Microdialysis Monitoring of 3,4‐Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus

Abstract

An on-line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4-dihydroxyphenylalanine (DOPA) accumulation in the extracellular fluid during perfusion of a n aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl-DOPA, which was shown to be more stable than NSD 10 15 or Ro 4-4602 in the perfusion fluid. A 1-h perfusion of a mol/L of difluoromethyl-DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates duringaromatic amino acid decarboxylase inhibition was confirmed by the disappearance ofthe chromatographic peak when DOPA formation was blocked by the administration ofa-methyl-p-tyrosine. Systemicadministration of the a,-antagonist piperoxane before difluoromethyl-DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady-state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons. Key Words: Tyrosine hydroxyla~e-3~4-Dihydroxyphenylalanine-Locus ceruleus-Difluoromethyl-3,4-dihydroxyphenylalanine-Microdialysis-Piperoxane. Tyrosine hydroxylase (TH; EC 1.14.16.2, tyrosine-3-monooxygenase) catalyzes the formation of 3,4-dihydroxyphenylalanine (DOPA) from L-tyrosine, the rate-limiting step in noradrenaline biosynthesis. Rapid increases in TH activity have been observed in response to different stimuli of catecholaminergic neurons (review in Zigmond et al., 1989). Long-lasting increases in TH activity and/or concentration have been demonstrated in noradrenergic neurons of the rat locus ceruleus (LC) in a variety of physiological and pharmacological models including cold stress (Zigmond et al., 1974), reserpine treatment (Reis et al., 1975), inactivation of serotonin afferents (McRaeDegueurce and Pujol, 1979), and RU 24722 administration (Labatut et al., 1988). To investigate whether these changes in TH activity and concentration are functionally relevant, an in vivo method for determination of TH activity is needed. One approach is the measurement of in vivo 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations in extracellular fluid, which have been shown to reflect changes in TH activity in noradrenergic neurons of the LC (Gonon et al., 1983). However, these measurements provide an indirect estimate of in vivo tyrosine hydroxylation. It was therefore more desirable to estimate changes in TH activity directly. For many years, such a determination has been achieved by measuring DOPA concentration in brain tissue homogenates after systemic ad-

Double Transduction with GTP Cyclohydrolase I and Tyrosine Hydroxylase Is Necessary for Spontaneous Synthesis ofl-DOPA by Primary Fibroblasts

“…Accumulation of L-DOPA after inhibition of AADC was used to isolate the in vivo TH function from other variables such as DOPA decarboxylation, dopamine uptake, and metabolism of dopamine by subsequent enzymatic steps (Carlsson et al, 1972;Westerink et al, 1990;Robert et al, 1993). Our data demonstrate that in the absence of its metabolism, L-DOPA accumulates in the striatum of rats with PFTHGC grafts, supporting the idea that TH function is enhanced by this genetic modification.…”

Estradiol Increases Tyrosine Hydroxylase Activity of the A15 Nucleus Dopaminergic Neurons during Long Days in the Ewe1

“…Conversely, the perfusion of 3 mmol/L NSD1015 induced the highest levels of L-DOPA with reduced variability between animals. Under these conditions, however, the inhibitor or its metabolites observed in the chromatogram [13,19] masked the identification of both DOPAC and HVA.…”

“…Nevertheless, numerous studies have shown that extracellular concentrations of L-DOPA are strongly correlated with TH activity when it is modified by pharmacological agents. Thus, the general consensus is that this method is valid as an in vivo measure of TH activity [13,19,20].…”

“…The stock solution contained 300 mmol/L and was kept at -20°C. This dose was chosen after a preliminary experiment on ewes in which we observed that after addition of NSD1015 at a concentration of 0.1 mmol/L (the quantity previously used on rats [13,19,20]), the output of L-DOPA was highly variable between animals; however, this dose allowed us to demonstrate a decrease in DOPAC and HVA concomitant with the increase in L-DOPA (Fig. 1).…”