Imidazoleacetic acid-ribotide induces depression of synaptic responses in hippocampus through activation of imidazoline receptors.

Imidazole-4-acetic acid-ribotide (IAA-RP), an endogenous agonist at imidazoline receptors (I-Rs), is a putative neurotransmitter/regulator in mammalian brain. We studied the effects of IAA-RP on excitatory transmission by performing extracellular and whole cell recordings at Schaffer collateral-CA1 synapses in rat hippocampal slices. Bath-applied IAA-RP induced a concentration-dependent depression of synaptic transmission that, after washout, returned to baseline within 20 min. Maximal decrease occurred with 10 µM IAA-RP, which reduced the slope of field extracellular postsynaptic potentials (fEPSPs) to 51.2 ± 5.7% of baseline at 20 min of exposure. Imidazole-4-acetic acid-riboside (IAA-R; 10 µM), the endogenous dephosphorylated metabolite of IAA-RP, also produced inhibition of fEPSPs. This effect was smaller than that produced by IAA-RP (to 65.9 ± 3.8% of baseline) and occurred after a further 5- to 8-min delay. The frequency, but not the amplitude, of miniature excitatory postsynaptic currents was decreased, and paired-pulse facilitation (PPF) was increased after application of IAA-RP, suggesting a principally presynaptic site of action. Since IAA-RP also has low affinity for α(2)-adrenergic receptors (α(2)-ARs), we tested synaptic depression induced by IAA-RP in the presence of α(2)-ARs, I(1)-R, or I(3)-R antagonists. The α(2)-AR antagonist rauwolscine (100 nM), which blocked the actions of the α(2)-AR agonist clonidine, did not affect either the IAA-RP-induced synaptic depression or the increase in PPF. In contrast, efaroxan (50 µM), a mixed I(1)-R and α(2)-AR antagonist, abolished the synaptic depression induced by IAA-RP and abolished the related increase in PPF. KU-14R, an I(3)-R antagonist, partially attenuated responses to IAA-RP. Taken together, these data support a role for IAA-RP in modulating synaptic transmission in the hippocampus through activation of I-Rs.

The experience of Wernicke's aphasia.

The authors induced a transient Wernicke's aphasia in a patient with left frontal arteriovenous malformation by superselective Wada injection exclusively into the lower division of the left middle cerebral artery. The patient was then asked to recall his experience, which the authors matched against his language during anesthesia. The patient's account showed that there was a more systematic attempt to respond appropriately than the authors could infer from his overt behavior. His narrative suggests that a thought process not measured by aphasia examinations may exist independent of language.

Vowel representations in the ventral cochlear nucleus of the cat: effects of level, background noise, and behavioral state.

Single-unit responses were studied in the ventral cochlear nucleus (VCN) of cats as formant and trough features of the vowel /epsilon/ were shifted in the frequency domain to each unit's best frequency (BF; the frequency of greatest sensitivity). Discharge rates sampled with this spectrum manipulation procedure (SMP) were used to estimate vowel representations provided by populations of VCN neurons. In traditional population measures, a good representation of a vowel's formant structure is based on relatively high discharge rates among units with BFs near high-energy formant features and low rates for units with BFs near low-energy spectral troughs. At most vowel levels and in the presence of background noise, chopper units exhibited formant-to-trough rate differences that were larger than VCN primary-like units and auditory-nerve fibers. By contrast, vowel encoding by primary-like units resembled auditory nerve representations for most stimulus conditions. As is seen in the auditory nerve, primary-like units with low spontaneous rates (SR <18 spikes/s) produced better representations than high SR primary-like units at all but the lowest vowel levels. Awake cats exhibited the same general response properties as anesthetized cats but larger between-subject differences in vowel driven rates. The vowel encoding properties of VCN chopper units support previous interpretations that patterns of auditory nerve convergence on cochlear nucleus neurons compensate for limitations in the dynamic range of peripheral neurons.

Measurements of active myocardial tension under a wide range of physiological loading conditions.

Active tension developed while cardiac muscle shortens has been studied extensively under afterloaded isotonic or isovelocity conditions. However, these are not true in vivo loading conditions. To obtain more physiological loading, we controlled sarcomere length to follow the time courses that we observed previously in a beating canine left ventricle. Sarcomere length was measured by laser diffraction in 12 rat cardiac trabeculae, superfused with Krebs-Henseleit solution (25 degrees C; [Ca] = 1.5 mM). Force was measured by a silicon strain gauge. Sarcomere length time courses were scaled slightly in time to account for temperature and species differences. We examined the relationships between active tension and sarcomere length under loading observed over a wide range of left ventricular preloads and afterloads, and at two sites. Under all loading conditions, active tension was not isotonic but declined steadily throughout the ejection period. While there were major differences in peak tension dependent on loading conditions and the incidence of 'pre-ejection' sarcomere shortening, these factors did not influence the relationship between sarcomere length and peak active tension. This study provides excellent illustrations of the potential differences in stress (1) within a ventricular wall, and (2) under different operating conditions. Moreover, it provides data for developing models of fiber contraction to be synthesized into a whole heart for predicting potential differences in stress at all sites and under all loading conditions.

Inhibitors of histamine methylation in brain promote formation of imidazoleacetic acid, which interacts with GABA receptors.

In brain, the precursor of imidazoleacetic acid (IAA), a GABAA agonist but a GABAC antagonist, is not known. In the periphery, IAA derives from oxidation of histamine. But in brain, histamine is thought to be metabolized solely by histamine methyltransferase (HMT), forming tele-methylhistamine (t-MH) and tele-methylimidazoleacetic acid (t-MIAA). We showed that [3H]-histamine (intracerebroventricularly) could be converted to IAA in brains of rats, a process increased by inhibition of HMT. This demonstrated that brain can oxidize histamine and suggested that endogenous histamine might also be oxidized if HMT activity were reduced. We examined in rat cerebral cortex, effects of the following HMT inhibitors (mg/kg i.p.): metoprine (10), tacrine (10), velnacrine (10, 30), and physostigmine (1,2). Tacrine was a potent inhibitor (Ki approximately 22 nM). To measure histamine in tissue that contained HMT inhibitors, we developed a gas chromatography-mass spectrometry method. After 2 h, all drugs reduced endogenous levels of t-MH and t-MIAA and increased levels of histamine and IAA. Our results show that inhibition of HMT promotes oxidation of histamine in brain, probably by shunting histamine to an alternative metabolic pathway. Formation of IAA provides a novel interaction between histaminergic and GABAergic systems in brain. Accumulation of IAA should be considered when inhibitors of HMT are used to probe brain histamine function.

Lack of a precursor-product relationship between histamine and its metabolites in brain after histidine loading.

Levels of histamine and its major metabolites in brain, tele-methylhistamine (t-MH) and tele-methylimidazoleacetic acid (t-MIAA), were measured in rat brains up to 12 h after intraperitoneal administration of L-histidine (His), the precursor of histamine. Compared with saline-treated controls, mean levels of histamine were elevated at 1 h (+102%) after a 500 mg/kg dose; levels of t-MH did not increase. Following a 1,000 mg/kg dose, mean histamine levels were increased for up to 7 h, peaked at 3 h, and returned to control levels within 12 h. In contrast, levels of t-MH showed a small increase only after 3 h; levels of t-MIAA remained unchanged after either dose. Failure of most newly formed histamine to undergo methylation, its major route of metabolism in brain, suggested that histamine was metabolized by another mechanism possibly following nonspecific decarboxylation. To test this hypothesis, other rats were injected with alpha-fluoromethylhistidine (alpha-FMHis; 75 mg/kg, i.p.), an irreversible inhibitor of specific histidine decarboxylase. Six hours after rats received alpha-FMHis, the mean brain histamine level was reduced 30% compared with saline-treated controls. Rats given His (1,000 mg/kg) 3 h after alpha-FMHis (75 mg/kg) and examined 3 h later had a higher (+112%) mean level of histamine than rats given alpha-FMHis, followed by saline. Levels of t-MH and t-MIAA did not increase. These results imply that high doses of His distort the simple precursor-product relationship between histamine and its methylated metabolites in brain. The possibility that some His may undergo nonspecific decarboxylation in brain after His loading is discussed. These findings, and other actions of His independent of histamine, raise questions about the validity of using His loading as a specific probe of brain histaminergic function.

pros-Methylimidazoleacetic acid in cerebrospinal fluid of patients with chronic schizophrenia: relationships to ratings of symptoms, ventricular brain ratios, and rates of urine excretion.

Concentrations of pros-methylimidazoleacetic acid (p-MIAA) were measured in cerebrospinal fluid of 30 patients with chronic schizophrenia. Levels of p-MIAA correlated negatively with mean scores of the Psychiatric Symptom Assessment Scale for positive symptoms (r = -0.48), but not negative symptoms, and with ventricular brain ratios (r = -0.48). Patients with abnormal ventricular enlargements had much lower concentrations of p-MIAA than those with normal ventricles. These results suggest that processes that reduce accumulation of p-MIAA in CSF may be associated with increased severity of symptoms among patients with chronic schizophrenia.

An open-label study of the therapeutic efficacy of high-dose famotidine adjuvant pharmacotherapy in schizophrenia: preliminary evidence for treatment efficacy.

Histaminergic projections innervate brain areas implicated in the pathophysiology of schizophrenia. In a previous open-label study, there was the suggestion that famotidine, and H2 histamine-receptor antagonist, possessed adjuvant therapeutic properties when added to the stable neuroleptic medications regimens of 10 treatment-refractory patients. In that study, the maximal dosage of famotidine was limited to 40 mg/day, the recommended maximal dosage for the treatment of peptic ulcer disease. In this study, we examined 18 patients fulfilling DSM-III-R criteria for schizophrenia and schizoaffective disorder who had famotidine (100 mg/day) added to their stable neuroleptic medication regimen. Patients were rated on baseline, weekly thereafter, and 1 week after famotidine discontinuation, by using the Brief Psychiatric Rating Scale (BPRS), Schedule for the Assessment of Negative Symptoms (SANS), and the Clinical Global Impression (CGI). On all of these outcome measures, statistically significant improvements suggestive of a beneficial adjunctive effect of famotidine were found. Famotidine (100 mg/day) was well tolerated by the study subjects. There was a wide range of famotidine blood levels achieved at the end of 3 weeks of famotidine adjunctive treatment, but these blood levels did not correlate with BPRS or SANS score changes. However, the patients with the greatest improvement in BPRS scores (and without concomitant deterioration in SAND scores) had some of the higher famotidine levels found in the study. Double-blind studies further assessing the potential adjunctive benefit of famotidine in the treatment of schizophrenia are indicated.

[3H]-thioperamide as a radioligand for the histamine H3 receptor in rat cerebral cortex.

1. The purpose of the present study was to characterize the binding of the histamine H3 receptor antagonist, [3H]-thioperamide, to rat cerebral cortical membranes. 2. The binding of [3H]-thioperamide to rat cerebral cortical membranes reached equilibrium after incubation with [3H]-thioperamide after 8-10 h at 4 degrees C. Equilibrium was maintained for up to 18 h of incubation. Addition of 1 microM (R)-alpha-methylhistamine rapidly dissociated [3H]-thioperamide from its binding sites. From these kinetic experiments a dissociation constant of 0.3 nM was obtained for [3H]-thioperamide. 3. Saturation experiments with [3H]-thioperamide using 1 microM (R)-alpha-methylhistamine to define nonspecific binding were best analysed according to a single site model. A dissociation constant (KD) of 0.80 +/- 0.06 nM (n = 3) and a maximal number of binding sites (Bmax) of 73 +/- 20 fmol mg-1 protein (n = 3) were obtained for the binding of [3H]-thioperamide to rat cerebral cortical membranes. 4. Saturation experiments with [3H]-thioperamide using 0.3 microM iodophenpropit to define nonspecific binding were best analysed according to a two site model. For the high affinity [3H]-thioperamide site a KD value of 1.1 +/- 0.3 nM (n = 3) and Bmax value of 162 +/- 108 fmol mg-1 protein (n = 3) were obtained whereas KD and Bmax values for the low affinity site were 96 +/- 19 nM and 4346 +/- 3092 fmol mg-1 protein (n = 3), respectively. 5. Using 5 nM [3H]-thioperamide, the binding was hardly displaced by H3 agonists within concentration-ranges expected to bind to the histamine H3 receptor. Under these conditions, [3H]-thioperamide binding was fully displaced by various H3-antagonists, yet most H3 antagonists showed Ki values different from those expected for the histamine H3 receptor. 6. Using 0.3 nM [3H]-thioperamide, 50-60% of the total binding was potently displaced by the H3 agonists histamine, (R)-alpha-methylhistamine, (S)-alpha-methylhistamine, imetit and immepip. Displacement of the binding of 0.3 nM [3H]-thioperamide binding exhibited clear stereoselectivity for the R and S isomers of alpha-methylhistamine. 7. Binding of 0.3 nM [3H]-thioperamide was completely displaced by several H3 antagonists (thioperamide, iodophenpropit, iodoproxyfan, and burimamide) and biphasic displacement curves were obtained; the Ki values for the high affinity site corresponded well with the expected values for the H3 receptor. Antagonists fully displaced the binding of 5 nM [3H]-thioperamide with affinities comparable to the low affinity site found with 0.3 nM [3H]-thioperamide. 8. Ondansetron and haloperidol did not displace binding of 5 nM [3H]-thioperamide at concentrations at which the former are known to bind to 5-HT3 or sigma receptors, respectively. On the other hand, nonselective cytochrome P450 inhibitors displaced the binding of 5 nM [3H]-thioperamide from both rat cerebral cortical membranes and rat liver microsomes. 9. It is concluded that the histamine H3 antagonist, [3H]-thioperamide, can be used as a radioligand to study the histamine H3 receptor in rat brain, provided that subnanomolar concentrations are used in displacement studies. Moreover, the specific binding should be defined with an H3 agonist, since most H3 antagonists share with [3H]-thioperamide a low affinity, high density, non-H3 receptor binding site(s) in rat brain. The latter is probably due to binding to cytochrome P450 isoenzymes.

Disposition of histamine, its metabolites, and pros-methylimidazoleacetic acid in brain regions of rats chronically infused with alpha-fluoromethylhistidine.

In mammalian brain, histamine is known to be metabolized solely by histamine methyltransferase (HMT), forming tele-methylhistamine (t-MH), then tele-methylimidazoleacetic acid (t-MIAA). We previously showed that imidazoleacetic acid (IAA), a GABA agonist, and histamine's metabolite in the periphery, is present in brain where its concentration increased after inhibition of HMT. Also, when [3H]histamine was given intracerebro-ventricularly to rats, a portion was converted to IAA, a process increased by inhibition of HMT. These results indicated that brain has the capacity to oxidize histamine but did not show whether this pathway is operative under physiological conditions. To address this question, rats were infused for > 4 weeks with alpha-fluoromethylhistidine (alpha-FMHis), an irreversible inhibitor of histamine's synthetic enzyme, L-histidine decarboxylase. Compared with controls (untreated and saline-treated rats), brain levels of histamine, t-MH, and t-MIAA in all regions were markedly reduced in treated rats. As a percentage of controls, depletion of t-MIAA > t-MH > histamine in all regions, and regional depletions of histamine co-responded to its turnover rates in regions of rat brain. In contrast, levels of IAA were unchanged as were levels of pros-methylimidazoleacetic acid, an isomer of t-MIAA unrelated to histamine metabolism. Results suggest that in brains of rats, unlike in the periphery, most IAA may not normally derive from histamine. Because histamine in brain can be converted to IAA under certain conditions, direct oxidation of histamine may be a conditional phenomenon. Our results also support the existence of a very slow turnover pool of brain histamine and use of chronic alpha-FMHis infusion as a model to probe the histaminergic system in brain.

The relationship between urine excretion and biogenic amines and their metabolites in cerebrospinal fluid of schizophrenic patients.

Concentrations of norepinephrine and metabolites of biogenic amines were measured in lumbar cerebrospinal fluid of 30 patients with chronic schizophrenia, nine of whom were polyuric. The mean level of norepinephrine was two-fold higher (p < or = 0.025) in polyuric patients than in patients whose excretion of urine was within the normal range. CSF levels of histamine's primary metabolite, tele-methylhistamine, an index of brain histaminergic activity, were positively correlated (p < 0.005) with daily urine volume. These results are consistent with the known influence of norepinephrine and histamine on fluid regulation and suggest that norepinephrine and histamine may be involved in psychogenic polydipsia-polyuria in schizophrenic patients.

Characterization of histaminergic H3 receptors in intraocular tuberomammillary transplants containing histaminergic neurons.

This study was performed to investigate the physicological properties of histaminergic neurons in intraocular hypothalamic transplants. Pieces of posterolateral hypothalamus containing the tuberomammillary nucleus were dissected from Embryonic Day 17 rat fetuses and transplanted into the anterior chamber of the eye of adult rat hosts. The hypothalamic transplants were left to mature for 2-5 months, after which in vivo electrophysiological recordings were performed. Extracellular recordings revealed spontaneously active neurons in the grafts, with a mean (+/- SEM) firing rate of 2.8 +/- 2.0 Hz and a mean action potential duration of 1.2 +/- 0.5 ms. When the surface of the grafts was superfused with histamine, the neuronal activity was depressed at concentrations above 30 microM. Superfusion with the H3 agonist (R)-alpha-methylhistamine also elicited depression of baseline firing rate, with an EC50 of 0.435 microM. This depression could be antagonized by superfusion with the H3-receptor antagonist thioperamide. In studies of histamine levels using a sensitive radioenzymatic assay, the mean (+/- SEM) level of histamine in the grafts was 73 +/- 28 ng/g tissue, i.e., about half the concentration of histamine in the adult rat hypothalamus in situ. Intracellular recordings in combination with biocytin labeling and histidine decarboxylase immunohistochemistry suggested that the grafted neurons from which recordings were made were histaminergic. Taken together, these data indicate that tuberomammillary neurons continue their development in intraocular transplants and develop physiological characteristics found in these neurons in situ.

Imidazoleacetic acid, a gamma-aminobutyric acid receptor agonist, can be formed in rat brain by oxidation of histamine.

It is generally accepted that in mammalian brain histamine is metabolized solely by histamine methyltransferase (HMT), to form tele-methylhistamine, then oxidized to tele-methylimidazoleacetic acid. However, histamine's oxidative metabolite in the periphery, imidazoleacetic acid (IAA), is also present in brain and CSF, and its levels in brain increase after inhibition of HMT. To reinvestigate if brain has the capacity to oxidize histamine and form IAA, conscious rats were injected with [3H]histamine (10 ng), either into the lateral ventricles or cisterna magna, and decapitated 30 min later. In brains of saline-treated rats, most radioactivity recovered was due to tele-methylhistamine and tele-methylimidazoleacetic acid. However, significant amounts of tritiated IAA and its metabolites, IAA-ribotide and IAA-riboside, were consistently recovered. In rats pretreated with metoprine, an inhibitor of HMT, labeled IAA and its metabolites usually comprised the majority of histamine's tritiated metabolites. [3H]Histamine given intracisternally produced only trace amounts of oxidative metabolites. Formation of IAA, a potent GABA-A agonist with numerous neurochemical and behavioral effects, from minute quantities of histamine in brain indicates a need for reevaluation of histamine's metabolic pathway or pathways in brain and suggests a novel mechanism for interactions between histamine and the GABAergic system.

Interaction of clozapine with the histamine H3 receptor in rat brain.

We examined possible interactions between neuroleptics and the histamine H3 receptor and found an interaction of clozapine with this receptor. In competition binding experiments, using the H3 antagonist, [125I]-iodophenpropit, we observed a Ki of 236 +/- 87 nM. Functionally, clozapine was studied on the H3-mediated inhibition of [3H]-5-hydroxytryptamine ([3H]-5-HT) release from rat brain cortex slices. Clozapine acts as an antagonist with an apparent KB value of 79.5 nM.

Release of glutamate from striatum of freely moving rats by pros-methylimidazoleacetic acid.

The effect of pros-methylimidazoleacetic acid (p-MIAA) was measured on the release of glutamate and aspartate from cerebral cortex, hippocampus, and striatum of freely moving rats, and on the uptake of 14C by striatal slices incubated in the presence of L-[14C]-glutamate. Twenty-four hours after implantation of a dialysis fiber, striatum, hippocampus, or cerebral cortex spontaneously released both glutamate and aspartate in the micromolar range. p-MIAA (1 microM to 1 mM), added to the dialysis perfusate, elicited a concentration-dependent increase of glutamate release from striatum with a maximal increase of about threefold. This effect did not occur in hippocampus or cortex. In none of these regions did p-MIAA increase aspartate release significantly. The p-MIAA effect was not mimicked by its isomer tele-methyl-imidazoleacetic acid. p-MIAA did not influence the uptake of glutamate by striatal slices. The glutamate-releasing action of p-MIAA may affect striatal function and explain the positive correlation between levels of p-MIAA in CSF and the severity of Parkinson's disease.

Histamine metabolites in cerebrospinal fluid of patients with chronic schizophrenia: their relationships to levels of other aminergic transmitters and ratings of symptoms.

Levels of the histamine metabolites, tele-methylhistamine (t-MH) and tele-methylimidazoleacetic acid (t-MIAA), and metabolites of other aminergic transmitters and of norepinephrine were measured in cerebrospinal fluid of 36 inpatients with chronic schizophrenia and eight controls. The mean t-MH level from controls was nearly identical to the levels seen previously in healthy volunteers. Compared with controls, the mean level of t-MH in the schizophrenic patients was 2.6-fold higher (p = 0.006); 21 of the patients had levels exceeding the range of controls. There was no significant difference (p > 0.05) in levels of other analytes, although the levels of t-MH correlated significantly with those of t-MIAA, homovanillic acid, 3,4-dihydroxyphenylacetic acid, norepinephrine, 3-methoxy-4-hydroxyphenylglycol and 5-hydroxyindoleacetic acid. The difference in levels of t-MH were not attributable to medication, since those taking (n = 10) or withdrawn from (n = 26) neuroleptic drugs had nearly the same mean levels of t-MH; each group had higher levels than controls (ANOVA: p < 0.05). Patients with or without tardive dyskinesia showed no significant differences in means of any analyte. Only levels of t-MH among those with schizophrenia correlated with positive symptom scores on the Psychiatric Symptom Assessment Scale (rs = 0.45, p < 0.02). The elevated levels of t-MH in cerebrospinal fluid, which represent histamine that was released and metabolized, suggest increased central histaminergic activity in patients with chronic schizophrenia.

Measurement of histamine metabolites in brain and cerebrospinal fluid provides insights into histaminergic activity.

Measurements of the concentrations of histamine's metabolites, tele-methylhistamine (t-MH) and tele-methylimidazoleacetic acid (t-MIAA), in brain have been used to evaluate histamine turnover in brains of animals, and the same measurements in CSF have been used to infer histaminergic activity in brains of man. In regions of rat brain, half-lives of histamine are shorter than those of dopamine, 5-hydroxytryptamine and norepinephrine. Studies of human CSF suggest that brain histaminergic activity increases with age and is higher in females than in males.

Effects of aminoguanidine on pre- and post-irradiation regional cerebral blood flow, systemic blood pressure and plasma histamine levels in the primate.

Exposure to ionizing radiation causes hypotension, cerebral ischemia and release of histamine (HA). To investigate the relationship among these three responses, rhesus monkeys (Macaca mulatta) received aminoguanidine (AG) (1 mg/kg), then were given either 50 Gy whole-body irradiation or sham-irradiation. Monkeys receiving AG had lower mean arterial blood pressure (MABP) than saline-treated controls. Compared to controls, rCBF was lower in irradiated monkeys but pre-treatment with AG did not influence this effect. Among untreated, irradiated monkeys, HA levels were increased only at two minutes post-irradiation, but among AG-treated, irradiated monkeys, HA levels were higher at all times postirradiation. Radiation-induced release of HA may be associated with radiation-induced hypotension and reduced rCBF, but failure of AG to alter rCBF suggests that released HA may not be the sole mediator of these effects. Because elevations in plasma HA are probably due to HA derived from degranulation of mast cells, release of other bioactive substances from mast cells may also influence these cardiovascular effects. Surprisingly, in sham-irradiated monkeys, AG alone had a slight but significant hypotensive effect.

Histamine metabolites and pros-methylimidazoleacetic acid in human cerebrospinal fluid.

In cerebrospinal fluid, levels of the histamine metabolites, tele-methylhistamine and tele-methylimidazole-acetic acid, were higher in elderly than in young people, and women had higher levels than men. Therefore, age and gender should be considered in studies of histamine metabolites as exemplified by their measurements in cerebrospinal fluid of patients with Huntington's disease. Levels of pros-methylimidazoleacetic acid, an isomer of tele-methylimidazoleacetic acid and not a metabolite of histamine, were higher in cerebrospinal fluid of men than of women. Levels of pros-methylimidazoleacetic acid in cerebrospinal fluid were highly positively correlated with the severity of Parkinson's disease in a group of non-medicated, mildly to moderately affected patients.