L-Dopa and the albino riddle: content of L-Dopa in the developing retina of pigmented and albino mice.

BACKGROUND: The absence or deficiency of melanin as in albinos, has detrimental effects on retinal development that include aberrant axonal projections from eye to brain and impaired vision. In pigmented retinal pigment epithelium (RPE), dihydroxyphenalanine (L-Dopa), an intermediate in the synthetic path for melanin, has been hypothesized to regulate the tempo of neurogenesis. The time course of expression of retinal L-Dopa, whether it is harbored exclusively in the RPE, the extent of deficiency in albinos compared to isogenic controls, and whether L-Dopa can be restored if exogenously delivered to the albino have been unknown. METHODOLOGY/ PRINCIPAL FINDINGS: L-Dopa and catecholamines including dopamine extracted from retinas of pigmented (C57BL/6J) and congenic albino (C57BL/6J-tyr(c2j) ) mice, were measured throughout development beginning at E10.5 and at maturity. L-Dopa, but not dopamine nor any other catecholamine, appears in pigmented retina as soon as tyrosinase is expressed in RPE at E10.5. In pigmented retina, L-Dopa content increases throughout pre- and postnatal development until the end of the first postnatal month after which it declines sharply. This time course reflects the onset and completion of retinal development. L-Dopa is absent from embryonic albino retina and is greatly reduced in postnatal albino retina compared to pigmented retina. Dopamine is undetectable in both albino and pigmented retinas until after the postnatal expression of the neuronal enzyme tyrosine hydroxylase. If provided to pregnant albino mothers, L-Dopa accumulates in the RPE of the fetuses. CONCLUSIONS: L-Dopa in pigmented RPE is most abundant during development after which content declines. This L-Dopa is not converted to dopamine. L-Dopa is absent or at low levels in albino retina and can be restored to the RPE by administration in utero. These findings further implicate L-Dopa as a factor in the RPE that could influence development, and demonstrate that administration of L-Dopa could be a means to rescue developmental abnormalities characteristic of albinos.

High oxygen prevents fetal lethality due to lack of catecholamines.

The catecholamine norepinephrine is required for fetal survival, but its essential function is unknown. When catecholamine-deficient [tyrosine hydroxylase (Th) null] mouse fetuses die at embryonic day (E)13.5-14.5, they resemble wild-type (wt) fetuses exposed to hypoxia. They exhibit bradycardia (28% reduction in heart rate), thin ventricular myocardium (20% reduction in tissue), epicardial detachment, and death with vascular congestion, hemorrhage, and edema. At E12.5, before the appearance of morphological deficits, catecholamine-deficient fetuses are preferentially killed by experimentally induced hypoxia and have lower tissue Po(2) levels than wt siblings. By microarray analysis (http://www.ncbi.nlm.nih.gov/geo; accession no. GSE10341), hypoxia-inducible factor-1 target genes are induced to a greater extent in null fetuses than in wt siblings, supporting the notion that mutants experience lower oxygen tension or have an enhanced response to hypoxia. Hypoxia induces a 13-fold increase in plasma norepinephrine levels, which would be expected to increase heart rate, thereby improving oxygen delivery in wt mice. Surprisingly, increasing maternal oxygen (inspired O(2) 33 or 63%) prevents the effects of catecholamine deficiency, restoring heart rate, myocardial tissue, and survival of Th null fetuses to wt levels. We suggest that norepinephrine mediates fetal survival by maintaining oxygen homeostasis.

Severe deficits in 5-HT2A -mediated neurotransmission in BDNF conditional mutant mice.

BDNF is thought to provide critical trophic support for serotonin neurons. In order to determine postnatal effects of BDNF on the serotonin system, we examined a line of conditional mutant mice that have normal brain content of BDNF during prenatal development but later depletion of this neurotrophin in the postnatal period. These mice show a behavioral phenotype that suggests serotonin dysregulation. However, as shown here, the presynaptic serotonin system in the adult conditional mutant mice appeared surprisingly normal from histological, biochemical, and electrophysiological perspectives. By contrast, a dramatic and unexpected postsynaptic 5-HT2A deficit in the mutant mice was found. Electrophysiologically, serotonin neurons appeared near normal except, most notably, for an almost complete absence of expected 5-HT2A -mediated glutamate and GABA postsynaptic potentials normally displayed by these neurons. Further analysis showed that BDNF mutants had much reduced 5-HT2A receptor protein in dorsal raphe nucleus and a similar deficit in prefrontal cortex, a region that normally shows a high level of 5-HT2A receptor expression. Recordings in prefrontal slice showed a marked deficit in 5-HT2A -mediated excitatory postsynaptic currents, similar to that seen in the dorsal raphe. These findings suggest that postnatal levels of BDNF play a relatively limited role in maintaining presynaptic aspects of the serotonin system and a much greater role in maintaining postsynaptic 5-HT2A and possibly other receptors than previously suspected.

Tyrosinase: a developmentally specific major determinant of peripheral dopamine.

L-3,4-dihydroxyphenylalanine, the immediate precursor of dopamine, can be formed by two enzymes: tyrosine hydroxylase (TH) in catecholamine-producing neurons and chromaffin cells and tyrosinase in melanocytes. In this study we examined whether tyrosinase contributes to production of dopamine. Deficiency of TH caused marked reductions in norepinephrine in albino and pigmented 15-day-old mice. In contrast, peripheral levels of dopamine were reduced only in albino TH-deficient mice and were higher in pigmented than in albino mice, regardless of the presence or absence of TH. We next examined age-related changes in dopamine and cutaneous expression of tyrosinase and melanin in albino and pigmented TH wild-type mice. We found that the differences in peripheral dopamine between pigmented and albino mice disappeared with advancing age following changes in expression and function of tyrosinase. In young animals, tyrosinase was present in epidermis but did not produce detectable melanin. With advancing age, tyrosinase was localized only around hair follicles, melanin synthesis became more pronounced, and dopamine synthesis decreased. The data reveal a previously unrecognized TH-independent major pathway of peripheral dopamine synthesis in young, but not adult, mice. The transient nature of this source of dopamine reflects a developmental switch in tyrosinase-dependent production of dopamine to production of melanin.

Catecholamines act via a beta-adrenergic receptor to maintain fetal heart rate and survival.

Mice lacking catecholamines die before birth, some with cardiovascular abnormalities. To investigate the role of catecholamines in development, embryonic day 12.5 (E12.5) fetuses were cultured and heart rate monitored. Under optimal oxygenation, wild-type and catecholamine-deficient fetuses had the same initial heart rate (200-220 beats/min), which decreased by 15% in wild-type fetuses during 50 min of culture. During the same culture period, catecholamine-deficient fetuses dropped their heart rate by 35%. Hypoxia reduced heart rate of wild-type fetuses by 35-40% in culture and by 20% in utero, assessed by echocardiography. However, catecholamine-deficient fetuses exhibited greater hypoxia-induced bradycardia, reducing their heart rate by 70-75% in culture. Isoproterenol, a beta-adrenergic receptor (beta-AR) agonist, reversed this extreme bradycardia, restoring the rate of catecholamine-deficient fetuses to that of nonmutant siblings. Moreover, isoproterenol rescued 100% of catecholamine-deficient pups to birth in a dose-dependent, stereo-specific manner when administered in the dam's drinking water. An alpha-AR agonist was without effect. When wild-type fetuses were cultured with adrenoreceptor antagonists to create pharmacological nulls, blockade of alpha-ARs with 10 microM phentolamine or beta-ARs with 10 microM bupranolol alone or in combination did not reduce heart rate under optimal oxygenation. However, when combined with hypoxia, beta-AR blockade reduced heart rate by 35%. In contrast, the muscarinic blocker atropine and the alpha-AR antagonist phentolamine had no effect. These data suggest that beta-ARs mediate survival in vivo and regulate heart rate in culture. We hypothesize that norepinephrine, acting through beta-ARs, maintains fetal heart rate during periods of transient hypoxia that occur throughout gestation, and that catecholamine-deficient fetuses die because they cannot withstand hypoxia-induced bradycardia.

Inhibition of caspases protects cerebellar granule cells of the weaver mouse from apoptosis and improves behavioral phenotype.

The homozygous mouse mutant weaver exhibits a massive loss of cerebellar granule neurons postnatally. The death of these cells is associated with a single amino acid mutation in the G protein-activated inwardly rectifying potassium channel, Girk2. Evidence suggests that both the mutated Girk2 channel and the calcium channel-associated N-methyl-d-aspartate receptor play important roles in the apoptotic death of weaver cerebellar granule cells, but the downstream events associated with this process are unknown. In this study, we demonstrate that the consequences of the mutation result in caspase activation. In addition, our results show that caspase inhibition in vivo decreases caspase activation and granule cell apoptosis and significantly improves behavioral deficits associated with the weaver's phenotype.