Retinal TUNEL-positive cells and high glutamate levels in vitreous humor of mutant quail with a glaucoma-like disorder.

PURPOSE: To investigate whether retinal cell death observed in an avian glaucoma-like disorder occurs by apoptosis and whether an increase in excitotoxic amino acid concentration in the vitreous humor is associated temporally with cell death in the retina. METHODS: Presumptive retinal apoptotic nuclei were identified by histochemical detection of DNA fragmentation (by TdT-dUTP terminal nick-end labeling [TUNEL]), and vitreal concentrations of glutamate and several other amino acids were determined by high-pressure liquid chromatography with fluorometric detection in the al mutant quail (Coturnix coturnix japonica) in which a glaucoma-like disorder develops spontaneously. RESULTS: TUNEL-labeled nuclei were located mostly in the ganglion cell layer (GCL) in the retina of mutant quails 3 months after hatching. However, labeled nuclei were also observed in the inner and outer nuclear layers. At 7 months, most TUNEL-positive nuclei were detected in the inner nuclear layer, whereas labeled cells in the GCL were reduced in number. No TUNEL-labeled nuclei were detected in the retina of control quails at any age. Vitreal concentrations of glutamate and aspartate were significantly increased in 1-month-old mutant quails compared with control animals. Concentrations decreased at 3 months, and no significant differences were observed between strains at 7 months. CONCLUSIONS: Presumptive apoptotic cell death is detected from 3 months after hatching in mutant quails and is not restricted to retinal ganglion cells. Cell death appears just after a significant increase in excitotoxic amino acid concentrations in the vitreous humor, suggesting a correlation between both events.

Day and night dysfunction in intraretinal melatonin and related indoleamines metabolism, correlated with the development of glaucoma-like disorder in an avian model.

As previous studies have suggested that melatonin and serotonin may be involved in the regulation of intraocular pressure, retinal concentrations of melatonin, 5-HT, and related indoleamines measured at day and at night were studied during the development of a glaucoma-like disorder with increased intraocular pressure in the al mutant quail. Indoleamine levels were determined by HPLC with electrochemical detection in 1-month-, 3-month-, and 7-month-old al mutant and control quails. Morphology and numbers of melatonin-synthesizing and 5-HT-containing cells, labelled immunohistochemically with an anti-hydroxyindol-0-methyltransferase (HIOMT) antibody and an anti-5-HT antibody, respectively, were studied. Major findings were that: (1) no significant changes in morphology of melatonin-synthesizing cells or in the morphology and density of 5-HT-containing amacrine cells were observed during the development of glaucoma: (2) 5-HT metabolism was modified during the night at 1 month of age and during the day after 3 months; and (3) melatonin metabolism was modified during the night at 7 months and during the day after 3 months. These results demonstrate a relationship between the temporal evolution of this avian glaucoma and a dysfunction in indoleamine retinal metabolism.

Determination of melatonin in rat pineal, plasma and retina by high-performance liquid chromatography with electrochemical detection.

A sensitive method for the routine measurement of endogenous melatonin (MEL) in pineal, retina and plasma rat tissues has been developed using reversed-phase high-performance liquid chromatography with electrochemical detection. Quantification limit for MEL was 0.2 ng/mg protein in pineal, 15 pg/ml in plasma and 2.0 pg/mg protein in retina. To improve both MEL quantification and the reproducibility of the assay, an internal standard was used when an extraction in organic solvent was required, in contrast with other available chromatographic methods. MEL values and the circadian profile obtained in this study from both rat pineal and plasma agree with those reported previously. This method allows MEL detection in mammal retina, particularly in rat, where MEL levels are very low.

Dopamine inhibits melatonin synthesis in photoreceptor cells through a D2-like receptor subtype in the rat retina: biochemical and histochemical evidence.

An intrinsic oscillator, using dopamine and melatonin as antagonist signals, controls rhythmic events in the retina of nonmammals. The purpose of the present work was to localize and characterize a dopamine receptor responsible for the nocturnal inhibition of melatonin synthesis in photoreceptor cells in a mammalian retina. An antibody against the D2 receptor stained photoreceptor cell inner segments of the rat retina. alpha-Methyl-p-tyrosine, a competitive inhibitor of tyrosine hydroxylase, enhanced the nocturnal content of melatonin, suggesting the dopamine control of melatonin synthesis as in non-mammals. Clozapine, a D2c/D4 antagonist, also enhanced the nocturnal level of melatonin, whereas raclopride, a D2A antagonist, did not. Taken together, these results support the control of melatonin levels by dopamine through a D2C/D4 receptor in photoreceptor cells of the rat retina. The presence of D4 receptors in the rat retina was confirmed by reverse transcription-PCR.

Changes in retinal dopaminergic cells and dopamine rhythmic metabolism during the development of a glaucoma-like disorder in quails.

PURPOSE: To examine the possible correlation between a dysfunction of the daily rhythm of retinal dopamine (DA) and the development of a glaucoma-like disorder in an animal model, the al mutant quail (Coturnix coturnix japonica). METHODS: The morphology and density of DA-containing cells labeled immunohistochemically with an anti-tyrosine hydroxylase (TH) antibody were correlated with the diurnal and nocturnal contents of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) determined by high-pressure liquid chromatography with electrochemical detection. RESULTS: The number of TH-immunoreactive cells was lower than normal in mutant quails suffering from the disorder. There were considerably fewer cells in the central retina, and the DA metabolism was reduced in parallel. The nocturnal DA content was lower than the diurnal level in normal quails, but there was no such circadian fluctuation in mutant quails. CONCLUSIONS: This glaucoma-like disorder in quails is correlated with the degeneration of DA-containing amacrine cells and a dysfunction of the circadian rhythmicity of DA synthesis.

Structure-activity relationships for substrates and inhibitors of pineal 5-hydroxytryptamine-N-acetyltransferase: preliminary studies.

Tryptamine, (1-naphthyl)ethylamine and phenethylamine derivatives were tested as substrates of ovine pineal serotonin-N-acetyl transferase (5-HT-NAT), a key enzyme involved in the synthesis of melatonin. Almost all of the indole derivatives possessed affinity similar to that of tryptamine (Km = 0.05 mM), while the substituted naphthalene and phenyl derivatives were less potent. However, the Km values seem be influenced by the steric hindrance and polar properties of the substituent. Vmax values for the naphthyl and phenyl derivatives were generally 10-20-fold higher than those of the indole derivatives and no clear structure-activity relationship was observed. Melatonin and several bioisosteric derivatives were shown to be inhibitors of 5-HT-N-acetyltransferase. Preliminary data suggested that over the 5-50-microM concentration range, melatonin was a competitive inhibitor (IC50 = 10 microM) with a concentration-dependent inhibitory effect on its own synthesis in the pineal gland. However, the bioisosteric naphthalene derivatives were characterized instead as mixed inhibitors. (1-Napthyl)ethylacetamido, a putative melatoninergic antagonist, was also shown to be an inhibitor of 5-HT-N-acetyltransferase (IC50 = 8 microM) and is a promising tool for the regulation of melatonin synthesis and the understanding of its role.

6-Fluoro-serotonin as a substrate for the neuronal serotonin transporter.

6-Fluoro-serotonin (6F-5-HT) was previously identified in the rat brain after peripheral administration of 6-fluoro-DL-tryptophan, a serotonin (5-HT) synthesis inhibitor. These present studies, performed with rat brain synaptosomes show that: i-neuronal 6F-5-HT uptake partly involved the 5-HT transporter since it was inhibited by clomipramine, a 5-HT uptake inhibitor, ii-6F-5-HT blocked the synaptosomal uptake of 3H-5-HT, with an IC50 value of 98 +/- 13 nM, and iii- 6F-5-HT induced 3H-5-HT release from preloaded synaptosomes, with an EC50 value of 95 +/- 6 nM; this release was decreased in the presence of clomipramine, suggesting the involvement of the 5-HT transporter. This release was also reduced when using synaptosomes from reserpinized rats, suggesting that the vesicular pool also participates to the 3H-5-HT release induced by 6F-5-HT. So, 6F-5-HT behaved as a substrate for the 5-HT neuronal transporter.

Changes in dopamine metabolism in rat forebrain regions after cessation of long-term fluoxetine treatment: relationship with brain concentrations of fluoxetine and norfluoxetine.

We examined the effects of repeated administration of the selective serotonin uptake inhibitor (SSRI) fluoxetine (Flx) (5, 10, or 15 mg/kg i.p., twice daily for 21 days) on brain and plasma concentrations of the parent drug and its active desmethyl metabolite, norfluoxetine (NFlx), in rats during the 21-day regimen as well as after cessation of drug treatment. We also measured dopamine (DA) levels in 2 midbrain regions (the striatum, St and nucleus accumbens, NAc) in rats killed 1-14 days after the last dose. NFlx concentrations in plasma and brain were ten times higher than those of Flx during the period of drug treatment. Although Flx accumulated more markedly in the rat brain than NFlx, it disappeared completely from plasma and brain after treatment stopped, while NFlx persisted up to Day P7. Chronic Flx treatment caused a persistent decrease in brain DA levels of -60% to -70% in St and NAc; this lasted for 7-14 days after cessation of treatment, depending on the dose used. The levels of DA metabolites decreased by 20-40%, and, except for 3-MT, tended to overshoot during the recovery period. Our data suggest that the long-term inhibition of DA neurons after cessation of Flx treatment parallels the inhibition previously observed for 5-HT neurons. Thus, besides blocking 5-HT uptake, Flx is likely to also inhibit in vivo DA uptake in forebrain regions, following prolonged administration.

Metabolism of 6-fluoro-DL-tryptophan and its specific effects on the rat brain serotoninergic pathway.

We administered 6-fluoro-DL-tryptophan (6F-Trp) to rats (50-200 mg/kg i.p.) and evaluated its neurochemical effects on central catechole and indole compounds; we also determined the time course of its action, together with its metabolism and kinetics in four rat brain areas. Neither norepinephrine nor dopamine and its major metabolites were affected by 6F-Trp. With regard to serotonin (5-HT), 6F-Trp induced a transient depletion in all the brain areas studied, with a maximum of about 60-65% obtained between 1 and 3 hr depending on the dose administered. After 6 hr, 5-HT levels generally returned to control values. 5-Hydroxyindolacetic acid (5-HIAA) levels were also reduced 3 hr after administration (-40 to -60%). A large dose-dependent increase in tryptophan (Trp) was observed in the four brain areas, possibly because of an inhibition of Trp incorporation into protein, as suggested by experiments with mouse neuroblastoma cells. The brain elimination half-life of 6F-Trp was estimated at 0.5-1 hr. Regarding 6F-Trp metabolism, three new compounds were detected in all four brain areas after 6F-Trp administration. They were identified by means of liquid chromatography with electrochemical detection and/or radioenzymology, in comparison with fluorinated standards, or after NSD 1015 or pargyline coadministration with 6F-Trp. The first two 6F-Trp metabolites detected were probably 6-fluoro-5-hydroxytryptophan and 6-fluoro-5-HIAA. The third, identified and quantified by means of the two analytical methods, was 6-fluoro-5-HT (6F-5-HT). These findings suggest that 6F-Trp could be used as the in vivo precursor of 6F-5-HT with a view to tracing neuronal serotoninergic pools, as has already been done with platelets.

Dopaminergic interplexiform cells in the retina of pigmented and hypopigmented quails (Coturnix coturnix japonica).

Interplexiform cells (IPCs) have not been previously described as a component of the population of tyrosine hydroxylase (TH) immunoreactive (presumably dopaminergic) cells in the avian retina. In this study, carried out in both pigmented and imperfect albino mutant quails (Coturnix coturnix japonica), we initially describe TH immunoreactive cells in the inner nuclear layer whose internal dendritic arborization extends into strata 1, 3 and 4/5 of the inner plexiform layer. Then, we describe ascending processes arising from the somata or proximal dendrites of these cells. These sclerally directed processes (100-1,000 microns long) run across the inner nuclear layer to terminate within the outer plexiform layer, sometimes even reaching the outer nuclear layer. Hence, the cells bearing such processes correspond well with the definition of IPCs. The number of scleral processes is higher in mutant (48 +/- 19/retina) than in normal (12 +/- 10/retina) quails and are distributed throughout the retina except the area surrounding the pecten. Comparison of biochemical assays for dopamine in the two strains reveals a significantly higher dopamine content in the mutant quails which could be related to its increased number of dopaminergic IPC processes.

Time course of brain serotonin metabolism after cessation of long-term fluoxetine treatment in the rat.

The effects of repeated fluoxetine (Flx) administration (5, 10, or 15 mg/kg i.p., twice daily for 21 days) on serotonin and 5-HIAA metabolism were examined in the hypothalamus, hippocampus, pons medulla and cerebral cortex of rats killed 1-28 days after the last dose. Dose-dependent weight loss was observed during treatment, followed by gradual and complete recovery of body weight over the following two weeks. Chronic Flx treatment caused a dose-dependent decrease in brain 5-HT levels (by between 10 and 50% depending on the region examined), lasting for 3-7 days after cessation of treatment with the lowest and intermediate doses, and for 7-14 days after cessation of the highest dose. 5-HIAA levels decreased more markedly (-20; -60% depending on the region examined) than those of 5-HT, and tended to overshoot during the recovery period. The prolonged reduction in brain 5-HT levels after chronic Flx treatment was similar to that seen in rats given very high doses of dexfenfluramine (d-fen), a drug which both blocks 5-HT uptake and increases its release. These data suggest that brain 5-HT and 5-HIAA depletion may reflect similar dose-related expressions of the drug's mechanisms of action.

Albumin binding and brain uptake of 6-fluoro-DL-tryptophan: competition with L-tryptophan.

We investigated potential competition between L-tryptophan (TRP) and 6-fluoro-DL-tryptophan (6-F-TRP) for binding to albumin and for passage through the blood-brain barrier (BBB). In experiments based on equilibrium dialysis, albumin (600 microM) bound about 80% of TRP and 50% of 6-F-TRP with affinity constants (Ka) of 3.7 +/- 0.04 x 10(4) and 0.62 +/- 0.01 x 10(4) M-1, respectively. Competitive inhibition was assessed as the decrease in the apparent Ka (K' a) of TRP in the presence of 6-F-TRP, with no modification of the N value. Competition between TRP, 6-F-TRP and L-valine (VAL) for passage across the BBB was demonstrated using two approaches. When administered concomitantly with TRP or 6-F-TRP to rats, VAL decreased brain uptake of TRP and 6-F-TRP and reversed their action on serotonin. In Oldendorf's model, 6-F-TRP and VAL decreased the brain uptake of TRP.

In vivo evidence for carrier-mediated brain uptake of a new 2-amino-2-oxazoline (COR3224) via the purine transport system in rat.

We studied the brain uptake of a new 2-amino-2-oxazolamines derivative (COR3224) in the rat by means of the rapid intracarotid injection technique described by Oldendorf. The brain uptake index (BUI) of labelled COR3224 decreased progressively from 10% to 5% when concentrations of unlabelled compound were increased. The effect of various compounds indicated that COR3224 is transported into the brain by the purine carrier. The affinity of COR3224 for this carrier (Km = 5.68 microM) was higher than that of adenine.