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.     

Developmental regulation of adenosine A1 receptors, uptake sites and endogenous adenosine in the chick retina.

Although adenosine A1 receptors mediate the inhibition of dopamine-dependent stimulation of adenylate cyclase activity in the developing chick retina, their localization and function are unknown. We have examined the localization of these receptors, and of endogenous adenosine and adenosine uptake sites at several stages of chick retinal development. A1 receptors were already localized predominantly to plexiform regions by embryonic day 12 (E12) with no gross changes at subsequent stages. Adenosine immunoreactivity was absent from retina at E8 but was detected at E12 in the ganglion cell layer, as well as cells in the inner nuclear cell layer and photoreceptors. At more advanced developmental stages the immunoreactivity was greater, but displayed similar localizations. Uptake sites labeled with [3H]nitrobenzylthioinosine (NBI) were detected even earlier using binding and autoradiographic methods. [3H]NBI binding was saturable, and Scatchard analysis demonstrated a single class of sites with a Kd of 0.91 nM and Bmax of 298 fmol/mg protein in E15 retinal membranes. The binding was displaced by unlabeled NBI and dipyridamole. NBI binding sites differentiated earlier than adenosine A1 receptors or endogenous adenosine immunoreactivity, showing a diffuse distribution at E8, but predominating in the plexiform layers of more developed retinas. The results indicate that elements of a putative purinergic system differentiate at specific localizations early in retinal development.     

Spatio-temporal patterns of differentiation of whole heads of the embryonic chick as revealed by binding of a FITC-coupled peanut-agglutinin (FITC-PNA)

The spatio-temporal expression of specific binding sites for FITC-labeled peanut agglutinin (Arachis hypogaea) in frozen sections of whole embryonic heads of the chick between embryonic day 2 (E2) and embryonic day 8 (E8) is described. PNA-binding sites are present already at E2 on the external limiting membrane and the ventricular boundary of the neural wall. With progressing differentiation fluorescent bands are formed from the outside of the neural wall radiating inside, forming second layers inside the CNS at later stages. The visual system (retina and tectum) shows remarkably little PNA-binding activity until about E7/8, a time when binding sites in the other brain areas are already most abundant. In the telencephalon a fuzzy distribution of fluorescence is found. A possible significance of this finding for fiber guidance from the retina to the tectum is briefly discussed. Local high activities can be observed in different parts of the brain. Intense bands of PNA-fluorescence on parts of the epidermis, along the eye cups, around the fore- and hindbrain and in the medial part of the mesenchyme delineate early biochemical compartments foreshadowing the succeeding ossification.     

Characterization of [125I]Tyr11-somatostatin binding sites in the rabbit retina.

We have identified specific receptors for somatostatin (SS) in the rabbit retina using the radioligand [125I]Tyr11-Somatostatin. [125I]Tyr11-SS bound with high affinity to retinal membranes as was ascertained by both kinetic and saturation experiments. Scatchard analysis of the saturation data for [125I]Tyr11-SS binding to retinal membranes suggest a single population of sites with an apparent affinity constant (KD) of 0.90 +/- 0.20 nM and a maximum number of binding sites (Bmax) of 104 +/- 52 fmol/mg protein. The specific binding of [125I]Tyr11-SS was displaced in a dose-dependent manner by SS, Tyr11-SS, SMS 201-995, SS-28 and D-Trp8-SS. The inactive SS analog SS28(1-14) as well as the peptides CRF and bombesin had no effect. In addition, the specific binding of [125I]Tyr11-SS was attenuated by GTPgS. These findings demonstrate the presence of a selective receptor for SS in the rabbit retina that is coupled to guanine nucleotide binding protein(s).     

Expression of functional N-methyl-D-aspartate receptors during development of chick embryo retina cells: in vitro versus in vivo studies

The N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the NMDA receptors was followed by monitoring the changes in the intracellular free calcium concentration ([Ca2+](i)), in response to NMDA or to L-glutamate. The expression of the subunits NMDAR1, NMDAR2A/B and NMDAR2C in the retinospheroids and in chick retinas were determined by Western blot analyses. The changes in [Ca2+](i) in response to 400 microM NMDA increased from 5 h in vitro to 3 days in vitro (DIV) and remained constant until 14 DIV, whereas the [Ca2+](i) response to 500 microM L-glutamate increased from 5 h in vitro to 3 DIV and decreased slightly until 14 DIV. In the retinospheroids, the expression of the NMDAR1 and NMDAR2A/B subunits increased from 5 h in vitro until 14 DIV, whereas the NMDAR2C subunit increased from 5 h in vitro until 10 DIV and remained constant until 14 DIV. In the retinas, the expression of NMDAR1 increased from embryonic day 8 (E8) until E15, decreased until E18, and increased again until day 22 (post-hatched 1, PH1). The NMDAR2A/B increased from E8 until E18 and decreased slightly until PH1, whereas the NMDAR2C subunit increased from E8 until E15, remained constant until E18, and increased again until PH1. The results suggest that NMDA receptors are expressed and functionally active at early embryonic stages in the retina and in retinospheroids, before synapse formation (E12). However, the calcium responses to NMDA were relatively constant from 3 DIV until 14 DIV, showing no correlation with the increase in the expression of the studied NMDA receptor subunit during the same period. Also, the patterns of NMDA receptor subunits expressed in chick embryo retina cells cultured in vitro and in retina cells developing in vivo were similar.     

Ontogeny of substance P receptors in rat spinal cord: quantitative changes in receptor number and differential expression in specific loci

Anatomic distribution and functional studies of substance P (SP) and its binding sites show a role for the peptide in sensory (nociception), autonomic and somatic motor control. These physiologic functions show postnatal developmental changes, which, if mediated by SP, suggest that the receptors for the peptide may also undergo postnatal changes. This hypothesis was tested by using light microscopic autoradiography and membrane homogenate binding of 125I-Bolton-Hunter-SP (125I-BH-SP) to study SP binding sites in the spinal cord of rats of different ages. In cervicothoracic segments of rat spinal cord, the autoradiographs showed that specific binding of 125I-BH-SP occurred predominantly in the grey matter and varied inversely to age. In pups, up to about 15 days old, binding sites were diffusely distributed over the grey matter, and became progressively more defined in specific nuclei as the rats aged. A novel nucleus which is located in the ventrolateral ventral horn of caudal cervical segments and contained a high density of SP binding sites has been identified. High densities of SP binding sites in this nucleus and the intermediolateral cell column were visualized from the first postnatal day; however, those in the phrenic motor nucleus and in the dorsal horn were not fully expressed until after the 8th postnatal day. The age-related binding was confirmed in a membrane homogenate binding study of whole spinal cord which showed that the ratio for the concentration (cpm/mg protein) of specific binding was 106:12:4:1, for rats 11 (26 g), 38 (145 g), 90 (329 g) and 260 (553 g) days old. The ratio for the specific binding to the spinal cord (uncorrected for tissue weight) for the same groups of rats was 6:3:2:1. These data suggest that SP receptors decreased as a function of age. Furthermore, the decrease in SP receptors was not entirely due to growth of the spinal cord.     

Pre- and postnatal development of opiate receptor subtypes in rat spinal cord

We have studied the developmental expression of opiate binding sites in the rat spinal cord at various prenatal and postnatal stages. For each developmental stage, we have compared the expression pattern of kappa receptors with that of mu and delta receptor subtypes. Both mu and kappa receptors appear relatively early during spinal cord ontogeny (from the 15th prenatal day), while delta sites are expressed later at the postnatal period (starting at the 1st postnatal day). The number of kappa sites predominates throughout the development (55-80% of total opiate sites) with two peaks of binding activity: one at the 20th gestational day, and the other around the 7th postnatal day. mu sites represent 20-38% of the total opiate receptor population with one peak of binding activity appearing at the 1st postnatal day. The densities of mu and kappa receptors at the adult stage are lower by 40-50% than the peak values observed at the early postnatal periods. The relative amounts of delta sites remain low throughout the ontogeny (4-8% of the total opiate sites). The binding properties of neonatal (1 day after birth) kappa sites (ligand binding affinities, regulation of agonist binding by guanosine triphosphate and various cations) are similar to those displayed by kappa receptors in adult spinal cord.     

Development of synapses between chick retinal neurons in dispersed culture

Morphological criteria allow several kinds of synapse to be recognized in the vertebrate retina. It is, however, not presently known if, or how, these morphological differences reflect physiological distinctions. Since a proper investigation of synaptic physiology in the intact retina is compromised by technical difficulties, we have examined dispersed cultures to discover if they are likely to provide a more tractable physiological preparation. The chief question addressed here concerns the extent to which normal synaptic development takes place in the impoverished conditions of dispersed cell culture. Cultures were established from embryonic day 8 chick retina and fixed for microscopy on embryonic equivalent (E.E.) days 12, 14, 16, and 18. Neuronal processes appeared shortly after plating and continued to increase in number and extent through E.E. 16. Cone cells were recognizable by virtue of their distinctive oil droplets. Two classes of cone could be distinguished on the basis of the density of their cytoplasmic staining. Presynaptic ribbons could be observed in cone cells on E.E. 12, but characteristic dyad and triad postsynaptic organization was seldom present at this stage nor was it often observed at subsequent times. An increase in the number of ribbon synapses in culture was seen on E.E. 18. These synapses may represent those of bipolar cells. Conventional synapses were found at all times examined but the number of these increased greatly between E.E. 14 and 16. Of these conventional synapses, we found some whose anatomy was characteristic of synapses made by amacrine cells as well as some whose anatomy was characteristic of synapses made by bipolar cells.     

Expression of AMPA/kainate receptors during development of chick embryo retina cells: in vitro versus in vivo studies

The activity and the subunit expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the AMPA/kainate receptors was monitored by following the changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to AMPA, kainate or to L-glutamate, and the expression of the receptor subunits GluR1, GluR2/3, GluR4 and GluR6/7 was determined in the retinospheroids and in chick retinas by immunodetection using polyclonal antibodies. The changes in [Ca(2+)](i) in response to 400 microM kainate increased from 5h in vitro to 3 days, and remained constant until day 14, whereas the [Ca(2+)](i) in response to 500 microM L-glutamate or 400 microM AMPA increased from 5h in vitro to 3 days, and thereafter decreased slightly until day 14. The [Ca(2+)](i) responses to kainate are mainly due to AMPA receptor stimulation, since the signals were abolished by LY303070, the AMPA receptor antagonist, and were not affected by MK-801, the NMDA receptor antagonist. In retinospheroids, the levels of expression of GluR1 subunit increased from 5h in vitro until day 7, then decreased until day 14. The levels of expression of GluR2/3 and GluR4 subunits increased from 5h in vitro until day 10, and remained constant until day 14. The levels of kainate receptor subunits GluR6/7 increased from 5h in vitro until day 3, and thereafter decreased slightly until day 14. In the retinas, the expression of GluR1 and GluR6/7 subunits increased from day 8 until day 15, and then decreased until day 22 (post-natal 1). The subunits GluR2/3 and GluR4 increased from day 8 until day 18, and remained constant until day 22. The results suggest that AMPA/kainate receptors are expressed at early embryonic stages, although at low levels and before synapse formation (E12). However, the AMPA receptors are not completely functional at the first stage studied since they do not respond to the agonist AMPA. Also, the patterns of AMPA/kainate receptor subunit expression in retinospheroids of chick embryo retina cells cultured in vitro and in retina cells developing in the embryo (in vivo) were similar, indicating that the AMPA/kainate receptor subunits expression in these primary cultures mimics their expression in the developing chick retina.     

Characterization of corticotropin-releasing factor receptors in dissociated brain cell cultures

Although corticotropin-releasing factor (CRF) receptors have been identified throughout the brain, relatively little is known about the regulation of CRF receptors. Recent investigations aimed at developing an in vitro model for studying the regulation of CRF receptors demonstrated CRF binding in brain cell cultures. To test the hypothesis that dissociated brain cell cultures contain CRF receptors and may provide a model for studying their regulation, studies characterizing binding of labeled CRF were performed. Dissociated cells derived from hypothalamus and extrahypothalamic forebrain (predominantly cortex) of day 17 fetal rats were maintained in chemically defined medium. We used a stable 125I-labeled analog of ovine CRF, 125I-Tyro-ovine CRF (125I-oCRF), to identify and characterize CRF receptors. Although specific binding of 125I-oCRF was demonstrated in both hypothalamic and extrahypothalamic cell cultures, the concentration of CRF receptors was much greater (3-5 fold) in extrahypothalamic cells. Binding of 125I-oCRF in extrahypothalamic cells was saturable and was composed of high affinity (Kd = 0.51 nM) and low affinity (Kd = 17.25 nM) sites. Pharmacological displacement of labeled CRF from cells with a variety of CRF fragments and analogs was similar to that in studies of pituitary and brain homogenates. Extrahypothalamic cells studied at several times between 4 and 13 days in culture revealed an increase in the number of CRF receptors; the concentration of CRF receptors at 13 days was 3.5 times that observed at 4 days. Studies directed toward determining whether CRF receptor concentration could be modulated by CRF, adrenocorticotropic hormone, atropine or a CRF antagonist showed a change (36% decrease) only in response to chronic exposure with CRF. Conclusions: (1) dissociated fetal rat brain cell cultures derived from extrahypothalamic forebrain and hypothalamus contain CRF receptors; (2) CRF receptors in brain cells exhibit a differential distribution and characteristics similar to those previously reported in brain and pituitary; (3) dissociated fetal rat brain cell cultures may provide a relatively simplified in vitro model for studying the regulation of CRF receptors; and (4) CRF down-regulates its own receptor in extrahypothalamic forebrain cells.     

Developmental expression patterns of bone morphogenetic proteins, receptors, and binding proteins in the chick retina

Bone morphogenetic proteins (BMPs), a large subfamily of the transforming growth factor-beta (TGF-beta) superfamily of growth factors, have been implicated in patterning of the central nervous system, but their role in the retina is much less well known. As an initial step in addressing this issue, we have investigated by in situ hybridization the expression patterns of BMP-2, -4, -5, -6, and -7, BMP receptor kinases (BRKs) -1, -2, and -3, and BMP binding proteins noggin and chordin, in the chick embryonic eye at embryonic day 3 (E3), and in isolated retinas at E6, E8, and E18. Strikingly, all mRNAs examined had spatially restricted patterns of expression in the early eye, with the receptors found primarily in the ventral portion of the retina and in the optic stalk, and the ligands and binding proteins localized to other regions of the retina and/or retinal pigment epithelium. Dorso-ventrally restricted patterns of expression persisted at E8, but were no longer apparent at E18, whereas layer-specific patterns of expression were detectable at both E8 and E18. This distribution of BMP family members, receptors, and binding proteins within the retina appears consistent with a possible role in patterning and/or differentiation of this tissue.     

Developmental expression of somatostatin receptors in the rat retina.

The ontogeny of somatostatin receptor binding was studied in developing rat retina using the iodinated derivative of the somatostatin analog, SMS 204-090. Specific binding of the ligand was seen as early embryonic day (E) 15 in the region of the inner neuroblastic layer. At E19 binding was localized to the ganglion cell and developing inner plexiform layers. At postnatal day (P) 2, there was diminished binding on autoradiography in this region. At P11, binding was more intense in the inner plexiform layer, and there was discernible binding in the outer plexiform layer. In the adult retina, the binding was seen clearly in two distinct bands corresponding to the inner plexiform layer and the outer plexiform layer. There was a single saturable binding site with the dissociation constant (Kd) of 0.25 +/- 0.04 nM. Binding sites were fairly constant throughout development except for a significant decline during the first postnatal week (Bmax = 1.8). These results demonstrate the early appearance of somatostatin receptors in the rat retina with high levels present embryologically followed by a brief decline in the early postnatal period with a return to high levels by synapse formation (P11). These receptor data parallel previous reports of the appearance of the somatostatin mRNA and peptide in rat retina.     

Characterization of neurotensin binding sites on rat mesencephalic cells in primary culture.

Many studies have reported the presence of high amounts of neurotensin (NT) binding sites in the mesencephalon of adult rat, and their possible role in mediating the effects of the peptide on the activity of mesencephalic dopaminergic neurons. In the present study, we demonstrate the presence of NT sites in primary cultures of embryonic rat mesencephalic cells. On these cells, a single class of high affinity 125I-NT binding sites was observed. The value of the apparent affinity constant (0.3 nM) did not show any significant change throughout time, from 3 to 14 days in culture. The number of sites, however, increased until day 11 and decreased thereafter. Acetylneurotensin (8-13), NT and neuromedin N were potent competitors of 125I-NT binding, while NT (1-10), NT (1-11) and levocabastine were uneffective. These results indicate that the sites detected in the mesencephalic cultures share common binding properties with the high-affinity NT sites already described in adult rat brain. The neuronal localization of the NT sites was suggested by their presence in neuron-enriched serum-free cultures and their absence in glial cultures. Autoradiographic studies confirmed the cellular localization of NT sites and indicated that, under our experimental conditions, cells labeled by 125I-NT represented 0.14% of the initially plated cell number. Taken together, these results show that the development of mesencephalic neurons in primary culture is associated with an increased expression of NT binding sites. Since such cultures have been shown previously to contain functional dopaminergic neurons, we suggest that they could provide a good model to investigate the modulation of the activity of these neurons by NT.     

Distribution and number of transferrin receptors in Parkinson's disease and in MPTP-treated mice

Transferrin is a glycoprotein that functions primarily to deliver iron to the cell. Recent studies suggest that the transferrin receptor mediates the intracellular delivery and transport of iron bound to transferrin in the CNS. Iron-catalyzed free radical generation has been proposed as a possible cause of nigral cell death in Parkinson's disease. Our hypothesis is that abnormal iron handling by the transferrin receptor may contribute to the formation of free radical species which catalyze the lipid peroxidation of nigral cell membranes. We have assessed the number of transferrin receptors on membrane fractions prepared from the human striatum from control subjects and patients with Parkinson's disease. Equilibrium-binding studies demonstrated a reversible, saturable, and high-affinity transferrin binding site (KD = 3 nM) in human brain membranes. Regional binding assays indicate that the number of transferrin receptors in the putamen was reduced significantly in Parkinson's disease. The density of transferrin receptors was unaltered in membranes prepared from the caudate nuclei and the globus pallidus. To address the possibility that transferrin receptors are located on dopaminergic terminals, we have examined the distribution and number of transferrin receptors in the striatum of MPTP-treated mice using in vitro autoradiographic methods. In these experiments, the loss of dopaminergic terminals in the striatum was visualized by differential [3H]mazindol uptake site autoradiography. A marked reduction in the density of both transferrin receptors and [3H]mazindol binding sites was observed in the mouse striatum 7 days post-MPTP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The ontogeny of transferrin receptors in the embryonic chick retina: an immunohistochemical study

Transferrin receptor (TfR) immunoreactivity in the developing chick retina was examined. Immunoreactivity was detectable in the ganglion cells of embryonic day (E) 4 retina. At E9, diffuse TfR immunoreactivity appeared in the outer portion of the inner nuclear layer. Amacrine cells were the most intensely TfR-positive cells in the inner nuclear layer. At E11, the inner segment of photoreceptor cells showed moderate immunoreactivity. With the appearance of the outer segments, positive immunoreactivity was observed in these structures. TfR's developmental distribution in the retina may reflect the developmental and physiological role of transferrin.     

Early appearance and transient expression of vasopressin receptors in the brain of rat fetus and infant. An autoradiographical and electrophysiological study

The development of vasopressin (AVP) receptors in the rat brain, spinal cord and pituitary gland was studied by in vitro light microscopic autoradiography. AVP binding sites were labeled using [3H]AVP in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the binding of [3H]AVP to oxytocin receptors was prevented by adding in the incubation medium a highly selective oxytocin agonist. Specific binding was first detected at E16 in the ventral pontine reticular formation. Many other brain areas were progressively labeled between E18 and PN5. The distribution of binding sites observed at PN5 remained unchanged until the beginning of the third postnatal week. Thereafter binding was markedly reduced or even disappeared in several areas, in particular in the facial nucleus. The adult distribution of AVP binding sites was established at the time of weaning. The properties of transient AVP binding sites in the facial nucleus were studied both by autoradiography and by electrophysiology. Non-radioactive AVP displaced [3H]AVP binding in this nucleus as efficiently as it did in the lateral septum of the adult. Single-unit extracellular recordings showed that AVP can excite facial motoneurones by interacting with receptors which are pharmacologically indistinguishable from V1 (vasopressor) type. Thus, AVP binding sites transiently expressed in the brain of fetal and infant rat probably represent functional neuronal receptors, having the same ligand selectivity and affinity than AVP binding sites present in the adult. This suggests that AVP acts not only as a neuropeptide in the adult brain but may play a significant role during maturation of the central nervous system.     

Expression of mineralocorticoid type I and glucocorticoid type II receptors in astrocyte glia as a function of time in culture.

In the present study we have examined the expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia maintained in culture for different periods of time. Cytosolic mineralocorticoid Type I receptors were labeled with [3H]aldosterone (ALDO) in the presence of a 500-fold molar excess of the potent Type II receptor ligand RU 28362. [3H]Dexamethasone (DEX) was used to label cytosolic Type II receptors. Both Type I and Type II receptor binding was saturable in astrocyte glia that had been maintained in culture for 20 and 30 days following final plating (i.e. 20- and 30-day-old cultures). Scatchard analysis of [3H]ALDO binding revealed a single class of Type I receptors, with dissociation constants (Kd) of 0.45 +/- 0.13 nM and 0.53 +/- 0.07 nM, respectively, in 20- and 30-day-old cultures. The number of Type I receptors in 30-day-old cultures was nearly half that found in 20-day-old cultures (22.06 vs 42.64 fmol/mg protein). Linear Scatchard plots were also obtained for [3H]DEX binding to cytosol prepared from 20- and 30-day-old cultures. There were no significant differences in the Kd or Bmax values for [3H]DEX binding in 20- or 30-day-old cultures, i.e. 2.06 +/- 0.15 nM and 247.36 +/- 18.16 fmol/mg protein for 20-day-old cells and 2.3 +/- 0.74 nM and 261.02 +/- 3.08 fmol/mg protein for 30-day-old cells. These Bmax values are more than double the Bmax value for [3H]DEX binding observed in our previous studies in 10-day-old astrocyte glial cultures. Switching cultured astrocyte glial from serum-supplemented to serum-free medium had no significant effects on the Kd values of Type I or Type II receptors in all the cultures tested. However, treatment with serum-free medium increased the number of Type I receptors in 30-day-old cultures to a level similar to that found in 20-day-old cultures. Taken together, these binding data suggest that Type I and Type II receptors are expressed differently in astrocyte glia as a function of time in culture.     

Differential ontogenesis of D1 and D2 dopaminergic receptors in the chick embryo retina

The differentiation of D1 and D2 dopamine receptors was investigated during the ontogenesis of the chick embryo retina. Our results reveal an interesting complexity in dopaminergic differentiation, with one major receptor system developing before synapses and another one developing after. The dopamine-dependent increase of chick retina cAMP level differentiates early during retina ontogeny. By the embryonic day 10-11 10(-4) M dopamine and ADTN elicit a 13-fold increase in cAMP content of the retina. However, [3H]spiperone (D2 ligand) binds very little to crude membrane preparation of retinas from embryos in the same developmental stage (12-13 fmol/mg protein). High specific binding of [3H]spiperone is only detected after the embryonic day 17-18, attaining 80 to 100 fmol of specific spiperone binding sites in the retinas from post-hatched animals. Apomorphine also promotes the accumulation of cAMP of retinas from early embryonic stages. However, it is only 20-30% as effective as ADTN or dopamine. In addition, while the dopamine responsiveness of the tissue decreases sharply during its ontogeny, the apomorphine effect remains practically constant throughout this period. Both dopamine and apomorphine are equally effective in eliciting cAMP accumulation of retinas from post-hatched animals. Moreover, apomorphine is a potent inhibitor of dopamine-induced cAMP level of the embryonic tissue. The results presented here indicate that D1 and D2 receptors differentiate independently from each other, and that apomorphine elevates retina cAMP levels via a subclass of D1 receptors that does not desensitize significantly during retina development.     

Role of nitric oxide in photoreceptor survival in embryonic chick retinal cell culture

The presence of nitric oxide synthase (NOS) in chick retina during development has allowed us to study the role of nitric oxide (NO) during retinal differentiation in dissociated chick retinal cell culture from embryonic day 6. We have demonstrated the presence of nicotinamide adenine dinucleotide phosphate diaphorase staining in these cultures after 3 days in vitro (Div), with a maximal intensity after 8 Div, corresponding to embryonic day 14. Immunohistochemistry studies confirmed the presence of the two isoforms of NOS, NOS-I and -III, in dissociated retinal cell cultures at 8 Div. Addition of NG-monomethyl-L-arginine, a NOS inhibitor, to retinal cell cultures prevented NO production but did not modify the appearance and the survival of ganglion and amacrine cells. However, immunohistochemical analysis with distinct markers for photoreceptor cells (rods and cones) showed that inhibition of endogenous NOS in retinal cell cultures prevented the developmental decrease of rod number between 5 and 8 Div, thus supporting the hypothesis that NO may be involved in the cell death of rods during the development of the retina.