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Human amyloid beta peptide (Aβ) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AβOs), which are known to accumulate in Alzheimer’s disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AβO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aβ as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AβOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AβO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AβOs, are linked to Alzheimer’s disease (AD). To test whether the AβOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AβO-selective antibody to prematurely lower the levels of AβOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AβOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.

Complex dynamic cellular networks have been studied in physiological and pathological processes under the light of single-cell calcium imaging (SCCI), a method that correlates functional data based on calcium shifts operated by different intracellular and extracellular mechanisms integrated with their cell phenotypes. From the classic synaptic structure to tripartite astrocytic model or the recent quadripartite microglia added ensemble, as well as other physiological tissues, it is possible to follow how cells signal spatiotemporally to cellular patterns. This methodology has been used broadly due to the universal properties of calcium as a second messenger. In general, at least two types of receptor operate through calcium permeation: a fast-acting ionotropic receptor channel and a slow-activating metabotropic receptor, added to exchangers/transporters/pumps and intracellular Ca2+ release activated by messengers. These prototypes have gained an enormous amount of information in dynamic signaling circuits. SCCI has also been used as a method to associate phenotypic markers during development and stage transitions in progenitors, stem, vascular cells, neuro- and glioblasts, neurons, astrocytes, oligodendrocytes, and microglia that operate through ion channels, transporters, and receptors. Also, cancer cells or inducible cell lines from human organoids characterized by transition stages are currently being used to model diseases or reconfigure healthy cells in terms of the expression of calcium-binding/permeable molecules and shed light on therapy.

Omega-3 (n-3) polyunsaturated fatty acids (PUFA) and the endocannabinoid system (ECS) modulate several functions through neurodevelopment including synaptic plasticity mechanisms. The interplay between n-3PUFA and the ECS during the early stages of development, however, is not fully understood. This study investigated the effects of maternal n-3PUFA supplementation (n-3Sup) or deficiency (n-3Def) on ECS and synaptic markers in postnatal offspring. Female rats were fed with a control, n-3Def, or n-3Sup diet from 15 days before mating and during pregnancy. The cerebral cortex and hippocampus of mothers and postnatal 1-2 days offspring were analyzed. In the mothers, a n-3 deficiency reduced CB1 receptor (CB1R) protein levels in the cortex and increased CB2 receptor (CB2R) in both cortex and hippocampus. In neonates, a maternal n-3 deficiency reduced the hippocampal CB1R amount while it increased CB2R. Additionally, total GFAP isoform expression was increased in both cortex and hippocampus in neonates of the n-3Def group. Otherwise, maternal n-3 supplementation increased the levels of n-3-derived endocannabinoids, DHEA and EPEA, in the cortex and hippocampus and reduced 2-arachidonoyl-glycerol (2-AG) concentrations in the cortex of the offspring. Furthermore, maternal n-3 supplementation also increased PKA phosphorylation in the cortex and ERK phosphorylation in the hippocampus. Synaptophysin immunocontent in both regions was also increased. In vitro assays showed that the increase of synaptophysin in the n-3Sup group was independent of CB1R activation. The findings show that variations in maternal dietary omega-3 PUFA levels may impact differently on the ECS and molecular markers in the cerebral cortex and hippocampus of the progeny.

Neurospheres prepared from multipotent progenitors in the retina obtained from postnatal mice differentiate into neurons and Müller glia (De Melo Reis et al., in Cell Mol Neurobiol 31:835-846, 2011). Here, we investigated whether neurospheres prepared from adult chickens (ciliary marginal zone, CMZ) or (ciliary body) retina could also lead to differentiated neurons and glia. Neurospheres were prepared from post-hatched chickens or from adult mice after 7 days in the presence of mitogenic factors (FGFb, insulin, and EGF), generating neurons and glial cells. In addition, Müller (2M6 or glutamine synthetase positive cells) derived from post-hatch chicken CMZ neurospheres displayed the dopaminergic phenotype. Furthermore, we observed that Müller cells derived from adult chickens and mice retina neurospheres released significant amounts of dopamine as well as of its metabolites. Taken together, our data lead us to conclude that as for embryonic (chick) or newborn (mouse), the dopaminergic phenotype is a default condition of Müller glial cells obtained from neurospheres prepared from mature retina. Our data raise the possibility that Müller cells from differentiated tissue could be used to ameliorate neurodegenerative diseases involving dopaminergic dysfunction as in Parkinson's disease as shown previously (Stutz et al., in J Neurochem 128:829-840, 2014).

Development of progenitors in the embryonic retina is modulated by signaling molecules, and cannabinoid receptors are highly expressed in the early developing retina. Here, we investigated whether the CB1/CB2 receptor agonist WIN 5212-2 (WIN) modulated the proliferation, viability, and calcium responses in chick embryo retinal progenitors in culture. A decline in [3H]-thymidine incorporation was observed when cultures were incubated with 0.5–1.0 μM WIN, an effect that was mimicked by URB602 and URB597, inhibitors of the monoacylglycerol lipase and fatty acid amide hydrolase, respectively. A reduction in the number of proliferating cell nuclear antigen-positive nuclei was also noticed in WIN-treated cultures, suggesting that activation of cannabinoid receptors decreases the proliferation of cultured retinal progenitors. WIN (0.5–5.0 μM), but not capsaicin, decreased retinal cell viability, an effect that was blocked by CB1 and CB2 receptor antagonists and by the P2X7 receptor antagonist A438079, implicating this nucleotide receptor in the cannabinoid-mediated cell death. Treatment with WIN also induced an increase in mitochondrial superoxide and P2X7 receptor-mediated uptake of sulforhodamine B in the cultured cells. While a high proportion of cultured cells responded to glutamate, GABA, and 50 mM KCl with intracellular calcium shifts, very few cells responded to the activation of P2X7 receptors by ATP. Noteworthy, while decreasing the number of cells responding to glutamate, GABA, and KCl, treatment of the cultures with WIN induced a significant increase in the number of cells responding to 1 mM ATP, suggesting that activation of cannabinoid receptors primes P2X7 receptor calcium signaling in retinal progenitors in culture.

Amyloid β oligomers (AβOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AβO-targeting diagnostics and therapeutics, the AβO structures contributing to AD-associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AβOs stabilized by the bifunctional crosslinker 1,5-difluoro-2,4-dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aβ in a soluble oligomeric conformation. With DFDNB, solutions of Aβ that would otherwise convert to large aggregates instead yield solutions of stable AβOs, predominantly in the 50-300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top-down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.-injected mice, the DFDNB-stabilized AβOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AβO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AβOs in structure-function studies.

The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Muller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212-2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.

We sought to examine interactions of the prion protein (PrPC) with monoaminergic systems due to: the role of PrPC in both Prion and Alzheimer diseases, which include clinical depression among their symptoms, the implication of monoamines in depression, and the hypothesis that PrPC serves as a scaffold for signaling systems. To that effect we compared both behavior and monoaminergic markers in wild type (WT) and PrPC-null (PrP−/−) mice. PrP−/− mice performed poorly when compared with WT in forced swimming, tail suspension, and novelty suppressed feeding tests, typical of depressive-like behavior, but not in the control open field nor rotarod motor tests; cyclic AMP responses to stimulation of D1 receptors by dopamine was selectively impaired in PrP−/− mice, and responses to serotonin, but not to norepinephrine, also differed between genotypes. Contents of dopamine, tyrosine hydroxylase, and the 5-HT5A serotonin receptor were increased in the cerebral cortex of PrP−/−, as compared with WT mice. Microscopic colocalization, as well as binding in overlay assays were found of PrPC with both the 5HT5A and D1, but not D4 receptors. The data are consistent with the scaffolding of monoaminergic signaling modules by PrPC, and may help understand the pathogenesis of clinical depression and neurodegenerative disorders.

Endocannabinoids are endogenous lipids that activate selective G protein coupled receptors (CB1 and CB2), mostly found at neuronal presynaptic sites in the nervous system. One of the main consequences of the activation of CB receptors is a decrease in GABA or glutamate release, controlling cell excitability. Here we studied the expression of CB1 and CB2 receptors in E8C8 cultured retina cells (embryonic day 8 and 8 days in vitro) using immunocytochemistry and western blot analysis. We also evaluated their functions in terms of cyclic AMP (cAMP) production, single cell calcium imaging (SCCI) and GABA release induced in basal conditions or activated by l -Aspartate (L-ASP) in cell cultures or under ischemia in young chick retina. We show that both cannabinoid receptors are expressed in retinal neurons and glial cells. WIN 55,212-2 (WIN, a CB1/CB2 agonist) decreased cAMP production in cultured avian embryonic retinal cells in basal conditions. WIN also led to a decrease in the number of glial cells that increased Ca2+ levels evoked by ATP, but had no effect in Ca2+ shifts in neuronal cells activated by KCl. Finally, WIN inhibited [3H]-GABA release induced by KCl or L-ASP, accumulated in amacrine cells, but had no effect in the amount of GABA released in an oxygen glucose deprivation (OGD) condition. Altogether, our data indicate that cannabinoid receptors function as regulators of avian retina signaling at critical embryonic stages during synapse formation.

The retina is a multilayered tissue that develops following a central-to-peripheral gradient. Its structure derives from multipotent precursors, as shown through clonal analysis of retinal cell lineage. These progenitors generate diverse cell types, controlled by complex influences of intrinsic and extrinsic factors (Hatakeyama and Kageyama, 2004). Several types of neurons and one main glia cell constitute the vertebrate retina, constructed in a highly organized manner. Muller cell is the predominant glia of the retina and the last cell type to differentiate. Upon damage or under the influence of growth factor stimulation, they proliferate, de-differentiate and become a source of new neurons for retinal regeneration in fish and, to a much less extent, postnatal chicks. Evidence indicates that some Muller glial cells in mammals also have the potential to proliferate in response to N-methyl D-aspartate (NMDA) treatment and produce bipolar cells and rod photoreceptors (reviewed in Goldman, 2014). Three cellular layers make the final organization of the retina, with each of the six types of neurons and the Muller glia. Selective markers in the adult vertebrate retina (mice for all, except GAT-3 for chick) can identify these elements as shown in Figure 1. Opsin and rhodopsin label photoreceptors, calbindin identifies horizontal cells, CHX10 (C. elegans ceh-10 homeodomain-containing homolog) identifies bipolar cells, GAT-3 transporter labels selective GABAergic amacrine cells, BetaIII tubulin (tuj-1) recognizes retinal ganglion cells (RGCs) and glutamine synthetase (GS) labels Muller glia. Figure 1 Immunohistochemistry of selective markers of the adult mouse retina. Anti-glial fibrilary acidic protein (GFAP) is used to identify reactive glial cells that involve the axons of RGCs that make the optic nerve. Retinal diseases affect millions of patients worldwide affecting photoreceptors, as in age-associated macular degeneration (AMD) and retinitis pigmentosa, or RGCs, as glaucoma or diabetic retinopathy. Several laboratories are looking for the possibility of retinal regeneration based on cellular and or molecular strategies. Normally, GFAP or nestin (a progenitor marker) do not label Muller cells (Figure 1) in the undamaged retina, as opposed to GS, a marker that shows Muller glia processes extend across all retinal layers going from the inner to the outer limiting membranes (Figure 1). Traumatic lesion of the retina induces Muller cell proliferation due to inflammatory processes. This condition is mimicked artificially by neurotoxins like NMDA or kainate injected into the eye of postnatal chick (Fischer and Reh, 2001). This procedure makes Muller glia to acquire neurogenic potential in response to injury providing a source of neural stem cells in this tissue. Several pathways seem to be involved with Muller glial proliferation and dedifferentiation such as Notch, initially discovered in zebrafish (reviewed in Goldman, 2014). Notch plays a central role in the conservation of stemness throughout retinal development. Notch-signaling components are expressed at low levels in healthy Muller glia in the postnatal retina, but upon stimulation with basic fibroblast growth factor (FGF2) and insulin, Muller glia proliferate and dedifferentiate. Tumor necrosis factor alpha (TNF-α) together with repression of Notch induce Muller glia to proliferate in the adult zebrafish retina, generating neuronal progenitor cells (reviewed in Goldman, 2014). Wnt/β-catenin also induces proliferation of Muller glia-derived progenitors and regeneration after damage, or during degeneration in the adult rodent retina (reviewed in Goldman, 2014). Finally, sonic hedgehog (Shh) has been shown to stimulate Muller glial proliferation through its receptor. Shh-treated Muller glial dedifferentiate through expression of progenitor-specific markers, leading to the fate of rod photoreceptor. Together, these results provide evidence that Muller glia operate on diverse signaling mechanisms (for a complete list of factors acting on Muuller cells, see Goldman, 2014) to reprogram and generate progenitors in zebrafish and perhaps give a clue as potential stem cells in mammalian retina. Examples depicting the phenotypic plasticity of Muller cells after injury have been described in different vertebrate models. For instance, spatiotemporal distribution of retinal cells induced by lesion is shown in the adult zebrafish (Yurco and Cameron, 2005). They show double labeling immunohistochemistry using proliferation (anti-BrdU or anti-PCNA) and Muller glial markers (carbonic anhydrase, or GS). Muller cell proliferation is also shown in postnatal chick (Fischer and Reh, 2001). Finally, in mammals, few Muller glial cells injected in the adult rat retina to stimulate proliferation produce bipolar cells and rod photoreceptors (reviewed in Goldman, 2014). Muller glia obtained from rat retina can generate clonal spheres capable of differentiating into functional neurons (Das et al., 2006); In addition, retinal neurospheres from postnatal mice have the potential to generate neurons and Muller glia as identified by calcium imaging protocols (De Melo Reis et al., 2011). The possibility to obtain different types of retinal cells from precursors raise the possibility of developing cell transplants methodologies to restore proper visual function lost in retina degeneration. The elimination of neurons from mixed retinal neuron-glia cultures makes Muller cells to express several markers found in neuronal cells. Among these, glutamate decarboxylase (GAD), TH, pituitary adenylate cyclase-activating peptide (PACAP) receptors (Kubrusly et al., 2005) and Nurr1, a transcriptional factor associated with dopaminergic phenotype were described. Dopamine D1 receptors are also functional as they generate cyclic AMP. Consequently, purified cultures of Muller cells develop the full complement of functional dopaminergic phenotype, including the release of dopamine. This seems to be due to a default pathway for Muller cells under this condition. This dopaminergic default occurs in Muller cells obtained from avian, mouse and monkey retina (Stutz et al., 2014). Dopaminergic Muller cells transplanted into the striatum of hemi-parkinsonian mice fully recover motor behavioral deficits (Stutz et al., 2014). Therefore, it is an attractive possibility to suggest that these dopaminergic Muller cells could be of potential use in cellular therapies for dopaminergic dysfunction. The fact that the dopaminergic default does not require hard manipulation for cells to express the dopaminergic phenotype makes it less likely to cause hazardous influence on healthy tissues. Muller cells are actively involved in the synaptic control of retinal neurons through the release of transmitters and trophic factors (de Melo Reis et al., 2008). These cells interact with most of the retinal neurons, ranging from RGC to photoreceptors. However, the majority of retinal synapses are glutamatergic and GABAergic in close association with glial cells. In this sense, recent data show that GAT-3, a GABA transporter found in purified Muller glia, is functionally regulated by glutamate. This response involves ionotropic glutamatergic receptors. In addition to GAT-3, GAT-1 is expressed in purified glial cells. However, only GAT-3 seems to be functional (De Sampaio Schitine et al., 2007). Glutamate decreases the levels of GAT-3 transporter in the plasma membrane of Muller cells as well as its mRNA. In the avian retina, GAT-3 is primarily expressed in the inner plexiform layer (IPL) and in some cell bodies in the INL, where most of the amacrine cells are located (Figure 1; Schitine et al., 2015). Muller glia also have their soma in the INL. Retinal lesion induced by NMDA injections provokes a large increase in GAT-3 immunoreactivity in Muller fibers (Figure 2), followed by damage to RGCs, and an increase in GFAP expression. Reactive gliosis is a hallmark in several neurologic diseases but not so well understood, and in the retina, it has been associated with several degenerative conditions such as hepatic retinopathy, macular edema, and retinitis pigmentosa. Figure 2 Schematic illustration showing γ-aminobutyric acid (GABA) signaling in avian retina. Evidence from Ortinski et al. (2010) shows that reactive gliosis artificially induced in hippocampal circuits leads to decreased expression of GS, implying a reduction in the glutamate production from glutamine. Therefore, a rapid decrease of GABA content in gabaergic synapses leads to a decreased inhibitory tonus on synaptic transmission in mouse CA1 pyramidal neurons. This seems to favor excitotoxicity. Our recent data suggest that in vivo lesions of the retina may be potentiated by decreased inhibitory tonus, due to increased GABA uptake by Muller cells overexpressing GAT-3 (Figure 2). Further investigations are necessary to reveal the molecular mechanisms involved in glutamate-dependent GAT-3 plasma membrane level reduction. Interestingly however, our observations open the possibility of using GABA transport inhibitors to prevent RGCs degeneration eventually caused by reactive gliosis that follow retina degeneration. This work was supported by grants from FAPERJ, CNPq (INCT- INNT), CAPES and PROLAB LARC/IBRO/CNPq. The authors thank Dr. Patricia Gardino, Dr. Silmara Lima (for the Tuj1 image) and Dr. Rodrigo Martins (for the CHK10 image) in Figure 1. CS is recipient of a CAPES- FAPERJ Postdoc fellowship.

Muller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Muller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Muller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor-related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Muller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Muller-derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Muller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Muller cells with the dopaminergic phenotype were transplanted into the striatum of hemi-parkinsonian mice generated by unilateral injection of 6-hydroxydopamine. These cells fully decreased the apomorphine-induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb-use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi-parkinsonian mice confirmed by measurement of striatal dopamine after Muller cell grafting. Muller cells are the main glial cells in the retina. When these cells are cultured in the absence of neurons, they spontaneously express proteins of the dopaminergic phenotype, including the enzymes tyrosine hydroxylase (TH), L-DOPA-decarboxylase (DDC) and the dopamine transport system (DAT). In this study, we show this phenomenon is observed with Muller cells obtained from different species, including primates, and address the therapeutic potential of these cells, using a mouse model of Parkinson's disease (PD). ‘Dopaminergic Muller cells’ synthesize dopamine and release most of this neurotransmitter to the extracellular space, constituting a natural dopaminergic ‘pump’. When transplanted to the striatum of PD mice, Muller cells decreased their apomorphine-induced rotational behavior and restored their overall motor functions, measured by rotarod and forelimb use asymmetry tests. Local restoration of dopaminergic signaling was also observed in grafted PD mice, by measuring striatum dopamine and its metabolite (DOPAC) levels (SB: 20µm).

Ampakines are chemical compounds known to modulate the properties of ionotropic a-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 lm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK ⁄ JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.

The most common neurodegenerative disorder afflicting the aging human population is Alzheimer's disease (AD). A major hallmark of AD is dementia from a loss of neuronal function, attributed to the presence and accumulation of β-amyloid (Aβ) peptide into senile plaques. Preceding senile plaque formation, abnormalities in axons can be observed as changes in morphologies and intracellular trafficking. Recently, it has been recognized that Aβ also accumulates within neurons and this intraneuronal Aβ accumulation has been reported to be critical in the disruption of synapses and cognitive function. Here, we report on the internalization of a fluorescently labeled Aβ peptide into cultured chick retinal neurons. The pattern of Aβ distribution during the time course of incubation is reminiscent of the endocytic pathway. Furthermore, the distribution of the internalized Aβ peptide converges with that of myosin Vb and both relocalize from the axon to cell body. These observations are consistent with the hypothesis that AD proceeds as a result of an imbalance between Aβ production and Aβ clearance, suggesting a role for myosin Vb in this process.

d -aspartate is present in significant concentrations throughout the nervous tissue but its physiological role is still under discussion. Here, we report the process of d -aspartate release in retinal cells. [ 3 H]- d -aspartate release occurs through a glutamate/aspartate exchange mechanism using excitatory amino acid transporters. This process is sodium-dependent and it is not prevented by glutamate receptor antagonists such as MK-801, DNQX or AIDA nor mimicked by glutamatergic agonists like kainate, NMDA or trans -ACPD. In vitro experiments indicate that the great majority of d -aspartate release is performed by neuronal cells and to a much lower extent by glial cells. This glutamate-mediated release process is mimicked by the competitive glutamate transporter antagonist l - trans -PDC and inhibited by the non-competitive transporter antagonist TBOA. Instead of the classical calcium-dependent exocytosis or transporter-reversal mediated neuronal release, d -aspartate efflux in the retina occurs mostly, if not exclusively, via an exchange of external l -glutamate by d -aspartate predominantly present in the cytoplasmatic compartment of neurons. These data also suggest that this process narrows down the specificity of excitatory signaling in the microenvironment of the synapses, reinforcing NMDA receptor activation by d -aspartate at the cost of reduction in the overall activation of excitatory amino acid receptors promoted by l -glutamate.

Polyamines are important regulators of growth and differentiation in a variety of cells, including parasitic protozoa. Promastigotes of Leishmania species have high levels of putrescine and spermidine and their growth can be inhibited by polyamine biosynthesis antagonists. The putrescine analogue 1,4-diamino-2-butanone (DAB) is microbicidal against Tritrichomonas foetus and Trypanosoma cruzi, so we tested its effects on Leishmania amazonensis proliferation, viability, organization, putrescine transport and synthesis as well as in vitro infectivity. DAB impaired promastigote proliferation dose-dependently (IC(50) 144 microM) and the parasite putrescine concentration was reduced by nearly 50 %. This analogue markedly inhibited both ornithine decarboxylase activity and [H(3)]putrescine uptake by promastigotes. Pre-treatment with DAB for 24 h led to compensatory enhancement of putrescine uptake, indicating an adaptive mechanism in DAB-treated parasites. Remarkably, DAB caused mitochondrial damage, assessed by transmission electron microscopy, and 3 h treatment with 1 mM DAB enhanced lipid peroxidation, whereas incubation with 10 mM DAB or for 24 h resulted in decreased peroxidation levels in the parasites. This effect was probably due to the loss of mitochondrial function, demonstrated by the diminished reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), not observed in macrophages. Murine macrophages infected with L. amazonensis amastigotes treated with DAB had parasite loads significantly (P

Purified retina glial Müller cells can express the machinery for dopamine synthesis and release when maintained in culture. Dopamine is detected in cell extracts of cultures exposed to its precursor, L-DOPA. A large portion of synthesized dopamine is recovered in the superfusing medium showing the tendency of the accumulated dopamine to be released. Müller cells purified from developing chick and mouse retinas express L-DOPA decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; EC 4.1.1.28) and the dopamine transporter DAT. The synthesis of dopamine from L-DOPA supplied to Müller cultures is inhibited by m-hydroxybenzylhydrazine, a DDC inhibitor. Dopamine release occurs via a transporter-mediated process and can activate dopaminergic D(1) receptors expressed by the glia population. The synthesis and release of dopamine were also observed in Müller cell cultures from mouse retina. Finally, cultured avian Müller cells display increased expression of tyrosine hydroxylase, under the influence of agents that increase cAMP levels, which results in higher levels of dopamine synthesized from tyrosine. A large proportion of glial cells in culture do express Nurr1 transcription factor, consistent with the dopaminergic characteristics displayed by these cells in culture. The results show that Müller cells, deprived of neuron influence, differentiate dopaminergic properties thought to be exclusive to neurons.

Müller cells represent the main type of glia present in the retina interacting with most, if not all neurons in this tissue. Müller cells have been claimed to function as optic fibers in the retina delivering light to photoreceptors with minimal distortion and low loss [Franze et al (2007) Proc Natl Acad Sci 104:8287-8292]. Most of the mediators found in the brain are also detected in the retinal tissue, and glia cells are active players in the synthesis, release, signaling and uptake of major mediators of synaptic function. Müller glia trophic factors may regulate many different aspects of neuronal circuitry during synaptogenesis, differentiation, neuroprotection and survival of photoreceptors, Retinal Ganglion Cells (RGCs) and other targets in the retina. Here we review the role of several transmitters and trophic factors that participate in the neuron-glia loop in the retina.

Previous work on in vitro culturing of silkmoth (Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.

GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na(+)/Cl (-) dependent transporters (GATs). It has been reported in the past that, in the avian retina, [(3)H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [(3)H]GABA in a Na(+) and Cl(+) dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [(3)H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K[(+) or veratridine stimulation, Müller glial cells did not release [(3)H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.

The role of dopamine in the retina has been studied for the last 30 years and there is now increasing evidence that dopamine is used as a developmental signal in the embryonic retina. Dopamine is the main catecholamine found in the retina of most species, being synthesized from the L-amino acid tyrosine. Its effects are mediated by G protein coupled receptors constituting the D 1 (D 1 and D 5 ) and D 2 (D 2 , D 3 and D 4 ) receptor subfamilies that can be coupled to adenylyl cyclase in opposite manners. Dopamine-mediated cyclic AMP (cAMP) accumulation, via D 1 -like receptors, is observed very early during retina ontogeny, before synaptogenesis and, in some species, before the expression of tyrosine hydroxylase (TH), the enzyme that characterizes the neuronal dopaminergic phenotype. D 2 -like receptors appear in the tissue days after D 1 -like activity is detected. In the embryonic avian retina, before the tissue is capable of synthesizing its own dopamine via TH, dopamine synthesis is observed from l -DOPA supplied to the neuroretina from retina pigmented epithelium which results in dopaminergic communication in the embryonic tissue before TH expression. Muller cells, the main glia type found in the retina, seem to actively contribute to dopaminergic activity in the retinal tissue. Understanding the dopaminergic role during retina development may contribute to novel strategies approaching certain visual dysfunctions such as those found in ocular albinism.

Background The early stages of central nervous system (CNS) development are extremely important. Key events such as neurogenesis, gliogenesis, synaptogenesis, and ontogenesis occur. Malnutrition promotes alterations in CNS development, including the retinal development. During retinal development, malnutrition can induce a delay in some important events, such as neurotransmitter expression and neurogenesis. Methodology/Principal findings Postpartum Wistar rats were fed either a commercial diet or a multideficient diet. Pups were breastfed by these rats, and from PND21 were kept with the same diet until PND45. We investigated the effects of malnutrition on adult retinal tissue with regard to (1) endogenous gamma-amino butyric acid (GABA) release induced by excitatory amino acids (EAAs) and (2) the expression of cellular markers related to degenerative events, such as reactive gliosis, microglial activation, cell proliferation and cell death. Endogenous GABA release induced by EAAs was higher in the retina of malnourished rats. The Muller cell population was reduced and displayed alterations in their phenotype profile compatible with reactive gliosis. The expression of glutamine synthetase and markers of cellular proliferation were higher in the retina of malnourished rats. Additionally, retinal dysplasia-like structures were present, indicating disturbance in the cell cycle machinery. Conclusion/Significance The current study provides evidence that the adult retina shows degenerative processes induced by long-term malnutrition during the postnatal development. These findings have high clinical significance with regard to the identification of possible targets for interventions in malnourished patients.

Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism.

In the chick retina, dopaminergic cells are generated between embryonic days 3 and 7 (E3/E7). However, the expression of tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthetic pathway, is only detected after E11/E12. During the interval comprising E7 to E12, signals conveyed by cAMP are important to determine the TH phenotype. The present study shows that pituitary adenylyl cyclase-activating polypeptide (PACAP), via cAMP, is a major endogenous component in defining the TH phenotype of retina dopaminergic cells during development. PACAP type 1 receptor and its mRNA were detected in retinas since E6. PACAP was also immunodetected in cells localized in the inner nuclear layer of retinas since E8. This peptide promoted greater than 10-fold increase in cAMP accumulation of retinas obtained from embryos since E8, an effect that was blocked by PACAP6-38 (PAC1 receptor antagonist). In cultured retina cells from E8 and E9, maintained for 6 days in vitro with 10 nM PACAP (for 5 days), the number of dopaminergic cells expressing tyrosine hydroxylase increased 2.4-fold. The cAMP analog, 8-Br-cAMP and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) also increased the number of tyrosine hydroxylase-positive cells by 4- to 6-fold. IBMX plus PACAP treatment resulted in 17-fold increase in the number of cells positive for tyrosine hydroxylase. Under this condition the amount of tyrosine hydroxylase expression, as detected by western blot analysis, was also increased. The protein kinase-A inhibitor, rp-cAMPS, significantly reduced the effect of PACAP. Our data show that this peptide is an important factor influencing the definition of the tyrosine hydroxylase phenotype of retina dopaminergic cells within a narrow window of development.

Glia represents the most numerous group of nervous system cells and CNS development and function depend on glial cells. We developed a purified Muller glia culture to investigate the expression of several neurotransmitter markers on these cells, such as dopaminergic, cholinergic, GABAergic and peptidergic receptors or enzymes, based on functional assays measuring second messenger levels or Western blot for specific proteins. Purified Muller cell culture was obtained from 8-day-old (E8) embryonic chick. Glial cells cultured for 15 days (E8C15) expressed D1A and D1B receptors mRNAs, but not D1D, as detected by RT-PCR. The binding of [3H]-SCH 23390 revealed an amount of expressed receptors around 40 fmol/mg protein. Dopamine (100 microM), PACAP (50 nM) and forskolin (10 microM) induced a 50-, 30- and 40-fold cAMP accumulation on glial cells, respectively, but not ip3 production. The dopamine-promoted cAMP accumulation was blocked by 2 microM SCH 23390. Carbachol stimulated a 3-fold ip3 accumulation. Western blot analysis also revealed the expression of tyrosine hydroxylase, L-dopa decarboxylase, PAC1 receptor, GAD67 and beta2-nicotinic receptor subunit by these cells. These results indicate that several components of neurotransmitter signaling and metabolism are found in cultured Muller cells.

The participation of eicosanoids and second messengers in the regulation of endocytosis by the ovaries was investigated using the uptake of Rhodnius heme binding protein (RHBP) as an experimental model. The rate of RHBP uptake decreased up to 40% in the presence of BWA4C and NDGA, 5 and 12-lipoxygenase inhibitors, respectively, suggesting the involvement of lipoxygenase products in endocytosis regulation. Addition of Leukotriene B4 (LTB(4); one product of the 5 lipoxygenase pathway) increased in vitro the uptake of RHBP by 30%. The content of cAMP in the Rhodnius' ovaries were monitored after treatment with different eicosanoids and inhibitors of eicosanoids synthesis. The amount of cAMP decreased in the presence of indomethacin (by 50%), while treatment with PGE(2) induced an increase of 85% of this messenger in the ovaries. The presence of LTB(4) in the medium inhibited in 60% the content of cAMP in the ovaries, while BWA4C induced a 100% increase of this messenger in the ovaries. Addition of 1 microM DBcAMP in the medium resulted in a 30% decrease in the rate of RHBP uptake. Taken together, these data show that cyclooxygenase and lipoxygenase products participate in the control of protein internalization by modulation of cAMP levels.

DOPA decarboxylase (DDC; aromatic-l-amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6-day-old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre-incubated with l-DOPA for 1 h, increased 250- and 600-fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre-incubated for 1 h with 1 mm l-DOPA, washed and followed by incubation in the presence of 0.5 mm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre-incubation with l-DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 micro m). The incubation of retinas in medium previously conditioned by retina-pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l-DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l-DOPA is supplied to the tissue. It also shows that RPE is a potential source of l-DOPA early in development.

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.

Inhibition of protein synthesis leads to apoptosis in the undifferentiated neuroblastic layer of the retina of newborn rats. We have shown previously that an increase in the intracellular concentration of cyclic AMP prevented apoptosis induced in the retinal neuroblastic layer by inhibition of protein synthesis. In this study, we tested the effects of dopamine on retinal apoptosis and related these effects to the intracellular concentration of cyclic AMP. Both dopamine (100 microM) and the D1-like agonists SKF-38393, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (6-Cl-PB), and (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (100 microM) blocked apoptosis induced in the neuroblastic layer by the protein synthesis inhibitor anisomycin. The antiapoptotic effects of the D1-like agonists were not reversed by the D1-like antagonist SCH-23390 (5-100 microM). Both dopamine and D1-like agonists induced a five- to sevenfold increase in the intracellular concentration of cyclic AMP in the retina of newborn rats. The concentration of cyclic AMP induced by the D1-like agonists in the presence of 100 microM SCH-23390 was still at least two- to threefold as high as control values, showing that the activation of adenylyl cyclase by D1-like agonists was reversed only partially by the specific antagonist. The isoquinolinesulfonamide H-89 (20 microM), an inhibitor of cyclic AMP-dependent protein kinase, partially prevented the antiapoptotic effect of 6-Cl-PB. The data show that an early effect of dopamine in the developing retina is the control of programmed cell death. The antiapoptotic effect of dopamine is mediated, at least in part, through an atypical D1-like receptor coupled to stimulation of adenylyl cyclase, followed by activation of cyclic AMP-dependent protein kinase.

Aging represents a major risk factor for numerous illnesses that are of increasing importance to society, including two of the most prevalent: diabetes and Alzheimer׳s disease. Studies have shown that diabetes is a risk factor for spontaneous Alzheimer׳s disease. While these studies suggest that diabetes can contribute to Alzheimer׳s disease, the implications of AD on diabetes are practically unexplored. The major mediator of the pathophysiological effects, the Aβ42 peptide, has been shown to enter neurons and lead to an alteration of the intracellular distribution of the molecular motor myosin Vb. Myosin Vb functions in memory and learning by participating in the strengthening of the long-term potentiation (LTP) of synaptic transmissions. It has also been implicated in the translocation of the glucose transporter, GLUT4, to the plasma membrane in response to insulin, a process that is defective in diabetes. Here, the effect on GLUT4 upon entry of the Aβ42 peptide into cultured chick retinal neurons was explored. The results suggest an alteration in distribution and a reduced level at the cell surface, as well as an increased colocalization with myosin Vb, which can partially explain the changes in glucose metabolism associated with AD. It is also shown that the presence of the Aβ40 peptide inhibits the internalization of the Aβ42 peptide in cultured cells. Together, the results provide additional targets for the development of therapeutics against the progression and effects of Alzheimer׳s disease.

Choline acetyltransferase (ChAT) activity was reduced by more than 85% in cultured retina cells after 16 h treatment with 150 µm kainate (T1/2 : 3.5 h). Glutamate, AMPA and quisqualate also inhibited the enzyme in equivalent proportion. Cell lesion measured by lactate dehydrogenase (LDH) release, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - thiazolyl blue (MTT) reduction and microscopic observation was not detected even after 48 h with kainate. Other retina neurochemical markers were not affected by kainate and full recovery of the enzyme was achieved 9 days after kainate removal. Moreover, hemicolinium-3 sensitive choline uptake and hemicolinium-3 binding sites were maintained intact after kainate treatment. The immunoblot and immunohistochemical analysis of the enzyme revealed that ChAT molecules were maintained in cholinergic neurons. The use of antagonists showed that ionotropic and group 1 metabotropic receptors mediated the effect of glutamate on ChAT inhibition, in a calcium dependent manner. The quisqualate mediated ChAT inhibition and part of the kainate effect (30%) was prevented by 5 mmNG-nitro-l-arginine methyl ester (l-NAME). Veratridine (3μM) also reduced ChAT by a Ca2+ dependent, but glutamate independent mechanism and was prevented by 1μM tetrodotoxin.

We examined the effects of dopamine and cAMP on the differentiation of dopaminergic retinal cells in the chick retina, using an in vitro system and tyrosine hydroxylase immunocytochemistry. Tyrosine hydroxylase-positive cells were detected in cultures prepared from embryonic day 10 retinas. These increased in number as a function of time in vitro and by treatment for 4 days with forskolin. Besides causing a 3.4-fold increase in the tyrosine hydroxylase-positive population, forskolin also caused these cells to developed morphogenetic features of more mature cells. As opposed to forskolin, cultures treated with dopamine exhibited a 55% reduction of the tyrosine hydroxylase-positive cell population, as compared to untreated cultures. Quinpirole was able to mimic the dopamine effect. This dopamine effect could only be blocked by clozapine, whereas raclopride and eticlopride were ineffective. Our results suggest the existence of a narrow window during development when undifferentiated dopaminergic cells are capable of being influenced by specific signals, possibly via cAMP production. The data also indicate that dopamine may act as a regulatory factor limiting the tyrosine hydroxylase-positive population in the retina.

The effects of excitatory amino acids (EAAs) upon transporter-mediated gamma-aminobutyric acid (GABA) release were investigated in cells containing tyrosine hydroxylase (TH) or nitric oxide synthase (NOS) in retina of the primate Cebus apella. Retinas were treated in vitro with 50 microM Kainate (KA) or 5 mM L-Glutamate (L-Glu), for 30 min at 37 degrees C, in an Mg2+-free Locke's solution with or without Ca2+. The effects of EAAs were measured immunocytochemically by determining the GABA content in TH or NOS-immunoreactive cells in the inner retina, after stimulation. L-Glu and KA induced a Ca2+-independent GABA release from most GABA-immunoreactive cells of the inner retina. Double label experiments indicated that this release occurs in NOS+/GABA+ cells, but not in TH+/GABA+ cells suggesting that these cell subpopulations may be differentiated in some functional aspects.

The effects of glutamate receptor antagonists on the toxicity of the b-amyloid peptide (Ab1‐42) in embryonic chick retina were investigated. When used alone or in combination, the N-methyl-d-asparate antagonist, MK-801, the (^)-aamino-3hydroxyl-5-methylisoxazole-4-propinic acid/kainate antagonist, DNQX, and the metabotropic receptor antagonist, (RS)1-aminoindan-1,5-dicarboxylic acid, blocked the neurotoxicity of Ab1‐42. Aggregation of Ab1‐42 was significantly increased in the presence of acidic glutamate solutions, but not in the presence of other neurotransmitters. These results point to a dual role of glutamatergic transmission in Alzheimer’s disease (AD): (i) Ab neurotoxicity requires activation of glutamate receptors and its blockade prevents cell death; (ii) high concentrations of glutamate in the synaptic cleft indirectly enhance Ab aggregation through acidification of the medium, resulting in increased amounts of neurotoxic amyloid fibrils. These results suggest that glutamatergic neurotransmission may represent a novel target for therapeutic approaches in AD. q 2001 Elsevier Science Ireland Ltd. All rights reserved.

The release of GABA from amacrine and interplexiform cells after exposure to excitatory amino acids (EAAs) agonists was investigated by immunohistochemistry. Cebus monkey retinas were treated in vitro with 50 microM kainate (KA) or 5 mM L-Glutamate (L-Glu), for 30 min at 37 degrees C. The effects of the EAAs were measured by detecting immunocytochemically the GABA remaining in the tissue after stimulation. L-Glu and KA reduced the number of GABA-immunoreactive perikarya in the innermost part of the inner nuclear layer by approximately 60% and 80%, respectively, as compared to controls. The cell processes in the inner plexiform layer (IPL) were restricted to only three defined bands in the strata 1, 3 and 5, as compared to an intense and homogeneous labeling in the IPL of the untreated retinas. The effect of KA was inhibited by 100 microM CNQX, 100 microM NNC-711, or when Na(+) was replaced by choline. The release of GABA was Ca(2+)-independent, suggesting the mobilization of GABA from the cytoplasmic pool of this neurotransmitter. At least two subsets of retinal neurons including amacrine and interplexiform cells retained GABA-immunoreactivity after stimulation with EAAs, as revealed by glutamic acid decarboxylase (GAD) immunocytochemistry. Our results suggest that non-NMDA receptor activation by KA and glutamate are associated with the efflux of GABA from cells of the inner retina (amacrine and interplexiform cells). The data also show that cells containing GAD-67 released GABA via its transporter, while cells containing exclusively GAD-65 apparently did not release the neurotransmitter by the reversal of the transporter.

In the present work, we have characterized the maturation of the GABAergic system in mammalian retina. Immunoreactivity for GABA, GAD (glutamic acid decarboxylase, EC 4.1.1.15) -65 and -67 in the adult rat retina was localized in cells in the inner nuclear and ganglion cell layers. This pattern was established around postnatal day 8 and included transient GABA and GAD-67 expression in horizontal cells. GAD activity was very low at P1 and P4, increasing after P8, reaching maximal activity by P21 and decreasing to attain adult values by P30. GABA content was approximately constant from P1 to P13, increasing thereafter to reach adult levels. GAD protein content increased progressively with postnatal development and the two isoforms could be distinguished at P8. The disparity between retinal GABA content vs presence and activity of the synthesizing enzyme, led us to investigate the alternative pathway for GABA synthesis that utilizes putrescine as a substrate. Highest levels of ornithine decarboxylase activity (the limiting step for putrescine synthesis) were found between P1 and P4, decreasing to very low levels after P13. The same pattern was observed for putrescine content in the retina. Highest amounts were found at P1, that decreased and remained constant after P13. Additionally, approximately 40% of tritiated putrescine incorporated by P1, P4 and adult retinas was converted into GABA. Our results suggest the existence of two different sources of GABA in mammalian retina, one that uses glutamate as a precursor and predominates in the mature nervous system and another that utilizes putrescine and is present transiently at early developmental stages.

1. Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G-protein-coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors. 2. Ionic currents induced by fast application of N-methyl-D-aspartate (NMDA) were recorded under whole-cell patch-clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 microM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms. 3. The D1 receptor agonist (+)-SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 microM on the NMDA response, while the enantiomer (-)-SKF38393 was ineffective at 50 microM. However, the D1 antagonist R(+)-SCH23390 did not prevent the inhibitory effect of (+)-SKF38393. 4. The degree of inhibition by dopamine and (+)-SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage-dependent block by dopamine was also observed in the presence of MgCl2 1 mM. 5. Single-channel recordings showed that the open times of NMDA-gated channels were shortened by (+)-SKF38393. 6. These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1-type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)-SKF38393 are NMDA channel ligands.

Cultured retina cells released accumulated [3H]GABA (gamma-aminobutyric acid) when stimulated by L-glutamate, N-methyl-D-aspartate (NMDA) and kainate. In the absence of Mg2+, dopamine at 200 microM (IC50 60 microM), inhibited in more than 50% the release of [3H]GABA induced by L-glutamate and NMDA, but not by kainate. This effect was not blocked by the D1-like dopamine receptor antagonist, R-(+)-7-chloro-8-hydroxy-3-methyl- -phenyl-2,3,4,5-tetrahydro- H-3-benzazepine hydrochloride (SCH 23390), neither by haloperidol nor spiroperidol (dopamine D2-like receptor antagonists). The dopamine D1-like receptor agonist R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,diol hydrochloride (SKF 38393) at 50 microM, but not its enantiomer, also inhibited the release of [3H]GABA induced by NMDA, but not by kainate; an effect that was not prevented by the antagonists mentioned above. (+/-)-6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e hydrobromide (SKF 812497) had no effect. Neither 8BrcAMP (5 mM) nor forskolin (10 microM) inhibited the release of [3H]GABA. Our results suggest that dopamine and (+)-SKF 38393 inhibit the glutamate and NMDA-evoked [3H]GABA release through mechanisms that seem not to involve known dopaminergic receptor systems.

Dopamine is the main catecholamine found in the retina of most species, being synthesized from the L-amino acid tyrosine. Its effects are mediated by G protein coupled receptors subfamilies that are commonly coupled to adenylyl cyclase in opposite manners. There is evidence that this amine works as a developmental signal in the embryonic retina and several distinct roles have been attributed to dopamine in the retina such as proliferation, synaptogenesis, neuroprotection, increased signal transmission in cone, gap junction modulation, neuronal-pigmented epithelium-glial communication, and neuron-glia interaction. Here we describe methods that have been used in the study of the dopaminergic function in the retina in the last 40 years. We emphasize the approaches used in the studies on the development of the avian and rodent retina. The dopaminergic system is one of the first phenotypes to appear in the developing vertebrate retina.

Pituitary Adenylyl Cyclase-Activating Polypeptide (PACAP) is a neuroactive peptide present in the avian retina where it activates adenylyl cyclase (AC) since early in development via PACAP receptors. The synthesis of cAMP in response to PACAP is observed since embryonic day 8/9 (E8/9). After E12, signaling via PACAP receptors desensitizes, reaching very low levels in the mature tissue. We show here that chronic administration of PACAP in vitro desensitizes PACAP-induced cAMP accumulation, while the administration of the PACAP antagonist (PACAP 6-38) re-sensitizes PACAP receptor/cyclase system in vitro and in vivo. Moreover, a twofold increase in the number of tyrosine hydroxylase positive (TH+) cells is observed after in vivo injection of PACAP6-38. NURR1, a transcription factor associated with the differentiation of dopaminergic cells in the CNS, is present in the chick retina in all developmental stages studied. The presence of NURR1 positive cells in the mature tissue far exceeds the number of TH+ cells, suggesting that these NURR1-positive cells might have the potential to express the dopaminergic phenotype. Our data show that if PACAP signaling is increased in mature retinas, plastic changes in dopaminergic phenotype can be achieved.

Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.

Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-β peptide (Aβ). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aβ oligomers (AβOs). AβOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AβOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AβOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AβOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AβO binding sites, fully prevented AβO-induced inhibition of ChAT. Interestingly, we found that AβOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AβO-targeted neurons. Reduction in ChAT activity instigated by AβOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.

The neurotoxicity of the amyloid-β peptide (Aβ) appears to be, at least in part, related to pathological activation of glutamate receptors by Aβ aggregates. However, the downstream signaling pathways leading to neurodegeneration are still incompletely understood. Hyperactivation of nitric oxide synthase (NOS) and increased nitric oxide (NO) production have been implicated in excitotoxic neuronal damage caused by overactivation of glutamate receptors, and it has been suggested that increased NO levels might also play a role in neurotoxicity in Alzheimer's disease. We have examined the effect of blockade of NO production on the neurotoxicity instigated by Aβ₄₂ and by elevated concentrations of glutamate in chick embryo retinal neurons in culture. Results showed that L-nitroarginine methyl ester, a potent inhibitor of all NOS isoforms, had no protective effect against neuronal death induced by either Aβ₄₂ (20 μM) or glutamate (1 mM). Surprisingly, at short incubation times both Aβ and glutamate decreased NO production in retinal neuronal cultures in the absence of neuronal death. Thus, excitotoxic insults induced by Aβ and glutamate cause inhibition rather than activation of NO synthase in retinal neurons, suggesting that cell death induced by Aβ or glutamate is not related to increased NO production. On the other hand, considering the role of NO in long term potentiation and synaptic plasticity, the decrease in NO levels instigated by Aβ and glutamate suggests a possible mechanism leading to synaptic failure in AD.