Calmodulin-dependent adenylate cyclase in rat retina and the response to dopamine

Adenylate cyclase activity from the rat neural retina was highly stimulated with Ca²⁺ and calmodulin. The retinal adenylate cyclase activity was also increased by dopamine, and the activation was not changed with or without Ca²⁺-calmodulin in fractions from the neural retina homogenate after sucrose density gradient centrifugation. The results suggest that the two regulation systems (i.e. dopamine and Ca²⁺-calmodulin) of adenylate cyclase in the rat retina appear to be independent.     

Heterogeneity of muscarinic cholinergic receptors in the developing chick embryo retina

Heterogeneity of muscarinic cholinergic receptors was investigated in chick embryo retina throughout development and in chicks immediately after hatching. The presence of a homogeneous receptor population was evidenced by antagonist binding. The affinity of antagonists increased up to day 14 of incubation, when synaptogenesis occurs. After this stage, it remained substantially unchanged. The number of receptors increased in embryos until hatching. On the contrary, agonists, such as acetylcholine and carbachol, bound to two (high- and low-affinity) binding sites. Through development, the affinity of both significantly increased until day 14, further substantiating the hypothesis of a maturation of the receptor pattern which precedes synapse formation. Muscarinic cholinergic binding seems to identify 3 critical steps in retinal neuronal development. The first is between 7 and 9 days of incubation, the second when synaptogenesis occurs and the third after initiation of function.     

Dopamine-stimulated adenylate cyclase and tyrosine hydroxylase in diabetic rat retina

The effects of streptozotocin-induced diabetes on the retinal dopaminergic system have been examined in Long-Evans (pigmented) rats. Tyrosine hydroxylase activity was significantly decreased while dopamine-stimulated adenylate cyclase was increased in 2-month-diabetic rats. The observed increase in dopamine-stimulated adenylate cyclase activity in diabetic retinae may be related to neurotransmitter receptor changes because postreceptor activation of adenylate cyclase by guanylyl imidodiphosphate was not altered.     

The effects of TGF-β1 on chick embryo retina development in vitro

This paper studies the effect exerted by TGF-β₁ on the development of chick embryo retina cultured in vitro. The addition of TGF-β₁ to retinal explants inhibited DNA synthesis, measured as ³H-thymidine incorporation into trichloroacetic acid-insoluble fraction, while it increased both wet weight and protein content, in particular that of extracellular matrix proteins. Lastly, in explants treated with TGF-β₁ an increment in the level of fibronectin was demonstrated by means of Western blotting analysis.     

Bidirectional regulation of cAMP generating system by dopamine-D1 and D2-receptors in the rat retina

Summary Accumulation or inhibition of cAMP formation in response to dopamine or dopamine related drugs in the absence or in the presence of forskolin and/or IBMX was investigated in isolated rat retina. While the existence of D₁-receptors (positively coupled with adenylate cyclase) was confirmed, D₂-receptors (negatively coupled to adenylate cyclase) were also revealed by using a selective D₁-antagonist (SCH 23390), a D₂-agonist (LY 171555) or two D₂)-antagonists (S-sulpiride, spiroperidol). These results indicate that rat retina may be used for the study of both types of dopamine-receptors.     

Muscarinic cholinergic binding in chick embryo retino-tectal system: effects of corticosterone

Muscarinic binding sites were measured using the radioligand [3H]quinuclidinyl benzilate (QNB) in the retina and tectum of 11-day-old chick embryos, after intracerebral administration of 0.02 microgram of corticosterone at 8 days of incubation. This age was chosen because the hormone preferentially accumulates in retinas at 8 days of development. Hormone treatment significantly affected the affinity of 3H-QNB-binding sites in retinas and slightly affected the affinity in treated tecta, whereas the number of binding sites remained unchanged. The specific binding was determined with either atropine or unlabeled QNB. Scatchard plot analysis of specific 3H-QNB binding revealed the presence of nonsaturable binding at high 3H-QNB concentrations (6-11 nM) in the treated retinas, but not in controls. It can be concluded from these data that the hormone has a primary effect on retinal cells during early growth in the chick embryo. The possibility that the hormone delays maturation of specific populations of retinal cells is considered in the discussion.     

Development of A1 adenosine receptors in the chick embryo retina

Adenosine inhibits cyclic AMP synthesis induced by dopamine in embryonic but not in post-hatched chick retinas. N6-Cyclohexyladenosine (CHA), which preferentially activates A1 receptors as well as 2-chloroadenosine, inhibits cyclic AMP accumulation induced by dopamine in retinas from 10-day-old embryos (E10) with IC50's of 0.1 and 0.5 microM, respectively, but this effect is not detectable after hatching. In order to verify if this developmental change reflects variations in the number or affinity of A1 adenosine receptors, their development during chick retina ontogeny was studied. Binding studies using 3(H)CHA revealed the presence of A1 receptors at all stages of development examined, including the post-hatched retina. The number of binding sites increased between E10 and E17, and then decreased in post-hatched animals. In the latter, 3(H)CHA binding was to a single site with a Bmax of 128.6 +/- 13.4 fmol/mg protein and a Kd of 2.1 + 0.2 nM. Various ligands showed similar hierarchies of affinity for the A1 receptor in embryonic and post-hatched retinas, namely, CHA greater than R-N6-phenylisopropyladenosine (1-PIA) greater than 5'-N-ethylcarboxamideadenosine (NECA) greater than isobuthylemethyl-xanthine (IBMX). Given that CHA inhibited forskolin-induced cyclic AMP production and Gpp(NH)p inhibited 3(H)CHA binding in both embryonic and post-hatched retinas, it appears that receptor coupling to adenylate cyclase is present since early embryonic stages. The results suggest that the A1 receptors may have different functions in the embryonic as compared to the mature chick retina.     

Neurotrophic effects of transferrin on embryonic chick brain and neural retina cell cultures

The viability and differentiation promoting effects of various transferrins [iron-saturated (holo) and iron-depleted (apo) human and chick ovo (conalbumin)-transferrins, and bovine apo-transferrin] were studied, using serum-free, flat-sedimented cell cultures of embryonic chick brain and neural retina. The effects of transferrin (Tf) on the cell cultures depended on the type of Tf used and the parameter measured.Significant differences between brain and neural retina cultures in the effects of apo-ovoTf and iron [supplemented as ammonium-iron (III) citrate] were detected. Maximal levels of mitochondrial activity were observed in the presence of 2 mg/l apo-ovoTf in neural retina cell cultures. In brain cell cultures, 40 mg ovoTf/l were needed to achieve maximal levels. In brain, but not in neural, retina cell cultures ovoTf and optimal concentrations of Fe³⁺ exhibited similar effects on biochemical parameters of cell function and differentiation. Although, in the absence of ovoTf, neuronal outgrowth on areas not covered by glial cells was inhibited in both cell cultures, the differences were more prominent in neural retina cell cultures. Our data strongly suggest that Tf plays a key role in processes not connected directly with its iron transport capability.     

The effects of intraocular injection of kainic acid on the synaptic organization of the goldfish retina

Kainic acid (KA), an extended analog of L-glutamate, was injected into the eyes of living goldfish. After survival times ranging from 15 min to 6 days, retinae were inspected for KA-induced degeneration at both the LM and EM levels. KA had little effect on photoreceptors, mixed rod-cone bipolar cells, Müller cells, at least two types of amacrine cells and the optic nerve. Reversible edema was seen in both rod and cone horizontal cells. Pure cone bipolar cells and the majority of amacrine cells appeared to be destroyed by KA. The effect of KA is selective not only on the cell types involved, but also in the location of KA-induced edema on the affected cells, i.e., soma and proximal portions of dendrites of cone horizontal cells as opposed to the distal ends of dendrites of rod horizontal cells. Implications of these data are discussed in regard to the use of KA as a probe for glutamatergic pathways in the retina. One hypothesis suggested by our results is that rods use glutamate whereas cones use aspartate as their neurotransmitter.     

Changes in ganglioside profile in chick embryo retina: Studies on tissue and cell cultures

The developmental profile of gangliosides in the neural retina of the chick embryo is characterized by a progressive decrease in the concentration of GD3 complex from a high level on day 6; by a continuous increase in GD1a concentration; and by less striking increases in GD1b and GT1b concentrations during the growth phase; GM1 increases in the post-mitotic retina. Gangliosides were analyzed by thin layer chromatography and by densitometry of the TLC plates. (Ganglioside nomenclature is according to Svennerholm.³⁷)We have examined comparatively ganglioside changes in organ cultures of retina tissue from 6 day embryos (R³⁶), in cell aggregates and in primary monolayer cultures of R²⁶ cells, all maintained for 6 days in vitro. In all cases, the pattern of ganglioside changes was qualitatively similar to that in the retina in vivo. These results suggest that, unlike some other aspects of retina differentiation, the progression of ganglioside changes in the 6–12 day embryonic retina is not critically dependent on histotypic cell organization or on specific contact-dependent cell interactions; these changes appear to be largely preprogrammed in the cells at some earlier phase of development.     

The organization of the visual hyperstriatum in the domestic chick. I. Topology and topography of the visual projection

The organization of the visual projection to the hyperstriatum, or Wulst, in the domestic chick has been investigated using extracellular recording with microelectrodes.The entire visual field of the contralateral eye projects retinotopically into the Wulst. The superior and inferior margins of the visual field are represented, respectively, in the posterior and anterior regions of the visual projection area. The projection of the nasal and temporal margins of the visual field is more complex, the superior and inferior parts of the temporal hemi-field being represented in the superficial and deep regions of the Wulst respectively, with an intervening projection of the nasal hemi-field at an intermediate depth.Visually responsive units appear to be restricted to the accessory hyperstriatum (HA) and possibly also to a narrow, medial region of the intercalated nucleus (HIS). Visual-evoked activity was never detected in the dorsal hyperstriatum (HD) or ventral hyperstriatum (HV).The organization found in the chick is discussed in relation to the previous physiological and anatomical findings of other workers in the visual Wulst of the pigeon and the owl.     

The organization of the visual hyperstriatum in the domestic chick. II. Receptive field properties of single units

The organization of the visual projection to the hyperstriatum, or Wulst, in the domestic chick has been investigated using extracellular recording with microelectrodes.The entire visual field of the contralateral eye projects retinotopically into the Wulst. The superior and inferior margins of the visual field are represented, respectively, in the posterior and anterior regions of the visual projection area. The projection of the nasal and temporal margins of the visual field is more complex, the superior and inferior parts of the temporal hemi-field being represented in the superficial and deep regions of the Wulst respectively, with an intervening projection of the nasal hemi-field at an intermediate depth.Visually responsive units appear to be restricted to the accessory hyperstriatum (HA) and possibly also to a narrow, medial region of the intercalated nucleus (HIS). Visual-evoked activity was never detected in the dorsal hyperstriatum (HD) or ventral hyperstriatum (HV).The organization found in the chick is discussed in relation to the previous physiological and anatomical findings of other workers in the visual Wulst of the pigeon and the owl.     

Evidence for selective loss of brain dopamine- and histamine-stimulated adenylate cyclase activities in rabbits with aging

Dopamine-stimulated adenylate cyclase activity in striatum and both dopamine-and histamine-stimulated adenylate cyclase activity in hypothalamus, frontal cortex and anterior limbic cortex declined by about 50% as rabbits aged from 5.5 months to 5.5 years of age. These changes were primarily in maximal response to amine although an additional component involving decreased affinity in the case of dopamine may also be present. In contrast, dopamine-stimulated adenylate cyclase of retina and both basal and guanyl-5′-yl-imidodiphosphate (Gpp(NH)p)-stimulated activity in these regions were not altered with age. There was no measurable decrease in the old animals in either dopamine or norepinephrine concentration in striatum, anterior limbic cortex or retina, or in choline acetylase activity or [³H]quinuclidinylbenzilate binding in striatum, anterior limbic cortex or frontal cortex. It is proposed that selective age-dependent decreases in transmitter receptors coupled to adenylate cyclase occur in the absence of or independent from neuronal cell loss, as evidenced by the retention of the other biochemical markers.     

Localization of α-bungarotoxin binding sites in synapses of the developing chick retina

Alpha-bungarotoxin binding sites in chick retina from 12 days in ovo to hatching, were visualized at the ultrastructural level by use of a 1:1 conjugate of horseradish peroxidase with α-bungarotoxin. At all stages binding sites in the inner plexiform layer were localized in synapses, predominantly on or near the postsynaptic membrane. Localization of binding sites was found in bipolar and amacrine-cell synapses which appeared morphologically immature as well as more well developed synapses. The results suggest that α-bungarotoxin-binding synapses, tentatively considered to be nicotinic cholinergic, are formed throughout the course of synaptogenesis, and that the aggregation of nicotinic receptors occurs early in the formation of these synapses.     

Photoreceptor plasticity in reaggregates of embryonic chick retina: rods depend on proximal cones and on tissue organization

Plasticity of photoreceptors and their integration into epithelial structures homologous to an outer nuclear layer (ONL), was investigated in embryonic chick retinal cell reaggregates by immunohistochemistry using an antibody specific for red plus green cones (RG-cones) and an antibody for rods. If reaggregates are raised in the presence of pigmented epithelium (RPE), completely reconstructed, stratified retinal spheres are produced, where all rods and cones are integrated into an outer laminar ONL, similar to a normal retina. In the absence of RPE, 'rosetted' spheres form which contain internal rosettes homologous to an ONL. Only a minor fraction of cones and rods of 'rosetted' spheres are located within rosettes, while a larger fraction is diffusely displaced in nonorganized areas, thus, not contributing to an ONL-like epithelium. In both types of spheres, the total percentage of RG-cones was similar to the in vivo retina, indicating that expression of cones is autonomous. Following cones, after about one day, rods developed only within already existing RG-cone clusters. Thereby, the ratio of rods to RG-cones increases as the tissue organization decreases: for stratified spheres this ratio is, 0.50 (1 rod/2 cones; similar to mature retina); for rosettes, 0.74 (3 rods/4 cones) and for nonorganized areas, 1.09 (1 rod/1 cone) -- a higher ratio under our conditions has never been detected. Thus, rod expression depends strictly on the presence of nearby cones; their relative numbers are distinctively adjusted according to the cytoarchitecture of the tissue environment. The biomedical implications of these findings are briefly discussed.     

β-Bungarotoxin-induced cell-death of neurons in chick retina

The cytotoxicity of β-bungarotoxin (β-BTX), a snake venom neurotoxin with phospholipase A₂ activity, for chick neurons was investigated using organ and monolayer cultures of retina. β-BTX led to a marked reduction in the total activities of choline acetyltransferase and glutamate decar☐ylase of retina cultures at concentrations as low as 100 pM. The total activity of lactate dehydrogenase was, however, much less affected by β-BTX. Also, the total activity of tyrosine hydroxylase of organ-cultured retina decreased only at 30–50 fold higher concentrations of the toxin. The total activity of the glial marker glutamine synthetase was not changed by β-BTX. In contrast to this selectivity for neurons displayed by β-BTX, non-neurotoxic phospholipases A₂ from bee venom and porcine pancreas led to a simultaneous loss of both neuronal and glial marker enzymes.

Light and electron microscopy of organ-cultured retina showed that only cells in the ganglion cell layer and the inner third of the amacrine cell layer degenerated after incubation with β-BTX. In the toxin-sensitive cells, the Golgi apparatus and the endoplasmatic reticulum appeared the first subcellular structures to be affected.

It is concluded that β-BTX preferentially recognizes and/or destroys cholinergic and GABAergic cells in the amacrine and ganglion cell layers of the developing chick retina. This toxin may thus be a useful probe to investigate cell surface properties of cholinergic and GABAergic neurons in the chick central nervous system.     

Effects of mood stabilizers on the inhibition of adenylate cyclase via dopamine D(2)-like receptors

OBJECTIVE: The mood stabilizing drugs lithium, carbamazepine and valproate modulate brain adenosine monophosphate (cAMP) levels, which are assumed to be elevated in bipolar disorder patients. The aim of this work was to investigate how these three mood stabilizing agents affect the regulation of cAMP levels by dopamine D(2)-like receptors in vitro in rat cortical neurons in culture and in vivo in the rat prefrontal cortex. METHODS: The production of cAMP was measured in the cultured cortical neurons or in microdialysis samples collected from the prefrontal cortex of freely moving rats using the [8-(3)H] and [(125)I] radioimmunoassay kits. RESULTS: In vitro and in vivo data showed that the treatment with the mood stabilizing drugs had no effect on basal cAMP levels in vitro, but had differential effects in vivo. Direct stimulation of adenylate cyclase (AC) with forskolin increased cAMP levels both in vitro and in vivo, and this effect was significantly inhibited by all three mood stabilizers. Activation of dopamine D(2)-like receptors with quinpirole partially inhibited forskolin-induced increase in cAMP in untreated cultures, but no effect was observed in cortical neuron cultures treated with the mood stabilizing drugs. Similar results were obtained by chronic treatment with lithium and valproate in the prefrontal cortex in vivo. However, surprisingly, in carbamazepine-treated rats the activation of dopamine D(2)-like receptors enhanced the responsiveness of AC to subsequent activation by forskolin, possibly as a consequence of chronic inhibition of the activity of the enzyme. CONCLUSIONS: It was shown that each of these drugs affects basal- and forskolin-evoked cAMP levels in a distinct way, resulting in differential responses to dopamine D(2)-like receptors activation.     

Muscarinic and purinergic Ca mobilizations in the neural retina of early embryonic chick

Acetylcholine and adenosine triphosphate (ATP) raise intracellular Ca²⁺ concentration via muscarinic receptors and P_2U purinoceptors by releasing Ca²⁺ from intracellular Ca²⁺ stores in the neural retina of early embryonic chick. The signal transduction mechanisms for the muscarinic and purinergic Ca²⁺ responses with fura-2 fluorescence measurements were studied. Li⁺ (1 mM), which inhibits phosphatidylinositol metabolism, enhanced both the Ca²⁺ rises to carbamylcholine (CCh, 30 μM), a muscarinic agonist, and ATP (200 μM). Thapsigargin (250 nM), an inhibitor of Ca²⁺-ATPase of inositol trisphosphate (IP₃)-sensitive Ca²⁺ stores, abolished both the Ca²⁺ rises to CCh (100 μM) and ATP (500 μM). U-73122 (2 μM), an inhibitor of phospholipase C_b, suppressed the Ca²⁺ rise to ATP (500 μM), but its analog U-73343 (2 μM) did not suppress it. In contrast, both U-73122 and U-73343 suppressed the Ca²⁺ rise to CCh (100 μM). Pertussis toxin (250 ng/ml) suppressed the ATP-induced Ca²⁺ rise at least partly, whereas no inhibition was observed on the CCh-induced Ca²⁺ rise. Cross-talk occurred between the muscarinic and purinergic Ca²⁺ mobilizations but they were not occlusive. This study suggests that the muscarinic and purinergic Ca²⁺ mobilizations utilize IP₃-sensitive Ca²⁺ stores, but different signal transduction pathways are involved in between the muscarinic and purinergic Ca²⁺ responses.     

Localization of dopamine-sensitive adenylate cyclase within the rat olfactory tubercle

There are three histological layers within the rat olfactory tubercle: plexiform, pyramidal, and polymorphic. We have assayed dopamine-sensitive adenylate cyclase in homogenates of frozen sections cut parallel to these layers. Consecutive sections (16 μm) were hooogenized in groups of 6. Every seventh section was stained with toluidine blue to monitor the depth and orientation of the plane of section. I report the laminar distribution of dopamine-sensitive adenylate cyclase at two locations within the tubercle where the local topographies are quite different from one another. In the caudomedial tubercle where the parallel alignment of the neuronal layers is clearest, there is a marked gradient in the activity of dopamine-sensitive adenylate cyclase. Its distribution corresponds very closely to the distribution of pyramidal cell bodies and their dendrites in this region of the olfactory tubercle.     

The adenylate cyclase inhibitor MDL-12330A has a non-specific effect on glycine transport in Müller cells from the retina

Müller glial cells express two transport systems for glycine (Gly): one with low affinity and another identified as GLYT1 with high affinity. The latter colocalizes with NMDA receptors in the CNS. Gly is considered as an obligatory coagonist at NMDA receptors, and, hence, the Gly transport system could contribute to the modulation of glutamate (Glu) excitatory transmission in the vertical pathways of the retina. For this reason, the regulation of Gly transport by cAMP was studied. We report here a non-specific effect of MDL-12330A, a compound reported to inhibit adenylate cyclase (AC), on Gly transport in Müller glia. This effect might be due to a toxic action on the cells, decreasing cell viability, and not to a specific inhibition of the adenylate cyclase. Non-specific effects of this drug should be considered when the participation of cAMP in any biological process is studied. We have clearly demonstrated that cAMP does not participate in the regulation of Gly transport in Müller glia.