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.

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.

Dopaminergic signaling in the developing retina.

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. Müller 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.

Exchange of extracellular L-glutamate by intracellular D-aspartate: the main mechanism of D-aspartate release in the avian retina.

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.

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.