Dopaminergic cell differentiation in the developing chick retina

The distribution and morphology of dopaminergic (DA) neurons in the chick retina was studied in the course of development. Fluorescent DA cells were first detected on the 13th day of incubation. They were always found in positions two or three cell rows externally from the junction between the inner plexiform layer (IPL) and inner nuclear layer (INL). On the 14th day, DA cells were found in the innermost row of the INL. Subsequently their processes extended not only bilaterally along the IPL-INL junction but also vertically into the IPL. As a result, three fiber layers were formed as laminae 1, 3 and 5 in the IPL. In the newly-hatched chick retina, a number of growth cone-like fluorescent structures with fine spikes were seen at the IPL-INL junction, indicating that DA fibers were still growing and elongating at least at hatching. On the 4th postnatal day, the ramification of dendritic processes was very prominent and they often showed a spiral configuration.     

Dopaminergic regulation of GABA release from the intact goldfish retina

The rate of release of [³H]GABA from intact goldfish retinas was studied using a modified superfusion technique. Small, significant increases in the rate of GABA release were observed when the retinas were exposed to dopamine (DA) (100–1000 μM); however, when free Ca²⁺ was removed from the medium, the basal rate of GABA release was increased and DA became inhibitory. Forskolin, a non-specific stimulator of adenylate cyclase in intact cells, also inhibited GABA release in the absence of Ca²⁺. There was no significant effect of forskolin in the presence of Ca²⁺; however, (+)-butaclamol, a dopamine antagonist, increased basal GABA release under these conditios. l-glutamic acid (l-Glu) (1–10 mM) causes up to a 10-fold increase in GABA release. In the presence of Ca²⁺, DA did not significantly alter the effects of l-Glu; however, in the absence of Ca²⁺ a significant inhibition of the effects of l-Glu by DA was observed. Forskolin, on the other hand, inhibited the effects of l-Glu both in the presence and absence of Ca²⁺. Finally, EGTA (0.3–1 mM) produced a large release of GABA: this release was inhibited by DA, forskolin, theophylline and 8-bromo cyclic AMP. These results suggest a model wherein DA stimulates Ca²⁺-dependent GABA release from one site and inhibits Ca²⁺-independent GABA release from another site via cyclic AMP-mediated event.     

The ontogenesis of the dopaminergic cell in the pre- and postnatal guinea pig retina

We have examined the development of the dopaminergic system of the guinea pig retina, a species in which retinal neuronal and synaptic differentiation occurs largely in utero. Fetal animals aged 42–69 days (full term), neonates, postnatal (pn) animals to 12 weeks, and mature animals were studied to determine retinal dopamine (DA) storage, metabolism (DOPAC), in vitro tyrosine hydroxylase (TH) activity, postsynaptic target activation (cAMP stimulation) and localization (formaldehyde-induced histofluorescence). DA-stimulated adenylate cyclase at 42 days of gestation was threefold over basal activity, preceding the onset of the accumulation of DA and DOPAC at 45 days, and the initial localization of DA in cell perikarya at 47 days and in processes at 50 days. At birth DA and DOPAC levels were 45 and 37%, respectively, of adult levels. DA levels remained stable during the first few days pn, although _{in vitro} TH activity was capable of stimulation by light in the neonate as in the mature animal. DA and TH activity increased from 1 week pn to reach adult levels by 10 weeks pn. Although a significant degree of development of the dopaminergic neurotransmitter system in the guinea pig occurs before birth the attainment of a fully mature system postnatally may require normal photic stimulation of physiologic activity.     

Diurnal metabolism of dopamine in the mouse retina

Dopamine is an important retinal neurotransmitter and neuromodulator that regulates key diurnal cellular and physiological functions. In the present study we carried out a comprehensive analysis of dopamine metabolism during the light phase of the diurnal cycle and evaluated the presence of diurnal and circadian rhythms of dopaminergic activity in the mouse retina. Steady-state levels of dopamine did not change significantly between the dark phase (night) and the light phase (day) of the diurnal cycle, nor did they change between early and late points in the day. Dopamine synthesis and utilization, however, revealed significant alterations between the night and day and between early and late time points in the day. A spike in synthesis and utilization was measured immediately after light onset at the end of the night. Subsequently, dopamine synthesis and utilization partially declined and remained stable throughout the remainder of the day at a level that was significantly higher than that at night. The burst of dopamine synthesis and utilization at the beginning of the day is entirely light evoked and not driven by a circadian clock. Similarly, there was no circadian rhythm in dopamine synthesis and utilization in mice kept in constant darkness. This daily pattern of dopaminergic activity may impact upon a variety of temporally regulated retinal events. Moreover, these data will provide a basis for evaluating the role of dopamine in retinal pathology in mouse models of retinal degeneration where mutations affect light perception.     

Quantitative spatial analysis of the distribution of NADPH-diaphorase-positive neurons in the developing and mature rat retina

NADPH-diaphorase (NADPH-d) histochemistry labels a subpopulation of nitric oxide-synthesizing amacrine cells in the inner nuclear layer of the rat retina. We have studied their morphology and distribution in postnatal and adult rats in whole-mounted retinae. NAPDH-d-positive neurons are detected as early as postnatal day (P)5, especially in the peripheral retina; intense labeling of somata and long lengths of dendrites is obtained between P10 and P18, after which only the somata exhibit NADPH-d activity. The density and number of these cells increase progressively from P7 to P14, with a significantly higher density in the central retina as compared to the periphery. The sociology of these cells was analyzed quantitatively studying the Voronoi domains: a polygon area can be drawn that delineates the territory of the map that is closer to the cell than to any other cell of the map. In addition, we calculated the conformity ratio of Cook, i.e., the mean nearest neighbor distance/standard deviation of all the nearest neighbor distances, in order to reveal whether or not these cells are regularly distributed through the retina. We find that the distribution of the NADPH-d-positive cells tends to be regular throughout the retina: the local coefficient of variation (obtained by comparing the size of each Voronoi polygon area to those of its neighbors) tends to regularity at P14 and remains unaltered through maturity. Therefore, as other cell types, NADPH-d-positive amacrine cells are almost regularly distributed from the time of eye opening and nitric oxide may play a role in the development of retinal circuitry and in the regulation of retinal blood flow.     

Effects of dopamine on cyclic AMP concentration in the anterior pituitary glandin vitro

Summary Effects of different concentrations of dopamine on the cyclic AMP concentration in the rat anterior pituitary gland were investigated in vitro. Low concentrations of dopamine (10^–9–10^–8 mol/l) were found to decrease, whereas the high concentration (10^–5 mol/l) increased the cyclic AMP concentration in pituitaries collected from ovariectomized and estradiol-treated females. In contrast, dopamine had no effect on the anterior pituitary cAMP concentration when pituitaries were collected from ovariectomized rats which had not received estrogen replacement. These data show that the action of dopamine on the anterior pituitary cAMP largely depends on the dopamine concentration and the hormonal state of animals.This paper was supported by the Polish Academy of Sciences Grant No. 10.4.2.01.5.5.     

Endogenous opioids regulate cell proliferation in the retina of developing rat

The role of endogenous opioids and opioid receptors (endogenous opioid systems) in modulating cell proliferation in the developing mammalian retina was examined in 1-day-old rats. In contrast to a labeling index (LI) of 35.8% in control animals, administration of the opioid peptide [Met5]-enkephalin (100 micrograms/kg) significantly reduced (10.6%) the proportion of cells incorporating [3H]thymidine; concomitant injection of 1 mg/kg naloxone blocked the inhibitory effects of [Met5]-enkephalin on cell division. Naloxone (1 mg/kg) alone did not alter the LI. The interruption of endogenous opioid-opioid receptor interaction by naltrexone (50 mg/kg), a potent opioid antagonist, was accompanied by a significant increase (6.4%) in the LI relative to control levels. Immunocytochemical experiments revealed the presence of enkephalin-like immunoreactivity, with staining of the cortical cytoplasm of proliferating and differentiating retinal cells recorded; no immunoreactivity was noted in the adult retina. In vitro autoradiography using 125I-[Met5]-enkephalin indicated that [Met5]-enkephalin binding sites were localized to the developing retina; no binding of the radiolabeled ligand was recorded in the adult retina. These results demonstrate the presence of growth-related endogenous opioids and opioid receptors in the developing mammalian retina, but not in adult retina, and suggest that endogenous opioids serve as natural inhibitory trophic factors that tonically regulate cell proliferation.     

Development of muscimol binding sites in chick embryo neural retina in vivo and in vitro: Regulatory effects of cyclic AMP

We report here the development in the chick embryo retina of binding sites for [³H]muscimol, a potent agonist of GABA receptors. In vivo studies were carried out with isolated neural retinas from different stages of development. High-affinity binding sites were absent before embryonic day (E) 8, but increased conspicuously between E10 and E16. Scatchard analysis indicated that this rise was due to an increase in the number of binding sites. Kinetic parameters of the embryonic binding sites were consistent with those typically found for mature muscimol receptors. Measurements of the low-affinity binding site showed a relatively similar developmental pattern although a pronounced decrease in binding to the low-affinity site was observed between E12 and E14. In vitro studies were carried out using glial-free, purified monolayers of retinal neurons, starting at E8. Cultured retinal neurons showed a developmental pattern for high-affinity muscimol binding sites resembling that observed in ovo. These binding sites were susceptible to regulation by cyclic AMP analogues. Increases of 100 to 200% in muscimol binding could be induced by a 24 hr treatment with dibutyryl cyclic AMP, 8-bromo cyclic AMP, or the phosphodiesterase inhibitor IBMX. Scatchard analysis showed that this increase was due to a change in receptor affinity. No effects were found with either butyric acid or with adenosine 5′-monophosphate. These results raise the possibility that cyclic AMP may be involved in the regulation of components of the GABA system.     

Presence of interplexiform dopaminergic neurons in the rat retina

TH-like immunoreactive amacrine neurons have been demonstrated in the rat and Cynomolgus monkey retinas, using a PAP-immunoperoxidase technique. In the rat, some of these neurons, which send processes to both the inner and the outer plexiform layers, are clearly dopaminergic interplexiform cells.     

D1 dopamine receptors in neurite regions of embryonic and differentiated retina are highly coupled to adenylate cyclase in the embryonic but not in the mature tissue

[³H]SCH 23390 binds stereospecifically and with high affinity to D₁ dopaminergic receptors in the developing chick retina. Autoradiographic experiments revealed that in retinas from 3-day-old chicken and embryos with 12, 14 and 16 days of development, specific labeling of [³H]SCH 23390 was mainly observed over the plexiform layers of the tissue, showing that dopaminergic D₁ receptors are localized in retina cell neurites since the initial stages of neurite formation. The total number of [³H]SCH 23390 binding sites increased 5-fold during the differentiation of the retina, while the dopamine-dependent cyclic adenosine monophosphate (AMP) accumulation was significantly decreased. Consequently, the ratio between dopamine-dependent cyclic AMP accumulation and [³H]SCH 23390 binding sites decreased 10-fold as retina differentiated, indicating that a significant portion of D₁ receptors in retinas from adult chicken are not effectively coupled to adenylate cyclase molecules.     

Synaptic development in semi-dissociated cultures of rat retina

Cultured neurons provide a simpler and more accessible environment to study the synaptic physiology. However, it is not clear if development of synapses in culture is similar to that in the in vivo condition. We studied the developmental sequence and morphological differentiation of chemical synapses in semi-dissociated rat retinal cultures that consisted of dissociated neurons as well as undissociated retinal aggregates. Synapses were quantified by synaptophysin immunoreactive puncta. During second week of in vitro development the average number of chemical synapses on the cell body decreased while that on the neurites increased significantly. Conventional synapses appeared both in aggregate and in dissociated neurons, with the developmental profile similar to that reported for in vivo retina. In contrast, the development of ribbon synapses was adversely affected by the in vitro microenvironment as suggested by following observations. The ribbon synapses were more frequently found in aggregate than in dissociated neurons, and were not associated with dyadic or triadic synaptic arrangement. The photoreceptor ribbons did not contact a postsynaptic process while bipolar ribbons made single (monadic) synapses. Further, photoreceptor ribbons in dissociated neurons were late to form and took more time to mature as compared to those in the aggregate cultures. Most of the rod bipolar cells, identified by their immunoreactivity to protein kinase C (PKC), had three or more neurites. Unlike in the in vivo retina, the dissociated rod bipolar cells did not show any PKC immunoreactive varicosities, suggesting that they failed to develop a well-differentiated synaptic terminal. Interestingly, we did not find any parvalbumin positive AII amacrine cells that are normally postsynaptic to rod bipolar cells. These results show that the conventional synapses of retina, which are similar to chemical synapses in other parts of the brain, develop normally both in aggregate and dissociated neurons. However, the highly specialized ribbon synapses have more stringent developmental requirements, and their normal development may require the presence of postsynaptic neurons in their close vicinity.     

Morphometry and distribution of displaced dopaminergic cells in rat retina

The majority of dopaminergic (DA) cells, labeled by tyrosine hydroxylase (TH) immunohistochemistry, are located in the amacrine cell layer (i.e., the innermost sublayer of the inner nuclear layer) in the rat retina. We describe a small population of DA cells, observed in retinal wholemounts, that are displaced to either the inner plexiform layer (DAIcs) or the ganglion cell layer (DAGcs). Contrary to some other species, such cells are few in number in the rat retina. Their systematic study was made in young and adult retinas by retinal mapping, camera lucida drawing, and computer-aided three-dimensional reconstruction. Located predominantly in the superior temporal quadrant, they are observed as soon as the second postnatal day. Most of the morphometric parameters studied were not significantly different between the two types of displaced DA cells, despite the characteristic appearance of interstitial cells. Two hypotheses are proposed for the origin of their displacement: either it is accidental or programmed. Our results favor the former possibility.     

Amino acid signatures in the developing mouse retina

This study characterizes the developmental patterns of seven key amino acids: glutamate, γ-amino-butyric acid (GABA), glycine, glutamine, aspartate, alanine and taurine in the mouse retina. We analyze amino acids in specific bipolar, amacrine and ganglion cell sub-populations (i.e. GABAergic vs. glycinergic amacrine cells) and anatomically distinct regions of photoreceptors and Müller cells (i.e. cell bodies vs. endfeet) by extracting data from previously described pattern recognition analysis. Pattern recognition statistically classifies all cells in the retina based on their neurochemical profile and surpasses the previous limitations of anatomical and morphological identification of cells in the immature retina. We found that the GABA and glycine cellular content reached adult-like levels in most neurons before glutamate. The metabolic amino acids glutamine, aspartate and alanine also reached maturity in most retinal cells before eye opening. When the overall amino acid profiles were considered for each cell group, ganglion cells and GABAergic amacrine cells matured first, followed by glycinergic amacrine cells and finally bipolar cells. Photoreceptor cell bodies reached adult-like amino acid profiles at P7 whilst Müller cells acquired typical amino acid profiles in their cell bodies at P7 and in their endfeet by P14. We further compared the amino acid profiles of the C57Bl/6J mouse with the transgenic X-inactivation mouse carrying the lacZ gene on the X chromosome and validated this animal model for the study of normal retinal development. This study provides valuable insight into normal retinal neurochemical maturation and metabolism and benchmark amino acid values for comparison with retinal disease, particularly those which occur during development.     

The melatonin analog 5-MCA-NAT increases endogenous dopamine levels by binding NRH:quinone reductase enzyme in the developing chick retina

NRH:quinone reductase (QR2) is present in the retinas of embryonic and post-hatched (PH) chicks. 5-Methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) is a QR2 ligand that increases cAMP levels in developing retinas, but it does not affect cAMP levels in CHO-QR2 cells. The dopamine quinone reductase activity of QR2 retrieves dopamine, which increases cAMP levels in developing retinas. The objective of the present study was to investigate whether 5-MCA-NAT increases endogenous dopamine levels in retinas from chick embryos and post-hatched chicks. Endogenous dopamine was measured by enzyme-linked immunosorbent assay (ELISA). 5-MCA-NAT increased retinal endogenous dopamine levels at all developmental stages studied and in PH chicks (−log EC50 = 11.62 ± 0.34 M). This effect was inhibited by non-selective antagonists of receptors and melatonin binding sites N-acetyl-2-benzyltryptamine (luzindole, 5 μM), but it was not inhibited by the Mel1b melatonin receptor antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT, 10 nM). The QR2 cosubstrate, N-methyl-dihydronicotinamide (NMH) (−log EC50 = 6.74 ± 0.26 M), increased endogenous dopamine levels in controls and in retinas stimulated with 5-MCA-NAT (3 nM). The QR2 inhibitor benzo[e]pyrene inhibited endogenous dopamine levels in both control (−log IC50 = 7.4 ± 0.28 M) and NMH-stimulated (at 100 nM and 1 μM benzo[e]pyrene concentrations) retinas. Theoretical studies using Molegro Virtual Docking software corroborated these experimental results. We conclude that 5-MCA-NAT increases the level of endogenous dopamine via QR2. We suggest that this enzyme triggers double reduction of the dopamine quinone, recovering dopamine in retinal development.     

Nitric oxide signaling via cGMP‐stimulated phosphodiesterase in striatal neurons

Nitric oxide (NO) acts in the nervous system to activate guanylyl cyclase and increase cGMP. One target for cGMP appears to be the cGMP‐stimulated phosphodiesterase (PDE2A), which is widely expressed in the brain and provides a molecular mechanism for NO to regulate cAMP levels. We have found that PDE2A is highly expressed in the medium spiny neurons of the striatum, which project to the pallidum and substantia nigra. These cells express dopamine‐stimulated adenylyl cyclase, and we have found that increases in cAMP in these neurons, produced by activation of the D1‐type dopamine receptor, are dramatically enhanced by the general phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine and the PDE2A‐selective inhibitor erythro‐p‐(2‐hydroxyl‐3‐nonyl)adenine (EHNA). These results indicate that PDE2A plays a major role in regulating dopamine‐stimulated cAMP production in striatal neurons. EHNA also enhances NO‐induced increases in striatal cGMP. In addition, dopamine appears to act via another receptor, activated by the agonist SKF83959, to increase striatal cGMP in a NO‐dependent manner. Together, these observations indicate that striatal NO producing interneurons can act via the PDE2A in the medium spiny neurons to regulate the cAMP response to dopamine stimulation. Synapse 64:460–466, 2010. © 2010 Wiley‐Liss, Inc.     

Retinal TrkB receptors regulate neural development in the inner, but not outer, retina

BDNF signaling through its TrkB receptor plays a pivotal role in activity-dependent refinement of synaptic connectivity of retinal ganglion cells. Additionally, studies using TrkB knockout mice have suggested that BDNF/TrkB signaling is essential for the development of photoreceptors and for synaptic communication between photoreceptors and second order retinal neurons. Thus the action of BDNF on refinement of synaptic connectivity of retinal ganglion cells could be a direct effect in the inner retina, or it could be secondary to its proposed role in rod maturation and in the formation of rod to bipolar cell synaptic transmission. To address this matter we have conditionally eliminated TrkB within the retina. We find that rod function and synaptic transmission to bipolar cells is not compromised in these conditional knockout mice. Consistent with previous work, we find that inner retina neural development is regulated by retinal BDNF/TrkB signaling. Specifically we show here also that the complexity of neuronal processes of dopaminergic cells is reduced in conditional TrkB knockout mice. We conclude that retinal BDNF/TrkB signaling has its primary role in the development of inner retinal neuronal circuits, and that this action is not a secondary effect due to the loss of visual signaling in the outer retina.     

Dopaminergic neurons in the human retina

The utilization of dopamine in the adult human retina was examined by using high-affinity uptake, localization, synthesis, and release as neurotransmitter-specific physiological probes. Autoradiographic and histochemical studies have shown that dopamine-accumulating and dopamine-containing cells of the human retina belong to a population of neurons whose somata are located in the proximal regional of the inner nuclear layer. Some of these are amacrine cells which are pre- and postsynaptic to other amacrine cells exclusively in the inner plexiform layer. However, evidence is presented which indicates the existence of interplexiform dopaminergic neurons which send processes to both plexiform layers of the retina. These neurons contain a high concentration of dopamine, take up 3H-dopamine by a hig-affinity mechanism, and release endogenous or accumulated dopamine by a Ca2+-dependent mechanism upon depolarization with high extracellular K+. An endogeneous level of about 20 pmoles dopamine per mg protein was measured in freshly isolated retina using high-pressure liquid chromatography with electrochemical detection. These results demonstrate that mechanisms for dopaminergic neurotransmission are present in the human retina.     

Tyrosine augments dopamine release in stimulated rat retina

Endogenous dopamine (DA) release was measured in perfused rat retinae. Perfusion with elevated potassium (40 mM K) resulted in a 5-6-fold increase in DA release over baseline or 11.6 +/- 0.9% of final tissue DA content. When the selective DA D2 receptor agonist quinpirole was added to the perfusion medium (at 1 and 10 microM), K-stimulated DA release was significant decreased compared to controls (to 7.0 +/- 1.6 and 6.14 +/- 1.4%, respectively). Addition of the D2 antagonist (+/-)-sulpiride (10 microM) significantly increased DA release to 19.1 +/- 1.3%. DA could be released with successive pulses of K; an initial 10 min pulse resulted in a 4-5-fold increase in endogenous DA release over basal levels or 11.4% of the final retinal tissue DA content and a 3-fold increase (a 9.3% fractional release) upon a second K stimulation given 50 min later. The ratio of stimulated DA release during the two K pulses was 0.82 +/- 0.04. When L-tyrosine (100 microM) was included in the medium throughout the perfusion, K2/K1 was increased to 1.14 +/- 0.13. Both tissue DA level and release were decreased by the tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine (AMPT). At 10 microM AMPT K-stimulated DA release was reduced by 50% during the first pulse and completely abolished during the second K pulse. At 100 microM both basal and K-stimulated release were significantly reduced. Exposure of dark-adapted retinae to light in L-tyrosine-supplemented perfusion medium resulted in an increased release of DA compared to retinae perfused with tyrosine-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of neuropeptides and neurotransmitter binding sites in the retina and brain of the developing chick to reduced visual input

The effect of 17 days of monocular suture of eyelids of day-old chicks upon levels of neuropeptides and high affinity binding sites was studied. A significant reduction of met-enkephalin, but not of substance P or neurotensin, was observed in the retina of the eye receiving dim and unpatterned light. Retinal muscarinic, dopaminergic, opiate, and benzodiazepine receptors appeared unaltered by the experimental procedure. Levels of neuropeptides were not significantly changed in optic lobes contralateral to and innervated by the sutured eyes relative to the optic lobes receiving afferentation from the open eyes. Seventeen days after unilateral enucleation of new-hatched chicks, the neuropeptide content of the smaller denervated optic lobes did not differ from that of the lobes receiving an intact neuronal input.