Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.     

Ontogenesis of lipids in chick embryo retina

PURPOSE: The effects of embryonic development on lipid composition in the retina were studied in 7, 11, 15, and 18-day-old chick embryos and newly hatched chicks. METHODS: The proportions of phospholipids, free and esterified cholesterol, diacylglycerides, and free fatty acids were determined using the Iatroscan TLC/FID procedure. Gas chromatography and mass spectrometry were used to determine the fatty acid composition. RESULTS: The major phospholipid species were phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, and sphingomyelin. Concentrations of the analyzed components have been related to the chronology of concrete stages of retinal development. The fatty acid composition of the total lipids, (n-6):(n-3) and saturated: unsaturated fatty acid ratios, and other parameters are reported. The proportions of total saturated and total monounsaturated fatty acids decreased very little from day 7 to hatching, whereas total polyunsaturated fatty acids nearly doubled over the same period. The increase in C18:2(n-6) from day 11 onwards was not followed by a similar increase in C20:4(n-6), hence the C20:4 to C18:2 ratio decreased with age. CONCLUSIONS: The cholesterol:phospholipid ratio decreased from day 7 to day 15 and increased from day 15 to hatching. High proportions of esterified cholesterol, very probably originating in the retinal pigment epithelium, were also recorded. Total saturated and monounsaturated fatty acids decreased, while polyunsaturated fatty acids increased during the period of initial retinal growth.     

Ultrastructure of the neural canal closure in the chicken embryo

We have studied the neuroectoderm of the chicken embryo, from the beginning of somitic segmentation up to the state at which it has seven somities, i.e. the period covering the passage from the 'neural canal' into the 'neural tube'. This paper was devoted mainly to the study of the ultrastructural cytodifferentiation which takes place during the stages in which the neural canal closes up, and at the level of the first area of contact between the 'neural crests'-roughly at the level of the third somite. We used eggs from a hen of the White Leghorn breed, incubated at 38 degrees C, from which we extracted chicken embryos after 24-30 h of incubation, corresponding to Hamburger-Hamilton's stages 7, 8 and 9. Thus we were able to obtain several series of embryos with three, six and seven somites. The neural canal, or tube, at the level of the third somite, was fixed in glutaraldehyde at 6.25% for 30 min and postfixed in 1% osmium tetroxide for 2 h embedded in Araldite, and the sections were then stained with lead citrate. We observed that the vacuoles in the free edge of the neural canal gradually disappear as the canal closes up, while we gradually witness the appearance of the 'closure apparatus' (or the safety or occlusion apparatus) of what is beginning to form the ependymal epithelium, and the first rudimentary outlines of the cilia. All these changes begin to be observed at the seven-somite stage, i.e. when the neural canal is beginning to close up. The 'closure apparatus' consists of a number of intercellular joint complexes, of the 'close-join't type, between which we observe a number of fine filaments, like a terminal velum', or veil, which we call 'interconnecting filaments'. In the 'raphe', whereby contact is established between the neural crests, we observe the initial stages of fusion between the vacuolated edges, with the plasmatic membrane of these cells forming very fine cytoplasmic 'tongues' which interdigitate with cells from the opposite neural crest and finally constitute the so-called close joints.     

Tryptophan is present in glial cells and photoreceptors in the chicken retina

Tryptophan is a large neutral amino acid which is utilized in the biosynthesis of neuroactive substances such as serotonin and melatonin. However, it has been unclear where pools of tryptophan might be localized. Using a specific antiserum against tryptophan, we demonstrate that in the chicken retina tryptophan is present in radial glial cells and photoreceptors, but not in other neuronal elements. These data suggest that serotonergic neurones are probably dependent upon the transfer of tryptophan from the glial cells in order to manufacture serotonin and other tryptophan derivatives in the brain. If glia do supply tryptophan to neurones then this process will have significant practical implications for our basic understanding of and pharmacological manipulation of serotonergic systems.     

Distribution of an endogenous 16-kd S-lac lectin in the chicken retina

PURPOSE: To examine by indirect immunofluorescence the distribution of an endogenous 16-kd S-lac lectin (soluble lactose binding lectin) during development of the chicken retina. METHODS: Cryosections of retinal tissue at different developmental stages and cultured retinal cells (either not permeabilized or permeabilized with acetone) were incubated with a rabbit antiserum that specifically reacts with the retinal 16-kd S-lac lectin. After incubation with a fluorescent-labeled secondary antibody, tissue sections and cultured cells were analyzed by fluorescence microscopy. RESULTS: Retina was weakly stained with the antiserum on early embryonic day 7, whereas on embryonic days 13 and 18 it showed a restricted "granular" staining in the outer retina. At embryonic day 18, in addition, there was widespread staining in all retinal layers. This pattern was maintained by postnatal day 5 and in the adult retina, although the intensity of the staining of the outer retina was weaker. In retinal cell cultures, glial-like flat cells and monopolar, bipolar, and multipolar neurons were stained with the antiserum, but only if they had been previously permeabilized with acetone. CONCLUSION: The results suggest that the distribution of a 16-kd S-lac lectin changes during retinal development. Cell culture experiments indicate that most often the lectin is localized intracellularly in the different retinal cell types.     

Neurokinin 1 receptor expression in the rat retina

Tachykinin (TK) peptides influence neuronal activity in the inner retina of mammals. The aim of this investigation was to determine the cellular localization of the neurokinin 1 receptor (NK1), whose preferred ligand is the TK peptide substance P (SP), in the rat retina. These studies used a polyclonal antiserum directed to the C-terminus of rat NK1. The majority of NK1-immunoreactive (IR) cells were located in the proximal inner nuclear layer (INL), and very rarely they were found in the distal INL. Some small and large NK1-IR somata were present in the ganglion cell layer. NK1-IR processes were densely distributed across the inner plexiform layer (IPL) with a maximum density over lamina 2 of the IPL. Immunoreactive processes also crossed the INL and ramified in the outer plexiform layer where they formed a sparse meshwork. NK1-IR processes were rarely observed in the optic nerve fiber layer. Double-label immunofluorescence studies with different histochemical markers for bipolar cells indicated that NK1 immunoreactivity was not present in bipolar cells. Together, these observations indicate that NK1 immunoreactivity is predominantly expressed by amacrine, displaced amacrine, interplexiform, and some ganglion cells. Double-label immunofluorescence experiments were also performed to characterize NK1-containing amacrine cells. Sixty-one percent of the gamma-aminobutyric acid (GABA)-IR cells, 71% of the large tyrosine hydroxylase (TH)-IR cells, and 100% of the small TH-IR cells contained NK1 immunoreactivity. In addition, most (91%) of the NK1-IR cells had GABA immunoreactivity. In contrast, vasoactive intestinal polypeptide-, TK-, choline acetyltransferase-, and parvalbumin-IR amacrine tells did not express NK1 immunoreactivity. Overall, the present findings suggest that SP acts directly upon several cell populations, including GABA-containing amacrine cells and ganglion cells, to influence visual information processing in the inner retina.     

Tyrosine hydroxylase expression in the Cebus monkey retina

Tyrosine hydroxylase (TH) expression was used as a marker to study the dopaminergic cells in the Cebus monkey retina. Two types of dopaminergic cells were identified by cell body size and location, level of arborization in the inner plexiform layer, and amount of immunolabeling. Type 1 cells displayed intense immunoreactivity and larger somata (12-24 microns) located in the inner nuclear layer or ganglion cell layer, whereas type 2 had smaller cell bodies (8-14 microns) found either in the inner plexiform layer or ganglion cell layer and were more faintly labeled. Interplexiform cells were characterized as type 1 dopaminergic cells. Immunoreactive axon-like processes were seen in the nerve fiber layer, and a net of fibers was visible in the foveal pit and in the extreme periphery of the retina. The population of TH+ cells was most numerous in the temporal superior quadrant and its density peaked at 1-2 mm from the fovea. Type 1 TH+ cells were more numerous than type 2 cells at any eccentricity. Along the horizontal meridian, type 1 cell density was slightly higher in temporal (29 cells/mm2) than in nasal (25 cells/mm2) retina, while type 2 cells had a homogeneous distribution (4.5 cells/mm2). Along the vertical meridian, type 1 cells reached lower peak density (average 17.7 cells/mm2) in the inferior retina (central 4 mm), compared to the superior portion (23.7 cells/mm2). Type 2 cell density varied from 4.5 cells/mm2 in the superior region to 9.4 cells/mm2 in the inferior region. The spatial density of the two cell types varied approximately inversely while the total density of TH+ cells was virtually constant across the retina. No correlation between dopaminergic cells and rod distribution was found. However, we suggest that dopaminergic cells could have a role in mesopic and/or photopic vision in this species, since TH+ fibers are present in cone-dominated regions like the foveola and extreme nasal periphery.     

CD44 expression in the developing human retina

OBJECTIVE: This study was conducted to evaluate the cosmetic use of botulinum toxin type A (Botox), which blocks the release of acetylcholine at the presynaptic neuromuscular junction leading to an irreversible, but temporary chemical denervation muscular paralysis and weakness. This produces a significant cosmetic improvement of wrinkling in the upper face due to hyperfunctional animation. METHOD: A prospective clinical study representing our experience with this new technique is presented. Patient selection and evaluation, classification of animation lines, techniques, results and complications are discussed. In a 15-month period, 23 patients with seven anatomic sites were injected. Twenty-three patients had the lateral aspect and the inferior aspect of their squint lines injected, and 26 patients had their glabellar frownlines injected. RESULTS: Significant improvement occurred to the average depth and length of the glabellar frownlines. The subjective improvement by the patients was also significant. Regarding the crow's feet, the lateral canthal lines showed more improvement than the inferior lateral canthal lines because the latter has a greater component of zygomaticus major and minor muscle, which contributes to the inferior lateral squint line. CONCLUSION: Botox is a safe, easy-to-use, effective modality for the temporary elimination of hyperfunctioning upper-facial muscles.     

Development of the enkephalin-, neurotensin- and somatostatin-like (ENSLI) amacrine cells in the chicken retina

The development of the enkephalin-, neurotensin- and somatostatin-like immunoreactive (ENSLI) amacrine cells in the chicken retina has been investigated by radioimmunoassay (RIA) and immunocytochemistry (ICC). By RIA, enkephalin-like immunoreactivity (ENK-LI) was detected at embryonic day (E) 5 at only very low levels, which gradually increased until E17. From E18 to E21, there was a relatively rapid increase in ENK-LI levels, and just after hatching, there was a very steep rise. By ICC, the cell bodies of the ENSLI amacrine cells were first detected in the inner nuclear layer on E18, with no immunostaining in the inner plexiform layer (IPL). On E21, more cells were detected and processes in the IPL were visible, but detailed arborisations were not clear. On postnatal day (P) 1, the ENSLI amacrine cells showed a morphology similar to that in mature retina in both the density of cell bodies and the ramification pattern of processes. Antibodies to neurotensin and somatostatin revealed a similar developmental pattern. Thus, the three peptides appear to follow a similar developmental pattern in the ENSLI amacrine cells, suggesting that the three peptides respond similarly to developmental stimuli, just as they are released in parallel in response to physiological stimulation from mature ENSLI amacrine cells. After hatching, higher levels of ENK-LI were detected by RIA and more ENSLI amacrine cell bodies and processes were detected by ICC in animals kept in the light than in those kept in the dark. In retinas kept in the light for 12 h, it was found that immunoreactive processes in the IPL formed strongly stained patches, but this was not observed in retinas kept in the dark for 12 h.     

Clonal patterns of cell proliferation, migration, and dispersal in the brainstem of the chicken embryo

Retroviral-mediated gene transfer was used to study clonal patterns of proliferation, migration, and dispersal in the brainstem of the chicken embryo. Clones were generated at stages 13-17 (Hamburger and Hamilton, 1951), a period of neurogenesis in the brainstem neural tube subsequent to the formation of rhombomeres. Clones were examined in separate experiments at stages 24-27, when many neurons migrate and differentiate; at stages 28-29, when brainstem nuclei begin to form; and at stages 34-35, when brainstem nuclei are fully formed. Stages 24-29 are characterized by a general variability in proliferative kinetics and migratory behavior. Clone sizes range from 1 to 29 cells, and migration patterns range from strictly radial (i.e., normal to the ventricular surface) to combined radial and tangential (i.e., perpendicular to the radial component). There is, however, an underlying systematic variation: (1) clones exhibiting tangential migration contain on average more cells than clones exhibiting only radial migration, and (2) the proportion of tangentially migrating clones increases from medial to lateral. By stages 34-35 some individual clones have apparently dispersed to disparate neuronal groups. The regional diversity observed among clones suggests that position along the mediolateral axis may determine the proliferative potential of progenitors and the migratory behavior and subsequent dispersal of their descendants.     

Blockade of glutamate-mediated activity in the developing retina perturbs the functional segregation of ON and OFF pathways

The dendrites of ganglion cells initially ramify throughout the inner plexiform layer of the developing retina before becoming stratified into ON or OFF sublaminae. This ontogenetic event is thought to depend on glutamate-mediated afferent activity, because treating the developing retina with the glutamate analog 2-amino-4-phosphonobutyrate (APB), which hyperpolarizes ON cone bipolar cells and rod bipolar cells, thereby preventing their release of glutamate, effectively arrests the dendritic stratification process. To assess the functional consequences of this manipulation, extracellular recordings were made from single cells in the A laminae of the dorsal lateral geniculate nucleus and from the optic tract in mature cats that had received intraocular injections of APB during the first postnatal month. Such recordings revealed that stimulation of the APB-treated eye evoked both ON as well as OFF discharges in 37% of the cells tested. (As expected, when the normal eye was activated, virtually all cells yielded only ON or OFF responses.) The proportion of ON-OFF cells found here corresponds closely to the incidence of multistratified dendrites observed previously in anatomical studies of APB-treated cat retinas. This suggests that the ganglion cells with multistratified dendrites receive functional inputs from ON as well as OFF cone bipolar cells. This interpretation is further supported by the finding that the proportion of ON-OFF cells was very similar in the geniculate layer innervated by the treated eye and in the optic tract. The cells activated by the APB-treated eye were also found not to show response suppression when flashing stimuli of increasing size were used. This suggests that exposing the developing retina to APB perturbs the neural circuitry mediating the antagonistic center-surround organization found in normal receptive fields. The functional changes evident after treating the developing retina with APB suggest that it should now be feasible to assess how the segregation of ON and OFF retinal pathways relates to organizational features at higher levels of the visual system, such as orientation selectivity in cortical cells.     

Light-induced c-fos expression in amacrine cells in the rabbit retina

Retinal neurons that express the immediate early gene c-fos after light exposure were characterized by neurotransmitter content using histochemical and immunocytochemical staining. In Northern blots the amount of c-fos mRNA peaked at 30 min, but remained detectable 60 min following light stimulation. Fos proteins were seen in the inner nuclear and ganglion cell layers, and the staining was most intense two and three hours after beginning the light exposure. In the ganglion cell layer 30-40% of Fos-immunoreactive cells were cholinergic displaced amacrine cells and 3-5% were ganglion cells. In the inner nuclear layer 24% of Fos-immunoreactive cells were Type I and 7% Type II NADPH-diaphorase-reactive (nitric oxide synthase) amacrine cells, 11% were tyrosine hydroxylase-containing cells, and 10-15% cholinergic amacrine cells. No Fos immunoreactivity was seen in serotoninergic, somatostatin- or VIP-immunoreactive cells, bipolar, horizontal or photoreceptor cells. Nicotine, kainic acid, NMDA and SCH 38393, a dopamine D1 receptor agonist, induced Fos immunostaining in the inner nuclear and ganglion cell layers, but administration of the corresponding receptor blockers mecamylamine, kynuretic acid, MK-801, haloperidol and SCH 23990 did not prevent light-induced Fos expression.     

Regional pattern of retinoid X receptor-alpha gene expression in the central nervous system of the chicken embryo and its up-regulation by exposure to 9-cis retinoic acid

We study characteristics of cell-differentiation rules that realize stable formation of regularly arranged checker-board patterns, exemplified by cone "mosaic" zebrafish retina, or the regular arrangement of cone photoreceptor cells. We consider the situation in which cells are arranged on a square lattice and are initially undifferentiated. Later each cell becomes one of the two differentiated states, affected by the state of the neighboring cells. The cells that undergo differentiation form a "morphogenetic cell row" which sweeps from one end to the other end of the lattice through time. This models an outward sweep of the margin of expanding mosaic region of the retina which occurs as undifferentiated photoreceptor cells become differentiated in concentric circles, joining the mosaic. We introduce an index to measure the ability of cell-differentiation rules to generate regular checker-board patterns from irregular initial patterns, and attempt to characterize the successful rules. We first show the importance of six "preservation conditions" which guarantee perfectly regular photoreceptor arrangement for all the rows after a regular row. Then we select an additional six "optimizing conditions" for responses to configuration that are consistently shown by the rules of high average scores. We also examine the effect of interaction between responses to different configurations. Finally we examine the concept of morphogenetic row precedence, i.e. that the successful rules generating a high score tend to treat the consistency with neighbors in the newly differentiated cells (those in the morphogenetic cell row) as more important that the consistency with previously differentiated neighbors.     

Infectious bursal disease virus changes the potassium current properties of chicken embryo fibroblasts

Homophobia is a socially accepted, culturally based belief, which is heavily influenced by an individual's or a community's inherent attitudes, beliefs and values. This conceptual analysis of homophobia has endeavoured to review existing literature on homophobia and subsequently identify and examine the phobic constituents of the concept. References to homophobia are mostly from the 1970-1980 period and there is much unacknowledged conceptual baggage that accompanies the term, which results in restrictive and inappropriate ideas about this concept. This is mainly the consequence of comparisons of homophobia to other phobias, which directly infers fear of homosexuals, while in reality homophobia is more of a biased disgust at homosexuals' lifestyles. This paper attempts to re-conceptualize homophobia so that empirical research can begin to test the critical attributes of the concept. This forms the basis for the development of a comprehensive social psychological theory of attitudes towards homosexuals. Such a theory would transcend the unilateral and unidimensional concept of homophobia as a fear and help the understanding of attitudes and feelings towards homosexuals.     

Unique retina cell phenotypes revealed by immunological analysis of recoverin expression in rat retina cells

OBJECTIVE: This study was undertaken to determine the impact of previous cardiac surgery on the presentation, management, and outcome of late dissection of the ascending aorta. PATIENTS AND METHODS: From 1976 to 1998, type A dissection developed in 56 patients with a history of previous cardiac surgery. Interval from first operation to type A dissection was 49 +/- 47 months (0.3-180 months). Previous operations were coronary artery bypass grafting (n = 40), aortic valve replacement (n = 8), and other (n = 8). RESULTS: Type A dissection was acute in 34 patients and chronic in 22. In acute dissection, aortic insufficiency occurred in 50%, malperfusion in 12%, and rupture in 18%; 2 patients (6%) were in hemodynamically unstable condition because of rupture. Of patients with previous coronary bypass grafting, 98% had preoperative coronary angiography. Type A dissection was treated by supracoronary tube graft (84%), Bentall procedure (14%), or local repair (2%). Strategies for managing previous coronary bypass grafting included reimplantation of proximal anastomoses with a button of native aorta (29 patients), interposition graft to pre-existing saphenous vein grafts (9 patients), and new saphenous vein grafts (20 patients). Eight hospital deaths occurred (14%). CONCLUSIONS: We conclude that (1) patients having type A dissection late after cardiac surgery infrequently have cardiac tamponade and hemodynamic collapse; (2) patients with previous coronary bypass grafting require coronary angiography, because operative management must account for pre-existing coronary artery disease; and (3) operative mortality is low, and this may be attributable to preoperative hemodynamic stability, delineation of coronary anatomy in those with previous coronary bypass grafting, and operative treatment of coronary artery disease.     

Opioid peptides activate phospholipase D and protein kinase C-epsilon in chicken embryo neuron cultures

The mu-opioid peptide morphiceptin stimulated a Ca(2+)-independent protein kinase C (PKC-epsilon) that is expressed both in embryonic day 6 chicken telencephalon and in derived neuronal cultures. This activation was seen as a 2-fold increase in the activity and level of cytosolic PKC-epsilon and as a transient increase in membrane-associated PKC-epsilon following morphiceptin treatment. Morphiceptin did not activate phospholipase C-mediated phosphatidylinositol hydrolysis but did transiently activate (2- to 3-fold) phospholipase D (PLD), as measured by phosphatidylethanol formation in neuron cultures derived from embryonic day 6 or day 7 cerebral hemispheres. This PLD activation could provide an alternative source of diacylglycerol for the activation of PKC-epsilon and was naloxone-reversible and at least partially blocked by the tyrosine kinase inhibitor herbimycin A. Addition of phorbol 12-myristate 13-acetate stimulated both PLD and PKC-epsilon activities to a greater extent than opioids. The phorbol ester and insulin stimulation of PLD was also blocked by herbimycin. Both morphiceptin (in a naloxone-reversible manner) and phorbol ester increased phosphorylation of similar cytosolic proteins in intact cells, demonstrating a functional role for the PKC-epsilon activation by opioids. This is evidence that opioid receptors are transiently coupled to tyrosine kinase, PLD and PKC-epsilon activation and, by implication, to neuronal cell growth during brain morphogenesis.