Visual conditions affecting eye growth alter diurnal levels of vitreous DOPAC.

In chicks, the diurnal patterns of retinal dopamine synthesis and release are associated with refractive development. To assess the within-day patterns of dopamine release, we assayed vitreal levels of DOPAC (3,4-dihydroxyphenylacetic acid) using high performance liquid chromatography with electrochemical detection, at 4-h intervals over 24 h in eyes with experimental manipulations that change ocular growth rates. Chicks were reared under a 12 h light/12 h dark cycle; experiments began at 12 days of age. Output was assessed by modelling using the robust variance structure of Generalized Estimating Equations. Continuous spectacle lensdefocus or form deprivation: One group experienced non-restricted visual input to both eyes and served as untreated "normal" controls. Three experimental cohorts underwent monocular visual alterations known to alter eye growth and refraction: wearing a diffuser, a negative lens or a positive lens. After one full day of device-wear, chicks were euthanized at 4-h intervals over 24 h (8 birds per time/condition). Brief hyperopic defocus: Chicks wore negative lenses for only 2 daily hours either in the morning (starting at ZT 0; n = 16) or mid-day (starting at ZT 4; n = 8) for 3 days. Vitreal DOPAC was assayed. In chicks with bilateral non-restricted vision, or with continuous defocus or form-deprivation, there was a diurnal variation in vitreal DOPAC levels for all eyes (p < 0.001 for each). In normal controls, DOPAC was highest during the daytime, lowest at night, and equivalent for both eyes. In experimental groups, regardless of whether experiencing a growth stimulatory input (diffuser; negative lens) or growth inhibitory input (positive lens), DOPAC levels were reduced compared both to fellow eyes and to those of normal controls (p < 0.001 for each). These diurnal variations in vitreous DOPAC levels under different visual conditions indicate a complexity for dopaminergic mechanisms in refractive development that requires further study.

Increased endogenous dopamine prevents myopia in mice.

Experimental evidence suggests that dopamine (DA) modulates refractive eye growth. We evaluated whether increasing endogenous DA activity using pharmacological or genetic approaches decreased myopia susceptibility in mice. First, we assessed the effects of systemic L-3,4-dihydroxyphenylalanine (L-DOPA) injections on form deprivation myopia (FDM) in C57BL/6 J (WTC57) mice. WTC57 mice received daily systemic injections of L-DOPA (n = 11), L-DOPA + ascorbic acid (AA, n = 22), AA (n = 20), or Saline (n = 16). Second, we tested transgenic mice with increased or decreased expression of vesicular monoamine transporter 2 (VMAT2HI, n = 22; WTHI, n = 18; VMAT2LO, n = 18; or WTLO, n = 9) under normal and form deprivation conditions. VMAT2 packages DA into vesicles, affecting DA release. At post-natal day 28 (P28), monocular FD was induced in each genotype. Weekly measurements of refractive error, corneal curvature, and ocular biometry were performed until P42 or P49. WTC57 mice exposed to FD developed a significant myopic shift (treated-contralateral eye) in AA (-3.27 ± 0.73D) or saline (-3.71 ± 0.80D) treated groups that was significantly attenuated by L-DOPA (-0.73 ± 0.90D, p = 0.0002) or L-DOPA + AA (-0.11 ± 0.46D, p = 0.0103). The VMAT2LO mice, with under-expression of VMAT2, were most susceptible to FDM. VMAT2LO mice developed significant myopic shifts to FD after one week compared to VMAT2HI and WT mice (VMAT2LO: -5.48 ± 0.54D; VMAT2HI: -0.52 ± 0.92D, p < 0.05; WT: -2.13 ± 0.78D, p < 0.05; ungoggled control: -0.22 ± 0.24D, p < 0.001). These results indicate that endogenously increasing DA synthesis and release by genetic and pharmacological methods prevents FDM in mice.

Circadian clock genes of goldfish, Carassius auratus: cDNA cloning and rhythmic expression of period and cryptochrome transcripts in retina, liver, and gut.

Clock genes are known to be the molecular core of biological clocks of vertebrates. They are expressed not only in those tissues considered central pacemakers, but also in peripheral tissues. In the present study, partial cDNAs for 6 of the principal clock genes (Period 1-3 and Cryptochrome 1-3) were cloned from a teleost fish, the goldfish (Carassius auratus ). These genes showed high homology (approximately 90%) with the respective cDNAs of zebrafish (Danio rerio), the only other teleost from which clock genes have been cloned. The daily expression pattern of each gene in retina, gut, and liver of goldfish was investigated using quantitative RT-PCR and cosinor analysis. All clock genes analyzed in the retina showed circadian rhythmicity; however, only Per 2-3 and Cry 2-3 were rhythmic in goldfish liver and gut. The amplitude and phase of the expression in liver and gut were different from those found in goldfish retina. Such differences suggest that other cues, such as feeding time, may contribute to the entrainment of oscillators in goldfish liver and gut. Our results support the use of goldfish as a teleost model to investigate the location and functioning of the circadian oscillators.

Light regulation of retinal dopamine that is independent of melanopsin phototransduction.

Light-dependent release of dopamine (DA) in the retina is an important component of light-adaptation mechanisms. Melanopsin-containing inner retinal photoreceptors have been shown to make physical contacts with DA amacrine cells, and have been implicated in the regulation of the local retinal environment in both physiological and anatomical studies. Here we determined whether they contribute to photic regulation of DA in the retina as assayed by the ratio of DA with its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), and by c-fos induction in tyrosine hydroxylase (TH)-labelled DA amacrine cells. Light treatment (approximately 0.7 log W/m(2) for 90 min) resulted in a substantial increase in DA release (as revealed by an increase in the DOPAC : DA ratio), as well as widespread induction of nuclear c-fos in DA amacrine cells in wild-type mice and in mice lacking melanopsin (Opn4(-/-)). Light-induced DA release was also retained in mice lacking rod phototransduction (Gnat1(-/-)), although the magnitude of this response was substantially reduced compared with wild-types, as was the incidence of light-dependent nuclear c-fos in DAergic amacrines. By contrast, the DAergic system of mice lacking both rods and cones (rd/rd cl) showed no detectable light response. Our data suggest that light regulation of DA, a pivotal retinal neuromodulator, originates primarily with rods and cones, and that melanopsin is neither necessary nor sufficient for this photoresponse.

Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types.

The avian retina and pineal gland contain autonomous circadian oscillators and photo-entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehyde-based photopigments than the retinaldehyde-based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0-4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.

Spatial, temporal, and intensive determinants of dopamine release in the chick retina.

The retinal dopaminergic system is a global regulator of retinal function. Apart from the fact that the rates of dopamine synthesis and release are increased by increasing illumination, the visual image parameters that influence dopaminergic function are mostly unknown. Roles for spatial and temporal frequency and image contrast are suggested by the effects of form-deprivation with a diffusing goggle. Form-deprivation reduces the rates of dopamine synthesis and release, and induces myopia, which is prevented by dopamine agonists. Our purpose here was to identify visual stimulus parameters that activate dopaminergic amacrine cells and elicit dopamine release. White Leghorn cockerels 4-7 days old were exposed to 2 h of form-deprivation, reduced light intensity, or stimuli of varied temporal or spatial frequency. Activation of dopaminergic neurons, labeled for tyrosine hydroxylase (TH), was assessed with immunocytochemistry for c-Fos, and dopamine release was measured by HPLC analysis of dopamine metabolite accumulation in the vitreous body. Form-deprivation did not reduce TH+ cell activation or vitreal dopamine metabolite accumulation any more than did neutral-density filters of approximately equal transmittance. TH+ cell activation and vitreal metabolite accumulation were not affected significantly by exposure to 2, 5, 10, 15, or 20 Hz stroboscopic stimulation on a dark background, or by sine-wave gratings of 0.089, 0.44, 0.89, 1.04, or 3.13 cycles/deg compared to a uniform gray target of equal mean luminance. These data indicate that the retinal dopaminergic system does not respond readily to short-term changes in visual stimulus parameters, other than light intensity, under the conditions of these experiments.

Regulation of cAMP by light and dopamine receptors is dysfunctional in photoreceptors of dystrophic retinal degeneration slow(rds) mice.

cAMP levels in dark and light were studied in dystrophic retinal degeneration slow(rds) mice, which carry a mutation in the rds/peripherin gene. cAMP levels were measured in vivo, in freshly isolated retinas, and in vitro in the presence of glutamate, which confines light modulation to photoreceptors. Experiments were conducted on young animals, when significant numbers of viable photoreceptor cells are present. In vivo levels of cAMP are higher in illuminated rds/rds retinas than levels measured in normal BALB/c retinas. Light-evoked down-regulation of cAMP levels was observed in vitro in normal photoreceptors. These measurements were made in the presence of the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine; therefore, they reflect an inhibition of cAMP formation. In contrast, light had no effect on cAMP formation in photoreceptors of mutant mice, measured under identical conditions. Thus, elevated levels of cAMP in rds/rds retinas in vivo result from abnormalities in cAMP synthesis in the mutant photoreceptor cells. In addition to regulation by light, cAMP formation in photoreceptor cells is regulated by dopamine, acting through dopamine D4 receptors. A dopamine D2/D4 receptor agonist, quinpirole, reduced cAMP levels in dark-adapted normal retinas in vitro, but not in rds/rds retinas. Our data indicate that alterations in a signal transduction pathway that leads to inhibition of adenylyl cyclase might underlie the abnormalities in cAMP levels in mutant rds/rds retinas. Heterozygous rds/+ photoreceptors demonstrated a normal pattern of light-evoked and quinpirole-mediated down-regulation of cAMP. Thus, partial expression of the normal phenotype is sufficient to render normal characteristics of cAMP regulation to the photoreceptors of the heterozygous mouse. The data obtained in the present study might be relevant to the understanding of photoreceptor pathology of patients with peripherin/rds mutations.

The role of dopamine in the locomotor stimulant effects and tolerance to these effects of caffeine.

Current evidence indicates that the acute locomotor stimulant effects of caffeine involve dopamine (DA) receptor activation; however, few studies have investigated the role of DA receptors in mediating the development of tolerance to caffeine. Therefore, the present study was designed to determine the degree to which DA receptors mediate the development of tolerance to the locomotor stimulant effects of caffeine. Caffeine was examined alone and in combination with haloperidol (HAL), GBR 12909, nisoxetine and fluoxetine. HAL dose-dependently and completely blocked the acute effects of caffeine on locomotor activity, and the highest dose of GBR 12909 enhanced the effects of caffeine. Neither nisoxetine nor fluoxetine altered the effects of caffeine. HAL was infused via osmotic pumps (0.1 mg/kg/day) during a 14-day regimen of chronic caffeine administered in a caffeinated drinking solution ( approximately 136 mg/kg/day). HAL did not block the development of tolerance to the locomotor stimulant effects of caffeine, but did impair the recovery from tolerance following withdrawal of caffeine. [3H]SCH 23390 (DA D(1)) binding sites were downregulated in the nucleus accumbens and striatum and were upregulated in the prefrontal cortex of caffeine-treated vs. control rats; however, the affinity of [3H]SCH 23390 for these binding sites was unaltered. There were no differences between the caffeine-treated and control rats in number or affinity of [3H]spiperone (DA D(2)) binding sites. These results suggest that, although HAL did not alter the development of tolerance to caffeine, changes in DA D(1) receptors could be one component of the mechanism underlying caffeine-induced tolerance.

Diurnal metabolism of dopamine in dystrophic retinas of homozygous and heterozygous retinal degeneration slow (rds) mice.

Dopamine metabolism was studied in dystrophic retinal degeneration slow (rds) mice which carry a mutation in the rds/peripherin gene. RDS mutations in humans cause several forms of retinal degeneration. Dopamine synthesis and utilization were analyzed at various time points in the diurnal cycle in homozygous rds/rds retinas which lack photoreceptor outer segments and heterozygous rds/+ retinas which have short malformed outer segments. Homozygous retinas exhibited depressed dopamine synthesis and utilization while the heterozygous retina retained a considerable level of activity which was, nevertheless, significantly lower than that of normal retinas. By one year, heterozygous rds/+ retinas which had lost half of the photoreceptors still maintained significant levels of dopamine metabolism. Normal characteristics of dopamine metabolism such as a spike in dopamine utilization at light onset were observed in mutant retinas. However, light intensity-dependent changes in dopamine utilization were observed in normal but not rds/+ retinas. The findings of this study suggest that human patients with peripherin/rds mutations, or other mutations that result in abnormal outer segments that can still capture light, might maintain light-evoked dopamine metabolism and dopamine-dependent retinal functions during the progression of the disease, proportional to remaining levels of light capture capabilities. However, visual deficits due to reduced light-evoked dopamine metabolism and abnormal patterns of dopamine utilization could be expected in such diseased retinas.

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.

Regulation of tryptophan hydroxylase activity in Xenopus laevis photoreceptor cells by cyclic AMP.

The aim of this study was to investigate the role of cyclic AMP in the regulation of tryptophan hydroxylase activity localized in retinal photoreceptor cells of Xenopus laevis, where the enzyme plays a key role in circadian melatonin biosynthesis. In photoreceptor-enriched retinas that lack serotonergic neurons, tryptophan hydroxylase activity is markedly stimulated by treatments that increase intracellular levels of cyclic AMP or activate cyclic AMP-dependent protein kinase, including forskolin, phosphodiesterase inhibitors, and cyclic AMP analogues. In contrast, cyclic AMP has no effect on tryptophan hydroxylase mRNA abundance. Experiments using cycloheximide and actinomycin D demonstrate that cyclic AMP exerts its regulatory effect via posttranslational mechanisms mediated by cyclic AMP-dependent protein kinase. The effect of cyclic AMP is independent of the phase of the photoperiod, suggesting that the nucleotide is not a mediator of the circadian rhythm of tryptophan hydroxylase. Cyclic AMP accumulation is higher in darkness than in light, as is tryptophan hydroxylase activity. Furthermore, the stimulatory effect of forskolin and that of darkness are inhibited by H89, an inhibitor of cyclic AMP-dependent protein kinase. In conclusion, cyclic AMP may mediate the acute effects of light and darkness on tryptophan hydroxylase activity of retinal photoreceptor cells.

Behavioral evaluation of hemiparkinsonian MPTP monkeys following dopamine pharmacological manipulation and adrenal co-graft transplantation.

Bradykinesia and rigidity are the symptoms that most directly correlate with loss of striatal dopamine in Parkinson's disease. In the hemiparkinsonian (HP) monkey, this is represented by paucity of movement as measured by coli puterized movement analysis, diminished manual dexterity on clinical examination, and diminished performance on operant behavioral tasks. The present study used an MPTP-induced HP model in rhesus monkeys to evaluate the effectiveness of adrenal medullary and peripheral nerve co-grafts in diminishing parkinsonian symptoms. Unoperated controls (N = 4), surgical controls with caudate lesioning (N = 4), and caudate co-grafted (N = 4) HP monkeys demonstrated diminished movement in the home cage following MPTP. This behavior persisted in unoperated controls, but improved in both surgical control and co-grafted monkeys. Functional hand dexterity evaluations demonstrated similar impairment in all three groups but only surgical controls and co-grafted monkeys demonstrated improvement. In general, rotational behavior in response to apomorphine was consistent with recovery of function in surgical controls and co grafted monkeys, but marked between-subject variability precluded group statistical analyses. None of the monkeys could perform the operant task using the affected limb following MPTP. However, the performance of two co-grafted animals demonstrated partial recovery. L-dopa improved operant performance, demonstrating a dopaminergic component to the task. The results demonstrate recovery of behavioral function after surgical treatment, with adrenal co-grafted monkeys showing the greatest degree of improvement.

Serotonin N-acetyltransferase mRNA levels in photoreceptor-enriched chicken retinal cell cultures: elevation by cyclic AMP.

Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is a key regulatory enzyme in the biosynthesis of melatonin. Previous studies have shown that the activity of this enzyme in the chicken retina is regulated by a cyclic AMP-dependent mechanism. In the present report, we investigated whether cyclic AMP can regulate the levels of AA-NAT mRNA in photoreceptor-enriched chick retinal cell cultures. AA-NAT mRNA levels were elevated by acute treatment with cyclic AMP protagonists, including forskolin; this response was blocked by H-89, a selective inhibitor of cyclic AMP-dependent protein kinase. Forskolin did not alter the rate of disappearance of AA-NAT mRNA in actinomycin D-treated cells, suggesting that cyclic AMP enhances transcription of the AA-NAT gene. Forskolin-induced elevation of AA-NAT mRNA levels was enhanced by cycloheximide, which decreased the degradation of the transcript in cells treated with actinomycin D. These studies indicate that the abundance of AA-NAT mRNA is regulated in part through a cyclic AMP-dependent mechanism.

Effects of muscarinic cholinergic receptor antagonists on postnatal eye growth of rhesus monkeys.

PURPOSE: To study a potential role for muscarinic receptors in the inhibition of deprivation-induced excessive axial elongation and myopia in a monkey model. METHODS: The right eyes of 20 newborn rhesus monkeys were occluded with a black contact lens. In seven monkeys each, either atropine or pirenzepine was topically applied daily to the occluded eyes. The nonoccluded fellow eyes and both the occluded and nonoccluded fellow eyes of another six monkeys were treated with vehicle solution. RESULTS: After 33 to 39 weeks, in 5 monkeys of the vehicle group, occluded eyes were longer and the myopic shift significantly greater than in the nonoccluded fellow eyes. In six atropine-treated monkeys, axial length and reduction of the initial hyperopia of occluded and nonoccluded fellow eyes were not different statistically. The myopic shift of the occluded eyes was significantly smaller than in the vehicle-treated occluded eyes. In the pirenzepine-treated group, axial length of the occluded eyes was similar to the nonoccluded eyes of controls and the occluded eyes of atropine-treated monkeys. There was a trend of pirenzepine to reduce the myopic shift of the occluded eye. No effect of atropine or pirenzepine was noted on muscarinic receptor density in retina, brain, or heart, but a small increase was observed in iris + ciliary body. CONCLUSIONS: The drug treatment results implicate muscarinic receptors in postnatal eye growth regulation. Because of interanimal differences our data do not indicate whether nonselective or selective muscarinic blockade is more effective in reducing deprivation-induced myopia.

Diurnal rhythms of tryptophan hydroxylase activity in Xenopus laevis retina: opposing phases in photoreceptors and inner retinal neurons.

Tryptophan hydroxylase (TPH) is the first enzyme in the biosynthetic pathways of melatonin in photoreceptor cells and of serotonin in amacrine cells. To assess the regulation of TPH activity in photoreceptor cells, we pretreated retinas with kainic acid. The neurotoxin selectively killed inner retinal neurons while sparing photoreceptors. TPH activity in both control and kainate-treated retinas undergoes a day-night rhythm. The rhythms in both preparations fit sinusoidal functions. However, the rhythm in intact retinas peaks at midday while that in kainate-lesioned retinas does so at midnight. The daily rhythm of tryptophan hydroxylase activity in photoreceptors parallels that of melatonin release. Comparing the mean level of activity in rhythms of intact and lesioned retinas, we calculate that the TPH activity in photoreceptors represents 24% of the total activity. Therefore, the TPH activity measured in intact retinas reflects mainly the enzymatic activity in serotonergic neurons, masking that from photoreceptors. In contrast, the levels and diurnal variation of TPH mRNA did not differ in intact and kainate-lesioned retinas indicating that measurements of TPH mRNA content reflect primarily that in photoreceptor cells. Thus, TPH mRNA levels and enzyme activity are differentially regulated in amacrine neurons and photoreceptor cells. This differential regulation markedly impacts the patterns of daily rhythms observed in the intact retina.

Dopamine mediates circadian rhythms of rod-cone dominance in the Japanese quail retina.

A circadian clock modulates the functional organization of the Japanese quail retina. Under conditions of constant darkness, rods dominate electroretinogram (ERG) b-wave responses at night, and cones dominate them during the day, yielding a circadian rhythm in retinal sensitivity and rod-cone dominance. The activity of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, also exhibits a circadian rhythm in the retina with approximately threefold higher levels during the day than at night. The rhythm of tyrosine hydroxylase activity is opposite in phase to the circadian activity of tryptophan hydroxylase, the first enzyme in the melatonin biosynthetic pathway. We tested whether dopamine may be related to the physiological rhythms of the retina by examining the actions of pharmacological agents that effect dopamine receptors. We found that blocking dopamine D2 receptors in the retina during the day mimics the nighttime state by increasing the amplitude of the b-wave and shifting the retina to rod dominance. Conversely, activating D2 receptors at night mimics the daytime state by decreasing the amplitude of the b-wave and shifting the retina to cone dominance. A selective antagonist for D1 dopamine receptors has no effect on retinal sensitivity or rod-cone dominance. Reducing retinal dopamine partially abolishes rhythms in sensitivity and yields a rod-dominated retina regardless of the time of day. These results suggest that dopamine, under the control of a circadian oscillator, has a key role in modulating sensitivity and rod-cone dominance in the Japanese quail retina.

Intracellular Ca2+ concentrations in cultured chicken photoreceptor cells: sustained elevation in depolarized cells and the role of dihydropyridine-sensitive Ca2+ channels.

PURPOSE: Retinal photoreceptor cells are tonically depolarized in darkness. Ca2+ influx in darkness plays a critical role in the regulation of neurotransmitter release and melatonin synthesis in these sensory cells. The purpose of the present study was to examine the dynamic changes of intracellular Ca2+ concentrations ([Ca2+]in ) in response to a tonic depolarizing stimulus and to determine the role of dihydropyridine-sensitive calcium channels in the response. METHODS: Photoreceptor cells were prepared from embryonic chick retina and cultured for 6-12 days. Cells were depolarized by exposure to 35 mM extracellular K+. [Ca2+]in of individual photoreceptor cell bodies/synaptic terminals was determined by ratiometric fura-2 image analysis. RESULTS: Chemical depolarization with 35 mM [K+]out greatly increased [Ca2+]in of inner segment/synaptic terminal regions of photoreceptors. The increase usually reached a plateau after the first few minutes of stimulation and was sustained for prolonged periods (>2 h) in the presence of high K+. When the extracellular K+ concentration was reduced, the [Ca2+]in rapidly returned to the basal level. Substitution of 1 mM CoCl2 for CaCl2 in the superfusion medium rapidly and reversibly reduced the [Ca2+]in of depolarized photoreceptor cells. Antagonists of L-type Ca2+ channels, nitrendipine and nifedipine, inhibited the K+-evoked increase of [Ca2+]in. Bay K 8644, a dihydropyridine Ca2+ channel agonist, potentiated the increase of [Ca2+]in elicited by high K+. In some cells, Bay K 8644 alone increased [Ca2+]in under basal conditions. CONCLUSIONS: The increase of [Ca2+]in elicited by depolarization with 35 mM extracellular K+ is due to influx of calcium through the dihydropyridine-sensitive voltage-gated channels. Intracellular [Ca2+] remains elevated for extended periods of time during tonic depolarization. This sustained response requires continuous Ca2+ channel activity.

The melatonin antagonist luzindole protects retinal photoreceptors from light damage in the rat.

PURPOSE: Systemic administration of melatonin can increase retinal light damage in the rat. The role of retinal melatonin receptors in modulating light-damage susceptibility was investigated by intravitreally injecting the melatonin receptor antagonist luzindole into rats. METHODS: Nine Sprague-Dawley albino rats 8 to 9 weeks of age were kept in 50 lux cyclic light for at least 7 days before receiving an intravitreal injection of 1 microl 1 mM luzindole in one eye and 1 microl vehicle in the other eye. The injection was given just before the beginning of the normal 12-hour dark phase. At the end of this dark period, animals were exposed to constant light of 2500 lux for 48 hours. Animals were returned to dim cyclic light for 7 days, and dark-adapted electroretinograms (ERGs) were then recorded from the two eyes simultaneously. The eyes were processed for retinal morphology. Photoreceptor nuclei were counted in the outer nuclear layer (ONL), and the thickness of the ONL and that of the rod outer-segment plus inner-segment layer were measured at several points along sections through the vertical meridian. Two age-matched control rats were maintained in dim cyclic light but received no injections. RESULTS: Luzindole-treated eyes had ERG b-wave thresholds of 2.7 +/- 0.5 (mean +/- SEM) log candela (cd)/m2 lower than the fellow eyes injected with vehicle (P < 0.001), and the maximum b-wave amplitude was 1.0 +/- 0.2 log microV greater in luzindole-treated eyes (P < 0.001). Thresholds of the scotopic threshold response were 0.5 +/- 0.1 log cd/m2 lower than those in vehicle-injected eyes (P < 0.05). Luzindole-treated eyes on average had twice as many photoreceptor cells remaining (P < 0.005). In some areas, several rows of photoreceptor nuclei and outer segments remained in the luzindole-treated eye, whereas the fellow control eye showed cells only occasionally and no outer segments. CONCLUSIONS: Eyes pretreated with the melatonin receptor competitive antagonist luzindole before the dark phase preceding constant light exposure were substantially protected from light damage to the retinal photoreceptors. These results implicate the intraocular melatonin-dopamine system in the regulation of light-damage susceptibility.

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.