Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System.

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 Müller 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.

Neuro-glial cannabinoid receptors modulate signaling in the embryonic avian retina.

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.

Functional plasticity of GAT-3 in avian Müller cells is regulated by neurons via a glutamatergic input.

GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.

Murine dopaminergic Müller cells restore motor function in a model of Parkinson's disease.

Müller 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, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller 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 Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller-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 Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Müller 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 Müller cell grafting.

Pituitary adenylyl cyclase-activating polypeptide receptor re-sensitization induces plastic changes in the dopaminergic phenotype in the mature avian 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.

Neurochemical phenotype and birthdating of specific cell populations in the chick retina

The chick embryo is one of the most traditional models in developing neuroscience and its visual system has been one of the most exhaustively studied. The retina has been used as a model for studying the development of the nervous system. Here, we describe the morphological features that characterize each stage of the retina development and studies of the neurogenesis period of some specific neurochemical subpopulations of retinal cells by using a combination of immunohistochemistry and autoradiography of tritiated-thymidine. It could be concluded that the proliferation period of dopaminergic, GABAergic, cholinoceptive and GABAceptive cells does not follow a common rule of the neurogenesis. In addition, some specific neurochemical cell groups can have a restrict proliferation period when compared to the total cell population.

O embrião de galinha é um dos mais tradicionais modelosde estudos da neurociência do desenvolvimento e seu sistema visual tem sido um dos mais exaustivamente estudado. Aretina tem sido utilizada como modelo para estudar o desenvolvimento do sistema nervoso. Aqui, nós descrevemos as características morfológicas que caracterizam cada estádio da retina em desenvolvimento e os estudos do período de neurogênese de algumas subpopulações de células neuroquímicamente específicas da retina usando uma combinação de imunohistoquímica e autoradiografia de timidina-tritiada. Conclui-se que o período de proliferação das células dopaminérgicas, GABAérgicas, colinoceptivas e GABAceptivas não segue uma regra comum. Além disso, alguns grupos celulares neuroquimicamente distintos podem ter um período de proliferaçãomais restrito quando comparado ao da população total destas células.

Neurochemical phenotype and birthdating of specific cell populations in the chick retina.

The chick embryo is one of the most traditional models in developing neuroscience and its visual system has been one of the most exhaustively studied. The retina has been used as a model for studying the development of the nervous system. Here, we describe the morphological features that characterize each stage of the retina development and studies of the neurogenesis period of some specific neurochemical subpopulations of retinal cells by using a combination of immunohistochemistry and autoradiography of tritiated-thymidine. It could be concluded that the proliferation period of dopaminergic, GABAergic, cholinoceptive and GABAceptive cells does not follow a common rule of the neurogenesis. In addition, some specific neurochemical cell groups can have a restrict proliferation period when compared to the total cell population.

Expression of functional dopaminergic phenotype in purified cultured Müller cells from vertebrate retina.

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.

Thyroid hormone action is required for normal cone opsin expression during mouse retinal development.

PURPOSE: The expression of S- and M-opsins in the murine retina is altered in different transgenic mouse models with mutations in the thyroid hormone receptor (TR)-beta gene, demonstrating an important role of thyroid hormone (TH) in retinal development. METHODS: The spatial expression of S- and M-opsin was compared in congenital hypothyroidism and in two different TR mutant mouse models. One mouse model contains a ligand-binding mutation that abolishes TH binding and results in constitutive binding to nuclear corepressors. The second model contains a mutation that blocks binding of coactivators to the AF-2 domain without affecting TH binding. RESULTS: Hypothyroid newborn mice showed an increase in S-opsin expression that was completely independent of the genotype. Concerning M-opsin expression, hypothyroidism caused a significant decrease (P < 0.01) only in wild-type animals. When TRbeta1 and -beta2 were T3-binding defective, the pattern of opsin expression was similar to TRbeta ablation, showing increased S-opsin expression in the dorsal retina and no expression of M-opsin in the entire retina. In an unexpected finding, immunostaining for both opsins was detected when both subtypes of TRbeta were mutated in the helix 12 AF-2 domain. CONCLUSIONS: The results show, for the first time, that the expression of S- and M-opsin is dependent on normal thyroid hormone levels during development.

Identification of neurons with acetylcholinesterase and NADPH-diaphorase activities in the centrifugal visual system of the chick.

The isthmo-optic nuclei (ION) and ectopic neurons, which constitute the centrifugal visual system (CVS), are thought to be cholinoceptive and nitrergic. However, it is not clear which neurons express these markers, namely the ones that project to the retina rather than in neurons that only participate in a local circuit. Therefore, to characterize the neurochemical patterns of the centrifugal visual system in the post-hatched chick, retinopetal cells of the isthmo-optic nuclei and the ectopic region were identified via immunolabeling for cholera toxin, a neuronal tracer, which has been injected in the ocular globe. Then, double labeled with acetylcholinesterase histochemistry to reveal cholinergic synapses, or NADPH-diaphorase histochemistry as a nitrergic marker. Briefly, acetylcholinesterase activity was present mainly in cholera toxin labeled cell bodies of the isthmo-optic nucleus and the ectopic region indicating that retinal projecting neurons of centrifugal visual system comprise a cholinoceptive pathway. On the other hand, NADPH-diaphorase histochemistry was present in the neuropile and sparse cell bodies inside of the isthmo-optic nucleus and in ectopic neurons which were not cholera toxin positive suggesting their role in an intrinsic circuit of the centrifugal visual system. These data support the idea that these two neurochemical systems are present in distinct neuronal populations in the centrifugal visual system.

Regulation of vesicular acetylcholine transporter by the activation of excitatory amino acid receptors in the avian retina.

1. Previous studies have shown that phorbol esters induce protein kinase C (PKC) mediated phosphorylation of the vesicular acetylcholine transporter (VAChT) and change its interaction with vesamicol. However, it is not clear whether physiological activation of receptors coupled to PKC activation can alter VAChT behavior. 2. Here we tested whether activation of kaianate (KA) receptors alters VAChT. Several studies suggest that the cholinergic amacrine cells display KA/AMPA receptors that mediate excitatory input to these neurons. In addition, KA in the chicken retina can generate intracellular messengers with the potential to regulate cellular functions. 3. In cultured chicken retina (E8C11) KA reduced vesamicol binding to VAChT by 53%. This effect was potentiated by okadaic acid, a protein phosphatase inhibitor, and was totally prevented by BIM, a PKC inhibitor. 4. Phorbol myristate acetate (PMA), but not alpha-PMA, reduced in more than 85% the number of L-[3H]-vesamicol-specific binding sites in chicken retina, confirming that activation of PKC can influence vesamicol binding to chicken VAChT. 5. The data show that activation of glutamatergic receptors reduces [3H]-vesamicol binding sites (VAChT) likely by activating PKC and increasing the phosphorylation of the ACh carrier.