Suzanne Roffler-Tarlov

3,4-dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid in the mouse striatum: a reflection of intra- and extra-neuronal metabolism of dopamine?

1 The administration of probenecid to mice increased the concentration of 4‐hydroxy‐3‐methoxyphenylacetic acid (HVA) in the striatum, but did not raise the concentration of 3,4‐dihydroxyphenylacetic acid (DOPAC). 2 After drug treatments which normally increase the concentration of HVA several‐fold, inhibition of catechol‐O‐methyltransferase (COMT) by tropolone greatly reduced the concentration of HVA but resulted in only a small increase in the concentration of DOPAC in the striatum of the mouse. 3 These results indicate that HVA and DOPAC do not occur at the same location in the tissue of the striatum and that DOPAC is not normally metabolized to HVA to any great extent in this tissue. 4 When mice were treated with reserpine, which is thought to prevent the intraneuronal storage of dopamine, there was an increase in the striatal concentration of DOPAC which preceded an increase in the concentration of HVA. Since non‐cholinergic nerve endings of rat brain contain mitochondria and show monoamine oxidase activity, this result suggests that DOPAC is formed intraneuronally. 5 It is concluded that the DOPAC in the striatum represents intraneuronal metabolism of dopamine and that only the HVA which is sensitive to the action of probenecid represents entirely extraneuronal metabolism of this amine. Some of the HVA is not sensitive to the action of probenecid. This suggests that part of the metabolism of dopamine involves both locations. 6 A group of drugs which are chemically related to amphetamine were tested for their effects on the concentrations of DOPAC and HVA in the striatum. It is suggested that d‐amphetamine, 2‐aminotetralin and 1,2,3,4‐tetrahydroisoquinoline reduced the intraneuronal metabolism of dopamine whereas adamantanamine did not.

Catecholamine Synthesis is Mediated by Tyrosinase in the Absence of Tyrosine Hydroxylase

Catecholamine neurotransmitters are synthesized by hydroxylation of tyrosine to l-dihydroxyphenylalanine (l-Dopa) by tyrosine hydroxylase (TH). The elimination of TH in both pigmented and albino mice described here, like pigmented TH-null mice reported previously (Kobayashi et al., 1995; Zhou et al., 1995), demonstrates the unequivocal requirement for catecholamines during embryonic development. Although the lack of TH is fatal, TH-null embryos can be rescued by administration of catecholamine precursors to pregnant dams. Once born, TH-null pups can survive without further treatment until weaning. Given the relatively rapid half-life of catecholamines, we expected to find none in postnatal TH-null pups. Despite the fact that the TH-null pups lack TH and have not been supplemented with catecholamine precursers, catecholamines are readily detected in our pigmented line of TH-null mice by glyoxylic acid-induced histofluorescence at postnatal day 7 (P7) and P15 and quantitatively at P15 in sympathetically innervated peripheral organs, in sympathetic ganglia, in adrenal glands, and in brains. Between 2 and 22% of wild-type catecholamine concentrations are found in these tissues in mutant pigmented mice. To ascertain the source of the catecholamine, we examined postnatal TH-null albino mice that lack tyrosinase, another enzyme that converts tyrosine to l-Dopa but does so during melanin synthesis. In contrast to the pigmented TH-null mice, catecholamine histofluorescence is undetectable in postnatal albino mutants, and the catecholamine content of TH-null pups lacking tyrosinase is 18% or less than that of TH-null mice with tyrosinase. Thus, these extraordinary circumstances reveal that tyrosinase serves as an alternative pathway to supply catecholamines.

Concentrations of glutamic acid in cerebellar cortex and deep nuclei of normal mice and Weaver, Staggerer and nervous mutants.

The concentrations of several free amino acids including glutamate were measured in cerebellar cortex and deep nuclei, and in cerebral cortex, from three neurological mutant mice, two of which lose most granule cell neurons in the cerebellar cortex (Weaver and Staggerer), and one of which loses cerebellar Purkinje cells (nervous). (1) In Weaver and Staggerer, glutamate concentration was reduced to less than two-thirds of control values in both cerebellar cortex, where granule cells with all their axonal and dendritic processes reside, and in cerebellar deep nuclei where no granule cells are found. (2) No other amino acids, including aspartate, were significantly reduced in cerebellar cortex and deep nuclei of either granuloprival mutant at 3 weeks of age. (3) Glutamate concentration was normal in the cerebral cortex of Weaver and Staggerer mice. (4) Glutamate concentration was normal in cerebella of heterozygous Weaver animals, in which 10–20% of the granule cells are missing. (5) Glutamate was not reduced in either cerebellar cortex or deep nuclei of the Purkinje cell deficient mutant, nervous; only GABA, the Purkinje cell transmitter, was reduced significantly and only in the deep nuclei. (6) An incidental finding was that the Staggerer mutation, previously recognized to modify CNS structure only in the cerebellar cortex, causes a reduction of deep nuclear weight and protein to 30% of normal. No reductions were found in weight or protein in the deep nuclei of Weaver mutant mice. We conclude that glutamate reduction in an area of cerebellum distant from granule cells and their processes cannot be explained by the absence of granule cells alone, and may indicate that a glutamate-utilizing component of cerebellar cortex and deep nuclei has become modified secondary to the granule cell deficit.

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.—Eisenhofer, G., Tian, H., Holmes, C., Matsunaga, J., Roffler‐Tarlov, S., Hearing, V.J. Tyrosinase: a developmentally specific major determinant of peripheral dopamine. FASEB J. 17, 1248–1255 (2003)

Neurochemical and morphological consequences of axon terminal degeneration in cerebellar deep nuclei of mice with inherited Purkinje cell degeneration.

The concentrations of free amino acids and the activities of transmitter-related enzymes, glutamic acid decarboxylase (GAD), choline acetylase (ChAC) and GABA-transaminase (GABA-t) were measured in cerebellar cortex and deep cerebellar nuclei from the mouse mutant Purkinje cell degeneration (pcd) at various times before and after Purkinje cell loss. Axosomatic synapses on target cells in pcd deep nuclei were quantified by electron microscopy during and after degeneration. The concentration of GABA (nmol/mg wet weight), the Purkinje cell transmitter, was normal in pcd cerebellar cortex and deep nuclei before onset of Purkinje cell degeneration on postnatal day 15. Just after the major period of Purkinje cell loss in cerebellar cortex, GABA concentration was unchanged in the cortical layers but fell to 50% of normal values in the deep nuclei of pcd animals killed either by decapitation or by microwave irradiation. No other measured free amino acid decreased. There were no long-term increases following Purkinje cell degeneration in the concentration of any transmitter amino acids or related enzymes, GAD, ChAC or GABA-t, and thus no indication of axonal sprouting reactions. Progressive losses occurred in wet weight and protein and in activity of GABA-t in both the cerebellar cortex and the deep nuclei of pcd animals. Electron microscopic analysis indicated that Purkinje cell axon terminals contact 30% or more of the somatic surface of principal neurons of the lateral nucleus of the normal cerebellum, but only about 2% of the corresponding sites in the pcd cerebellum. Glial leaflets, rather than other synaptic terminals take their place. Axon terminals may degenerate earlier than Purkinje somata in the pcd disease.

Comparison of structural and stereoisomers of apomorphine on stereotyped sniffing behavior of the rat

N-n-propylnorapomorphine (NPA) is 35 times more potent than apomorphine (APO) in producing stereotyped behavior in rats. The effect of NPA is blocked by perphenazine but unaltered by alpha-methyltyrosine pretreatment and is accompanied by a decrease in central dopamine turnover. The activity of APO and NPA appears to be primarily in the (-)-isomer, and is diminished but not lost by removal of either hydroxyl group from the catechol ring system.

Quantitative examination of the deep cerebellar nuclei in the staggerer mutant mouse.

Quantitative morphological techniques have revealed several new aspects of the action of the Staggerer mutant gene. Staggerer is an autosomal recessive gene which causes ataxia and severe malformation of the cerebellar cortex in mice. The Purkinje cells of the cerebellar cortex are small, abnormal in morphology and reduced in numbers. The close synaptic and developmental relationship of Purkinje cells with the cells of the deep cerebellar nuclei (dcn) lead us to look for effects of the Staggerer mutation on the dcn neurons. The volume of the deep nuclear region is shrunken in Staggerer and there is a reduction in the volume of the white matter. These findings account for the reduced wet weights and protein concentration found by Roffler-Tarlov and Sidman. In contrast to the cells of the cortex, where 75% of the medium-to-large neurons are missing, the number of cells present in Staggerer dcn is identical to wild-type. The dcn neurons are not completely spared, however. Measurements of cross-sectional cell area revealed a 30% shrinkage of neurons in Staggerer dcn. The most likely interpretation of previous work and the current findings is that the Staggerer gene acts early in development but exerts its effects directly only on those derivatives of the ventricular zone in the roof of the fourth ventricle which are destined to become Purkinje and Golgi cells.

DNA regulatory sequences of the rat tyrosine hydroxylase gene direct correct catecholaminergic cell-type specificity of a human growth hormone reporter in the CNS of transgenic mice causing a dwarf phenotype

Transgenic mice bearing 4.8 kilobases (kb) of upstream rat tyrosine hydroxylase (TH) sequences linked to a human growth hormone gene (hGH) exhibited cell-specific expression of hGH in all the appropriate catecholaminergic neurons in the central nervous system (CNS), although with different penetrance in two different mouse lineages. No ectopic expression was observed in any brain or peripheral region in one founder and its progeny. In another founder there was some ectopic expression in addition to appropriate and high levels of tissue-specific expression in all catecholaminergic areas. These results identify regulatory sequences that are sufficient for targeting expression to all catecholaminergic CNS neurons. Also, expression of exogenous hGH in the hypothalamus caused a dwarf phenotype, generating a novel genetic model for GH deficiency of hypothalamic origin.

The content of amino acids in the developing cerebellar cortex and deep cerebellar nuclei of granule cell deficient mutant mice.

Glutamic acid is the only free amino acid to be clearly reduced in mature granule cell deficient cerebellum. The correlation between concentration of glutamic acid and extent of granule cell loss suggests that it may serve as a neurotransmitter. Curiously, in two neurological mouse mutants, glutamic acid is also decreased in the deep cerebellar nuclei where there are no granule cells. We have now examined the amino acid content of cerebellar cortex and deep cerebellar nuclei of the granule cell deficient mutants, weaver and staggerer, during the postnatal period in which granule cell development takes place. We have found: (1) an early and transient deficit in taurine in weaver cerebellar cortex during the period of granule cell migration, (2) deficits during the second postnatal week in taurine, aspartic and glutamic acids in both weaver and staggerer cerebellar cortex, (3) that aspartic and glutamic acid deficits result from failure to increase concentrations at the normal rate after birth rather than from a fall from normal levels, (4) decreased concentrations of glutamic acid but not of taurine and aspartic acids apparent in the deep nuclei of both weaver and staggerer at about the same time as in cerebellar cortex, (5) amino acid changes in weaver heterozygote cerebellum which result in values intermediate in magnitude between normal and homozygous weaver animals and (6) an early and persistent reduction in staggerer deep nuclei of gamma-aminobutyric acid (GABA), the Purkinje cell transmitter, indicating early denervation or lack of full innervation of deep nuclei by Purkinje cells.

Effects of Purkinje cell degeneration on the noradrenergic projection to mouse cerebellar cortex.

We have examined the effects of a genetically programmed target cell death on the noradrenergic afferent projection to mouse cerebellar cortex. We have observed that the noradrenergic axon terminals originating in in locus coeruleus are maintained in the cerebellar cortex of the Purkinje cell degeneration (pcd) mutant mouse in spite of the absence of Purkinje cells, the targets for the noradrenergic projection. The number of noradrenergic terminals in the atrophic mutant cerebellar cortex is approximately normal as assessed by counts of fibers exhibiting catecholamine fluorescence and by measurement of high affinity uptake of tritium-labeled norepinephrine (NE) by synaptosomes prepared from cerebellar cortex. An increased density of NE fibers is observed which appears to be a consequence of reduced cerebellar mass in the mutant. Although the number of noradrenergic terminals is unaffected, morphological and biochemical alterations are observed in this system. The fibers are more brightly fluorescent in mutant than in normal mice and their pattern is less orderly. The content of the endogenous transmitter, NE, is increased from 150 to 170% whereas the activity of the rate-limiting enzyme tyrosine hydroxylase (TH) is reduced to about 60% of normal values. These changes appear to be permanent as they are still present in 6 month-old mutant animals, the oldest studied. No alterations in either NE content or TH activity are found in pcd/pcd hippocampus, another target for the locus coeruleus axons.