Male Wistar rats received fenfluramine in subacute (5 mg/kg b.i.d. i.p. for 4 days) or escalating (0.5, 1, 1.5, 2, 3, 4 and 5 mg/kg b.i.d. i.p., each dose given for 4 days) doses. Saline-treated controls received food ad libitum, or were pair-fed with the fenfluramine-treated animals. Rats were killed 1, 15 and 30 days after drug withdrawal. On day 1, plasma and brain fenfluramine levels were higher, and hypothalamus norfenfluramine levels were lower following escalating compared to subacute dosing, although total active drug levels were unaltered. Both treatment regimes, and pair-feeding reduced food intake and body weight. Subacute fenfluramine reduced brain 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels for up to 30 days. Brain 5-HT and 5-HIAA levels were unaltered following escalating-doses of fenfluramine. Additionally, pair-feeding transiently decreased hippocampal 5-HT levels. These data suggest that escalating-doses of fenfluramine prevent the 5-HT-depleting effect of a sub-cute challenge without altering the anorexic action of the drug.

The physiological role of nitric oxide in the control of striatal dopamine release has not been fully established, therefore, the effect of neuronally produced nitric oxide (NO) on striatal dopamine (DA) efflux were investigated using in vivo microdialysis in anaesthetised and conscious rats. In the anaesthetised rat, the nitric oxide synthase inhibitors NG-nitro-L-arginine methylester (L-NAME) and 7-nitroindazole monosodium salt (7-NINA) produced concentration-dependent increases in DA efflux. The L-NAME (1 mM)- and 7-NINA (1 mM)-induced increase was reduced by co-administration with the NO precursor, L-arginine (L-ARG; 1 mM) by 37% and 54% respectively, and was prevented by tetrodotoxin (TTX, 1 μM). Similarly, in conscious rats, L-NAME (1 mM) and 7-NINA (1 mM) increased DA efflux to 161% and 166% of basal efflux respectively. These data suggest that neuronally produced NO inhibits striatal DA efflux through an indirect mechanism.

Nigral cell degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tertrahydropyridine (MPTP) or its metabolite 1-methy1-4-phenyl pyridinium (MMP+) may involve toxicity induced by nitric oxide. In the present study a microdialysis procedure incorporating salicylate hydroxylation was used to measure striatal hydroxyl radical production through the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA). MPP+ (5–20 mM for 20 min) increased 2,3-DHBA formation in the rat striatum in a concentration-dependent manner with a concomitant increase in dopamine release and decrease in 3,4-dihydroxyphenyl acetic acid (DOPAC) formation. Inhibition of NO synthesis following NG-nitro-L-arginine methyl ester (L-NAME; 1 mM) and 7-nitroindazole monosodium salt (7-NINA; 1 mM), but not NG-nitro-D-arginine methyl ester (D-NAME; 1 mM) attenuated the MPP+-induced increase in hydroxyl radical formation. However, neither L-NAME nor 7-NINA had any effect on the MPP+-induced increase in dopamine efflux measured in vivo by microdialysis or in vitro using superfused striatal slices, although nomifensine (10 μM) abolished the MPP+-evoked dopamine efflux in vitro. These data suggest that NO formation is necessary for the production of hydroxyl radical following MPP+ treatment, but is not involved in the MPP+-evoked dopamine release.

THE effect of L-arginine (L-ARG; 10-100 mM) on dopamine efflux from rat striatum was investigated using in vivo microdialysis. L-ARG (50 mM-100 mM), but not D-arginine (100 mM) nor L-citrulline (100 mM), produced a biphasic effect on dopamine efflux with an initial small reduction, followed by a large sustained increase. The effect of L-ARG was not prevented by nitric oxide synthase inhibition with N-G-nitro-L-arginine methyl ester or 7-nitroindazole monosodium salt. Efflux of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was reduced by L-ARG (10-100 mM), D-arginine (100 mM) and L-citrulline (100 mM). These data suggest that changes in dopamine, DOPAC and HVA efflux produced by high concentrations of L-ARG occur independently of NO, and that the use of high L-ARG concentrations are inappropriate when investigating the role of NO in striatum.

Rats were treated for 28 days with increasing doses of dexfenfluramine (0.5, 1, 1.5, 2, 3, 4 and 5 mg/kg bid ip, each dose given for 4 days before being increased) and subsequently studied at intervals between 1 and 60 days following the cessation of treatment. Control rats received vehicle and were allowed foodad libitumor were pair fed with dexfenfluramine-treated animals. Immediately following drug treatment 5-HT immunoreactivity was increased in cortical areas compared to control animals. Subsequently, there was a persistent decrease in fine fibre density and the appearance of coarse truncated fibres. 5-HT levels in cortex were decreased 1 day following dexfenfluramine treatment but recovered to control values by 15 days. GFAP and GAP 43 immunoreactivity was unaffected by dexfenfluramine treatment compared to control animals, indicating a lack of evidence for neuronal degeneration and regeneration. Dexfenfluramine treatment decreased the density of 5-HT uptake sites in the cortex, labelled with [3H]-citalopram, but this partially recovered towards control values at 60 days. These alterations in 5-HT terminal networks conflict with the return of 5-HT levels to normal and the lack of evidence for degenerative changes or neuronal regrowth. On the basis of these results, it cannot be concluded that dexfenfluramine is neurotoxic.

The role of nitric oxide (NO) in striatal dopamine release has been controversial. Most NO synthase inhibitors affect more than one isoform of the enzyme and exert vasoconstrictor effects which may also affect striatal dopamine function. We now report on the effect of a soluble monosodium salt of the selective brain NO synthase inhibitor 7-nitro indazole (7-NINA). Using 7-NINA the first study of selective inhibition of the brain isoform of NO synthase on dopamine efflux in rat striatum was undertaken by use of in vivo microdialysis. Perfusion with 7-NINA (1 mM) increased striatal dopamine efflux. The effect of 7-NINA was partially antagonized (67%) by co-perfusion with L-arginine (1 mM), the precursor of NO formation in vivo. This suggests that 7-NINA induces a competitive inhibition of NO synthase activity. These data show that endogenous NO has an inhibitory effect on striatal dopamine efflux in vivo.

Plasma levels of homovanillic acid (pHVA) following debrisoquine (DBQ) administration may be indicative of central dopaminergic activity. The effect of DBQ (10-20 mg) administration on pHVA in young healthy volunteers was studied to establish a protocol for use in de novo patients with Parkinson's disease. Subsequently, pHVA in de novo patients with Parkinson's disease were measured and compared to young healthy volunteers. Following DBQ (10 mg) administration to healthy volunteers, pHVA fell with time to a maximum of 62% of control values at 6 h. The decrease in pHVA was not affected by loading with DBQ (10 mg) 10 h previously (pHVA: 67.6 +/- 5.8% of preDBQ levels) or increasing the dose to 20 mg (56.1 +/- 11.8% of preDBQ levels) compared to a single 10 mg dose of debrisoquine (66.5 +/- 4.5% of preDBQ levels). pHVA was reduced in both de novo patients with Parkinson's disease and in healthy volunteers following DBQ (10 mg) administration. However, there was no difference in pHVA before or after DBQ administration when comparing the two groups. These results suggest that, following DBQ administration, pHVA does not reflect dopamine neuronal loss in de novo patients with Parkinson's disease, so it is unlikely to detect the disease before the clinical symptoms manifest themselves.

Brain tissue from normal individuals with incidental Lewy bodies and cell loss in pigmented substantia nigra neurons (asymptomatic Parkinson's disease) and age-matched control subjects without nigral Lewy bodies was examined biochemically. There was no difference in dopamine levels or dopamine turnover in the caudate and putamen of individuals with incidental Lewy body disease compared to control subjects. There were no differences in levels of iron, copper, manganese, or zinc in the substantia nigra or other brain regions from the individuals with incidental Lewy body disease compared to those from control subjects. Similarly, ferritin levels in the substantia nigra and other brain areas were unaltered. There was no difference in the activity of succinate cytochrome c reductase (complexes II and III) or cytochrome oxidase (complex IV) between incidental Lewy body subjects and control subjects. Rotenonesensitive NADH coenzyme Q(1) reductase activity (complex I) was reduced to levels intermediate between those in control subjects and those in patients with overt Parkinson's disease, but this change did not reach statistical significance. The levels of reduced glutathione in substantia nigra were reduced by 35% in patients with incidental Lewy body disease compared to control subjects. Reduced glutathione levels in other brain regions were unaffected and there were no changes in oxidized glutathione levels in any brain region. Altered iron metabolism is not detectable in the early stages of nigral dopamine cell degeneration. There may be some impairment of mitochondrial complex I activity in the substantia nigra in Parkinson's disease. The marked reduction in nigral reduced glutathione levels suggests this to be an important early change in the process of oxidative stress underlying Parkinson's disease.