Role of convergent activation of glutamatergic and dopaminergic systems in the nucleus accumbens in the development of methamphetamine psychosis and dependence.

Methamphetamine (Meth) abuse can result in long-lasting psychosis and dependence. The nucleus accumbens (NAc), which controls psychomotor and reward behaviours, is an important interface between the limbic system and receives convergent projections from dopaminergic and glutamatergic terminals. This study investigated the involvements of dopaminergic and glutamatergic transmission in the development of Meth psychosis and dependence by using tyrosine hydroxylase heterozygous mutant (TH+/-) mice and N-methyl-d-aspartate receptor knockout (NR2A-/-) mice. Repeated treatment with Meth (1 mg/kg s.c.) for 7 d in wild-type mice led to the development of behavioural abnormalities such as hyperactivity, sensory motor gating deficits and place preference. Associated with the behavioural changes, repeated treatment with Meth led to protein kinase A activation and phosphorylation of Ca2+/calmodulin kinase II and cyclic AMP response element binding protein in the NAc. In contrast, TH+/- and NR2A-/- mice displayed neither behavioural abnormalities nor activation of intracellular signalling pathways in the NAc. These results suggest that both dopaminergic and glutamatergic transmission play a crucial role in the development of Meth psychosis and dependence, which are associated with convergent activation of intracellular signalling pathways in the NAc.

In vivo catecholaminergic metabolism in the medial prefrontal cortex of ENU2 mice: an investigation of the cortical dopamine deficit in phenylketonuria.

OBJECTIVE: Phenylketonuria (PKU) is an inherited metabolic disease characterized by plasma hyperphenylalaninemia and several neurological symptoms that can be controlled by rigorous dietetic treatment. The cellular mechanisms underlying impaired brain functions are still unclear. It has been proposed, however, that phenylalanine interference in cognitive functions depends on impaired dopamine (DA) transmission in the prefrontal cortical area due to reduced availability of the precursor tyrosine. Here, using Pah(enu2) (ENU2) mice, the genetic murine model of PKU, we investigated all metabolic steps of catecholamine neurotransmission within the medial preFrontal Cortex (mpFC), availability of the precursor tyrosine, synthesis and release, to find an easy way to reinstate normal cortical DA neurotransmission. METHODS AND RESULTS: Analysis of blood and brain levels of tyrosine showed reduced plasma and cerebral levels of tyrosine in ENU2 mice. Western blot analysis demonstrated deficient tyrosine hydroxylase (TH) protein levels in mpFC of ENU2 mice. Cortical TH activity, determined in vivo by measuring the accumulation of l-3,4-dihydroxyphenylalanine (L-DOPA) in mpFC after inhibition of L-aromatic acid decarboxylase with NSD-1015, was reduced in ENU2 mice. Finally, a very low dose of L-DOPA, which bypasses the phenylalanine-inhibited metabolic steps, restored DA prefrontal transmission to levels found in healthy mice. CONCLUSION: The data suggests that a strategy of using tyrosine supplementation to treat PKU is unlikely to be effective, whereas small dose L-DOPA administration is likely to have a positive therapeutic effect.

Tyrosine hydroxylase deficiency in Taiwanese infants.

We analyzed the clinical manifestations, genetic mutations, treatment responses to L-dopa, and long-term neurologic outcomes in Taiwanese infants with tyrosine hydroxylase deficiency. From 1999 to May 2011, we enrolled six infants who had been diagnosed with tyrosine hydroxylase deficiency by identifying point mutations on the tyrosine hydroxylase gene. Two patients manifested fetal distress during the perinatal period. Four patients exhibited generalized tremor as their first observed neurologic sign at age 3 months. All presented brisk reflexes, hypokinesia, rigidity, distal chorea, and athetosis. We identified a novel missense mutation, I382T, and report on the first patient, to the best of our knowledge, with a homozygous R153X nonsense mutation. Five of six patients responded to L-dopa at a dose of 4.2-34.7 mg/kg/day combined with biperiden or selegiline or both. Long-term neurologic outcomes (median follow-up, 5 years and 10.5 months) revealed two patients demonstrated slightly low intelligence quotients, three demonstrated mild to moderate psychomotor retardation, and one died of respiratory failure. A higher dose of L-dopa, together with alternative therapies, may lead to improvements in motor function. However, several years of observation may be needed to reach definitive conclusions about neurologic outcomes.

Repression of the 14-3-3 gene affects the amino acid and mineral composition of potato tubers.

Recently, transgenic potato plants were created showing underexpression of the 20R isoform of the 14-3-3 protein. The transgenic plants grown in tissue culture showed a significant increase in nitrate reductase activity and a decrease in nitrate level. The transgenic line with the lowest 14-3-3 quantity was field-trialed (1997-2000) and analyzed. The reduction in the 14-3-3 protein level consistently resulted in a starch content increase and in an increase in the ratio of soluble sugars to starch in the tubers, although the latter was only barely visible. The determination of amino acid composition in the tubers showed a significant increase in methionine, proline, and arginine content and a slight but consistent increase in hydrophobic amino acid and lysine content in the cells of the transgenic potato plants. We also observed an increase in the crude protein content, from 19 to 22.1% of the control value in consecutive years. It is proposed that all of these changes might have resulted from the downregulation of nitrate reductase and sucrose phosphate synthase activities by 14-3-3, although other potential mechanisms cannot be excluded (e.g., an increase in enzyme protein level). 14-3-3-repressed transgenic plants showed a significant increase in calcium content in their tubers. It is thus proposed that a function of the isolated 14-3-3 isoform is in the control of amino acid synthesis and calcium metabolism. However, the mechanism of this control is as yet unknown.

Genetic evidence for noradrenergic control of long-term memory consolidation.

Memory formation involves dynamic interactions among many brain structures and their linking pathways. The noradrenaline (NA) system in the CNS plays an important role in a wide variety of neurological and psychological functions. Alteration in the NA system is implicated in the pathological states of some neuropsychiatric disorders. Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme for the biosynthesis of catecholamines. The regulatory mechanism of the TH reaction is generally considered to play a key role in controlling the catecholaminergic actions. Mice heterozygous for the mutation of the gene encoding TH exhibit the reduced TH activity in tissues. These mice have a moderate reduction in NA accumulation and release in brain regions. The mutant mice exhibit deficits in the water-finding task associated with latent learning performance, suggesting the impairment in memory formation. Spatial learning performance measured by the water maze task is normal in the mutants. However, they display deficits in long-term memory formation of conditioned learning evaluated with three distinct behavioral paradigms, including active avoidance, cued fear conditioning, and conditioned taste aversion, without affecting short-term memory. These memory deficits are restored by the drug-induced stimulation of NA activity at the postconditioning phase. Analysis of the mutant mice indicates that the central NA system is essential for the consolidation process in long-term memory of conditioned learning. The process appears to be implicated in the NA activity in the cerebral cortex and/or amygdaloid complex.

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.

Hypothalamic dopaminergic neurons in transgenic dwarf mice: histofluorescence, immunocytochemical, and in situ hybridization studies.

Spontaneous dwarf mice, in which both growth hormone (GH) and prolactin (PRL) are undetectable, are severely deficient in the PRL-inhibiting catecholamine dopamine (DA), as well as its synthetic enzyme, tyrosine hydroxylase (TH), in the basal hypothalamus (Phelps et al., Cell Tissue Res., 240:19-25, 1985; Phelps, Brain Res., 416:354-358, 1987). In contrast, transgenically constructed dwarf mice (Behringer et al., Genes Dev., 2:453-461, 1988) show complete ablation of pituitary GH cells, but PRL cells are retained at a level of approximately 10% of normal. In order to determine the feedback effect of this reduced, rather than absent, PRL on hypothalamic DA neurons, brains of transgenic dwarf mice were examined for catecholamine transmitters by histofluorescence, for the synthetic enzyme TH by immunocytochemistry, and for TH mRNA expression by in situ hybridization. DA histofluorescence in transgenic dwarfs was comparable to that of normal littermate mice in nonpituitary regulating areas (perikarya of zona incerta [A13] of hypothalamus and in midbrain substantia nigra area [A9]). Arcuate nucleus (A12) DA neurons that inhibit PRL secretion, however, showed dim to absent fluorescence in perikarya and in external median eminence terminals in dwarfs. There were reduced (P less than 0.05) numbers of A12 TH-immunoreactive neurons in transgenic dwarfs, to approximately 60% of those in normal mice. In contrast, TH-positive neurons in other hypothalamic areas (A13, A14) had average populations equivalent to those in normal mice. Quantification of TH mRNA abundance by in situ hybridization using both image analysis of hybridization over the arcuate nucleus, and grain counts per individual A12 cell in this nucleus, indicated that relative mRNA levels were the same in normal and transgenic dwarfs. The observations indicate that reduction in pituitary PRL is accompanied by defective expression in hypothalamic tuberoinfundibular neurons, which is severe at the DA neurotransmitter level, significant regarding observable TH immunoreactivity, and undetectable with regard to TH mRNA expression. Collectively, the findings suggest that posttranscriptional processes are involved with the mediation of PRL feedback upon hypothalamic neurons. Technically and quantitatively, the report presents the feasibility of simultaneous evaluation of transmitter histofluorescence, synthetic enzyme immunocytochemistry, and mRNA expression in individual animals.

Isolated deficiency of tyrosine hydroxylase immunoreactivity in tuberoinfundibular neurons in pituitary prolactin-deficient Snell dwarf mice.

Light microscopic morphology and relative numbers of tyrosine hydroxylase (TH)-immunoreactive neurons were assessed in hypothalamus of hypopituitary Snell dwarf mice, compared with normal mice of the same strain. Qualitatively, a specific deficit in staining of tuberoinfundibular neurons was noted in dwarf hypothalamus; TH-positive terminals were absent in dwarf median eminence, and morphology of axons in this region was aberrant. Qualitative observations were supported by quantitative assessment of hypothalamic dopaminergic neuron groups. Dorsal and intermediate TH-positive cells numbered 80% of those normal mouse brain; tuberoinfundibular (A12) TH-positive neurons in dwarf hypothalamus were 2% of those in normal mouse brain. The results imply that absence of pituitary hormone feedback impedes both structural and biochemical development of hypophysiotropic hypothalamic neurons.