this process begins during the first postnatal week and proceeds during the second and third postnatal weeks as spontaneous retinal activity and finally use-dependent fine tuning takes place. Reelin is a large extracellular matrix glycoprotein able to affect several steps of brain development, from neuronal migration to the maturation of dendritic spines and use-dependent synaptic development. In the present study, we investigated the role of reelin on the topographical refinement of primary sensory connections studying the development of retinal ganglion cell axon terminals in the rat superior colliculus. We found that reelin levels in the visual layers of the superior colliculus are the highest between the second and third postnatal weeks. Blocking reelin signaling with a neutralizing antibody (CR-50) from PND 7 to PND 14 induced a non-specific sprouting of ipsilateral retinocollicular axons outside their typical distribution of discrete patches of axon terminals. Also we found that reelin blockade resulted in reduced levels of phospho-GAP43, increased GluN1 and GluN2B-NMDA subunits and decreased levels of GAD65 content in the visual layers of the superior colliculus. The results suggest that reelin signaling is associated with the maturation of excitatory and inhibitory synaptic machinery influencing the development and fine tuning of topographically organized neural circuits during postnatal development.

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.

Intravitreous Injection of Interleukin-6 Leads to a Sprouting in the Retinotectal Pathway at Different Stages of Development.

OBJECTIVE: The development of retinotectal pathways form precise topographical maps is usually completed by the third postnatal week. Cytokines participate in the development and plasticity of the nervous system. We have previously shown that in vivo treatment with interleukin 2 disrupts the retinocollicular topographical order in early stages of development. Therefore, we decided to study the effect of a single intravitreous injection of IL-6 upon retinotectal circuitry in neonates and juvenile rats. MATERIALS AND METHODS: Lister Hooded rats received an intravitreous injection of IL-6 (50 ng/ml) or vehicle (PBS) at either postnatal day (PND)10 or PND30 and the ipsilateral retinotectal pathway was evaluated 4 or 8 days later, respectively. RESULTS: Our data showed that, at different stages of development, a single IL-6 intravitreous treatment did not produce an inflammatory response and increased retinal axon innervation throughout the visual layers of the superior colliculus. CONCLUSIONS: Taken together, our data provide the first evidence that a single intravitreous injection with IL-6 leads to sprouting in the subcortical visual connections and suggest that small changes in IL-6 levels might be sufficient to impair the correct neuronal circuitry fine-tuning during brain development.

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.

Expression of GAP-43 during development and after monocular enucleation in the rat superior colliculus.

The retinotectal projection of rodents presents a precise retinotopic organization that develops, from diffuse connections, from the day of birth to post-natal day 10. Previous data had demonstrated that these projections undergo reorganization after retinal lesions, nerve crush and monocular enucleation. The axonal growth seems to be directly related to growth-associated protein-43 (GAP-43) expression, a protein predominantly located in growth cones, which is regulated throughout development. GAP-43 is presented both under non-phosphorylated and phosphorylated (pGAP-43) forms. The phosphorylated form, has been associated to axon growth via polymerization of F-actin, and synaptic enhancement through neurotransmitter release facilitation. Herein we investigated the spatio-temporal expression of GAP-43 in the rat superior colliculus during normal development and after monocular enucleation in different stages of development. Lister Hooded rats ranging from post-natal day 0 to 70 were used for ontogeny studies. Another group of animals were submitted to monocular enucleation at post-natal day 10 (PND10) or PND21. After different survival-times, the animals were sacrificed and the brains processed for either immunohistochemistry or western blotting analysis. Our data show that GAP-43 is expressed in retinotectal axons in early stages of development but remains present in adulthood. Moreover, monocular enucleation leads to an increase in pGAP-43 expression in the deafferented colliculus. Taken together these results suggest a role for pGAP-43 in retinotectal morphological plasticity observed both during normal development and after monocular enucleation.

Nutritional tryptophan restriction impairs plasticity of retinotectal axons during the critical period.

The use-dependent specification of neural circuits occurs during post-natal development with a conspicuous influence of environmental factors, such as malnutrition that interferes with the major steps of brain maturation. Serotonin (5-HT), derived exclusively from the essential aminoacid tryptophan, is involved in mechanisms of development and use-dependent plasticity of the central nervous system. We studied the effects of the nutritional restriction of tryptophan in the plasticity of uncrossed retinotectal axons following a retinal lesion to the contralateral retina during the critical period in pigmented rats. Litters were fed through their mothers with a low tryptophan content diet, based on corn and gelatin, a complemented diet with standard tryptophan requirements for rodents or standard laboratory diet. The results suggest a marked reduction in the plasticity of intact axons into denervated territories in the tryptophan restricted group in comparison to control groups. Tryptophan complementation between PND10-21 completely restored retinotectal plasticity. However, the re-introduction of tryptophan after the end of the critical period (between PND28-P41) did not restore the sprouting ability of uncrossed axons suggesting a time-dependent effect to the reversion of plasticity deficits. Tryptophan-restricted animals showed a reduced activity of matrix metalloproteinase-9 and altered expressions of phosphorylated forms of ERK1/2 and AKT. Our results demonstrate the influence of this essential aminoacid as a modulator of neural plasticity during the critical period through the reduction of serotonin content which alters plasticity-related signaling pathways and matrix degradation.

Neonatal tryptophan dietary restriction alters development of retinotectal projections in rats.

The specification of sensory neural circuits includes the elimination of transitory axon collaterals/synapses that takes place during early post natal life, an important step for the acquisition of topographical order of sensory systems. Serotonin has been implicated in the patterning of connections in subcortical and cortical circuits. We investigated the effects of the dietary restriction of the only serotonin precursor, tryptophan, on the development of the uncrossed retinotectal pathway in pigmented rats. Litters were fed through their mothers with either a tryptophan restricted, corn and gelatin based diet or a similar control diet complemented with tryptophan during the lactation period. The developmental status of the uncrossed retinotectal terminal fields was studied after the anterograde transport of horseradish peroxidase injected into one eye. We also studied the effects of tryptophan restriction on 5-HT immunoreactivity of raphe neurons, on cAMP levels in the visual layers of the superior colliculus and on protein synthesis among retinal neurons. We found that tryptophan restriction resulted in reduced weight gain among tryptophan restricted rats, without differences in protein synthesis between tryptophan complemented and restricted groups. Tryptophan restriction was also associated with a reduction of serotonin immunoreactive cells in the raphe nuclei and increased cAMP levels in the superior colliculus. Finally we found that neonatal tryptophan restriction resulted in an abnormal patterning of retinotectal topography, which was consistent with a developmental delay in axonal elimination and fine tuning of central connections. These results suggest, therefore, that dietary tryptophan is crucial for the influence of serotonin in the maturation of central visual connections.