Effects of low doses of intracerebroventricular 6-OHDA on the levels of monoaminergic neurotransmitters in rat brain structures

Pharmacological Reports 2010, 62, 1225–1230 ISSN 1734-1140

Copyright © 2010 by Institute of Pharmacology Polish Academy of Sciences

Short communication

Effects of low doses of intracerebroventricular 6-OHDA on the levels of monoaminergic neurotransmitters in rat brain structures Anna Sadakierska-Chudy, Anna Haduch, Krystyna Go³embiowska, W³adys³awa A. Daniel Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

Correspondence:

Anna Sadakierska-Chudy, e-mail: [email protected]

Abstract: The objective of this study was to determine the degree and specificity of noradrenergic lesions in different areas of the rat brain after intracerebroventricular administration of low doses of 6-hydroxydopamine (6-OHDA) into both lateral ventricles. Our interest focused on the induction of an effective hypothalamic lesion. The results suggest that small doses of 6-OHDA (25 or 50 μg per ventricle) could effectively damage the noradrenergic system in the hypothalamus without significant interfering with the dopamine level and with only a modest reduction in the serotonin concentration. Key words: 6-OHDA, noradrenaline, dopamine, serotonin, brain structures, intracerebroventricular injection

Abbreviations: CA – catecholamine, DA – dopamine, DOPAC – 3,4-dihydroxyphenylacetic acid, 5-HIAA – 5-hydroxyindoleacetic acid, 5-HT – 5-hydroxytryptamine/serotonin, HVA – homovanillic acid, icv – intracerebroventricular injection, ip – intraperitoneal injection, NA – noradrenaline, 6-OHDA – 6-hydroxydopamine

Introduction 6-Hydroxydopamine (6-OHDA) is a hydroxylated analogue of natural dopamine (DA), a neurotransmitter, that selectively destroys catecholamine (CA) neurons [1, 9]. 6-OHDA has a high affinity for the noradrenaline (NA) and DA transporters, which allows easy entry of the neurotoxin into neurons. 6-OHDA is one of the most common neurotoxins used in animal

models of Parkinson’s disease [1, 11]. 6-OHDA may undergo enzymatic degradation by MAO (monoamine oxidase) [6] or non-enzymatic auto-oxidation generating reactive oxygen species (ROS) under physiological conditions [13]. The oxidative stress induced by 6-OHDA reduces the cellular antioxidant potential [8], and 6-OHDA-derived ROS (hydroxyl radicals, superoxide radicals and hydrogen peroxide) can damage proteins, lipids and DNA [16]. In addition, through inhibition of mitochondrial complexes I and IV, 6-OHDA leads to mitochondrial impairment and ATP deficiency [1, 3]. 6-OHDA does not cross the blood-brain barrier. Therefore, neuronal lesions can only be produced after direct intracerebral administration [16]. A previous study carried out on whole rat brains demonstrated that the neurotoxic effects of 6-OHDA after intracere-

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broventricular (icv) infusion were more visible in the case of NA than DA neurons, and low doses of the neurotoxin (25–50 μg) were sufficient to deplete mainly NA level [2, 14]. Interestingly, one study found that icv administration of 6-OHDA at a relatively high dose of 200 μg significantly decreased the 5-hydroxytryptamine/ serotonin (5-HT) content in different areas of rat brain [10], while such an effect was not observed by others [5, 12, 15]. Therefore, it seems that icv injection of 6OHDA may be an effective approach for the destruction of noradrenergic neurons in the brain, but the regional effects of the neurotoxin on the NA level depend on parameters of the experiment (dose, simultaneous protection of other neurotransmitter systems, the time elapsed between neurotoxin administration and neurotransmitter analysis, and a brain structure). The aim of our present study was to estimate the degree and specificity of noradrenergic lesions in different areas of rat brain after icv administration of low doses of 6-OHDA. In particular, we were interested in inducing hypothalamic lesions to produce an animal model for studying the contribution of noradrenergic innervation of the hypothalamus to the expression of liver cytochrome P450. The hypothalamic-pituitaryadrenal/thyroid axis is known to be involved in the regulation of cytochrome P450 expression. In the present experiment, the levels of NA, DA, 5-HT and their main metabolites in nine areas of rat brain were measured after icv injection of two low doses of 6-OHDA.

Materials and Methods Animals

Twenty male Wistar Han rats (Charles River Laboratories, Germany) weighing 249–300 g were housed individually under standard laboratory conditions (22 ± 2°C room temperature; 55 ± 5% room humidity; 12-h light/dark cycle, light from 6:00 a.m. to 6:00 p.m.). The animals had free access to water and food, but for the 18 h before decapitation, they were deprived of food because ingestion of food might affect enzymatic activity. The experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Bioethical Committee at the Institute of Pharmacology, Polish Academy of Sciences, Kraków. 1226

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Drugs

The following compounds were used in this study: 6-OHDA hydrochloride and fluoxetine hydrochloride (Sigma), Bioketan (ketamine hydrochloride, Vetoquinol Biowet, Poland), Sedazin (xylazine hydrochloride, Biowet, Poland) and ascorbic acid (Riedel-de Haën). Solutions were prepared fresh on the days of experimentation. Fluoxetine was dissolved in water and injected intraperitoneally (ip), while 6-OHDA was dissolved in isotonic saline with 0.05% ascorbic acid and was injected icv. Surgery

Rats were anesthetized with ketamine HCl (65 mg/kg, ip) and xylazine HCl (5 mg/kg ip) and were placed in a Kopf stereotaxic apparatus. Thirty minutes before the 6-OHDA or vehicle infusion, rats were pretreated with the 5-HT reuptake blocker fluoxetine (10 mg/kg, ip) to protect serotonergic terminals from 6-OHDA. The 6-OHDA hydrochloride solution contained 5 or 10 μg of base in a 1-μl vehicle solution composed of 0.9% NaCl containing 0.05% ascorbic acid. Rats were divided into three groups: the control group (n = 6), which was treated with vehicle (icv), and two groups of seven rats that received bilateral injections of 6-OHDA (25 μg or 50 μg per ventricle). The following coordinates were used in accordance with the Paxinos and Watson atlas (2007): AP –0.8, L ± 1.5 from the bregma and V –4.0 from the surface of the dura. Five microliters of vehicle or 6-OHDA solution were administered into the lateral ventricles using a Hamilton syringe (with a flow rate of 1 μl/min), which was left in place for 5 min after injection before being slowly removed. Two weeks after the injection, the rats were killed by decapitation. The brains were rapidly removed and dissected into the cerebellum, the nucleus accumbens, the striatum, the frontal cortex, the hippocampus, the rest of the cortex, the hypothalamus and the brain stem. The brain structures were frozen on dry ice and stored at –70°C until further analysis. Analysis of neurotransmitters and their metabolites by HPLC

The level of endogenous DA, NA, 5-HT, 3,4dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) were measured using high-performance liquid

Effects of low doses of 6-OHDA Anna Sadakierska-Chudy et al.

chromatography with electrochemical detection (HPLC-EC) using a method based on that of Go³embiowska et al. [4]. Tissue samples were homogenized in 7–20 volumes (v/w) of ice-cold 0.1 M HClO" and centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was filtered through a 0.2-μm membrane filter, and 5-μl aliquots were injected into an HPLC system. The external standard consisted of NA, DA, 5-HT, DOPAC and 5-HIAA (Sigma) at concentrations of 50 ng/ml and HVA (Sigma) at a concentration of 100 ng/ml. The chromatography system consisted of an LC-4C amperometric detector with a cross-flow detector cell (BAS, IN, USA), a 626 Alltech pump and a GOLDHypersil analytical column (3 μm, 100 × 3 mm, Thermo Scientific, USA). The mobile phase consisted of 0.1 M KH PO", 0.5 mM Na EDTA, 80 mg/l sodium 1-octanesulfonate and 4% methanol, adjusted to pH 3.7 with 85% H!PO". The flow rate was 0.6 ml/min. The potential of a 3-mm glassy carbon electrode was set at 0.7 V with sensitivity of 5 nA/V. The temperature of the column was maintained at 30°C. The identification and quantification of the chromatographic peaks were made by comparison with the reference standard peaks. Chromax 2007 (Pol-Lab, Warszawa, Poland) was used for data collection and analysis. Statistical analysis

Values are expressed as the mean ± SEM. Differences between the experimental and control groups were estimated by one-way analysis of variance (ANOVA)

followed by the Newman-Keuls test; p < 0.05 was considered statistically significant.

Results and Discussion In the present work, we studied the effect of icv injection of 6-OHDA on the levels of monoaminergic neurotransmitters (NA, DA and 5-HT) and their main metabolites (DOPAC, HVA and 5-HIAA) in various brain structures of Wistar rats. The level of neurotransmitters were measured two weeks after neurotoxin administration and demonstrated the effectiveness and relative neurotransmitter-selectivity of the noradrenergic hypothalamic lesion induced by 6-OHDA. Bilateral icv injection of a single dose of 25 μg or 50 μg per ventricle of 6-OHDA resulted in a structuredependent decrease in NA, DA and 5-HT levels (Figs. 1–3). The content of NA was significantly reduced in eight areas of the brain (from –22% to –86%), whereas in the nucleus accumbens, the level of NA decreased only slightly (from –12% to –19%). The damage of NA neurons induced by both 6-OHDA doses was similar in the same brain regions (Fig. 1). The NA level in the hypothalamus was significantly reduced by 56% in both 6-OHDA-treated groups (p < 0.05). However, the most pronounced decrease in the level of NA was observed in the hippocampus (–86%). This change is probably due to the location of the 6-OHDA injection site, which was close to the hippocampus

Fig. 1. Effects of 6-OHDA on the content of NA in nine areas of the rat brain. HT – hypothalamus, HP – hippocampus, NA – nucleus accumbens, ST – striatum, BS – brain stem, FCX – frontal cortex, RCX – the rest of the cortex, CB – cerebellum, RB – the residual parts of the brain. * p < 0.05, *** p < 0.001, significant differences between the 6OHDA-treated groups and the control group. # p < 0.05, ### p < 0.001, significant differences between the doses of 25 µg and 50 µg

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Fig. 2. Effects of 6-OHDA on the content of DA in nine areas of the rat brain. HT – hypothalamus, HP – hippocampus, NA – nucleus accumbens, ST – striatum, BS – brain stem, FCX – frontal cortex, RCX – the rest of the cortex, CB – cerebellum, RB – the residual parts of the brain. ** p < 0.01, *** p < 0.001, significant differences between the 6-OHDA-treated groups and the control group

HT

HP

NA

ST

BS

FCX

RCX

and allowed for the easier penetration and accumulation of the neurotoxin in this structure [16]. We observed a significant decrease in the level of DA in the hippocampus (–77%), the nucleus accumbens (–19%) and the striatum (–29%). These three brain structures are located close to the ventricle, and even a low dose of 6-OHDA could cause DA depletion. Moreover, the nucleus accumbens and the striatum are rich in dopaminergic nerve terminals, providing a specific uptake system for the neurotoxin into the neuron. The pronounced decrease in the DA level in the hippocampus (which contained approximately 10 times more NA than DA) could be partly attributed to the damage of noradrenergic terminals in that structure, leading to a decrease in the amount of the NA precursor DA. In other regions of the brain, the level

CB

RB

of DA was virtually unchanged. However, the content of DA was not reduced in the hypothalamus in the 6OHDA-treated rats (Fig. 2). Surprisingly, the administration of 6-OHDA led to a statistically significant decrease in the 5-HT level in all of the brain structures studied with the exception of the cerebellum. Similarly, as in the case of the DA level, the greatest reductions in the 5-HT level were observed in the hippocampus, the nucleus accumbens and the striatum (from –23% to –55%) (Fig. 3), i.e., in the structures located close to the ventricle. In addition, the lower dose of 6-OHDA (25 μg) decreased the 5-HT level to a greater extent than the higher dose (50 μg) in some structures, including the striatum and the residual parts of the brain. The levels of the metabolites HVA and 5-HIAA declined slightly. The metabolite/neurotransmitter ratios

Fig. 3. Effects of 6-OHDA on the content of 5-HT in nine areas of the rat brain. HT – hypothalamus, HP – hippocampus, NA – nucleus accumbens, ST – striatum, BS – brain stem, FCX – frontal cortex, RCX – the rest of the cortex, CB – cerebellum, RB – the residual parts of the brain. * p < 0.05, ** p < 0.01, *** p < 0.001, significant differences between the 6-OHDAtreated groups and the control group. ## p < 0.01, significant differences between the doses of 25 µg and 50 µg

HT

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HP

NA

ST

BS

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FCX

RCX

CB

RB

Effects of low doses of 6-OHDA Anna Sadakierska-Chudy et al.

(DOPAC/DA, HVA/DA and 5-HIAA/5-HT) were generally similar in the 6-OHDA-treated and control groups (data not shown). These results may indicate that the partial neuronal damage caused by 6-OHDA had no great effect on brain neurotransmitter turnover or on the adaptation of enzymatic system, allowing for unchanged monoamine synthesis and metabolism in the remaining neuronal cells. However, in a few cases, increases in the ratios of DOPAC/DA (in the hippocampus and cerebellum) and 5-HIAA/5-HT (in the striatum) were observed. Thus, the obtained data indicate that the low doses of icv-administered 6-OHDA decrease the brain levels of NA to a higher degree than that of DA, with an exception of the nucleus accumbens. Previous studies have also shown that the effect of icv-administered 6OHDA is more pronounced on the noradrenergic system than on the dopaminergic system [7, 10, 14]. Interestingly, small doses of 6-OHDA efficiently reduced the NA level with little or no effect on the DA level in rats [2, 14]. This result indicates that NA terminals are more sensitive than are other CA terminals to the toxic effects of 6-OHDA [16]. However, in the above-mentioned studies, higher doses of 6-OHDA were used and/or neurotransmitter levels were measured in the whole brain, which did not allow the observation of selective noradrenergic damage in individual brain regions produced by low doses of the neurotoxin. Previous studies carried out on the whole brain have shown that the level of 5-HT was unchanged after icv administration of higher 6-OHDA doses (250 or 500 μg) [12, 15]. In contrast, our data demonstrate that the level of 5-HT was significantly reduced, despite fluoxetine pretreatment. We cannot exclude the possibility that a single dose of fluoxetine (10 mg/kg) was not able to efficiently protect serotonergic neurons from damage. The most potent effect of 6-OHDA was found in small brain regions such as the hippocampus, the nucleus accumbens and the striatum. It is conceivable that the relative proximity of these structures to the site of 6-OHDA infusion favored the nonspecific transport of the neurotoxin and its metabolites into the serotonergic neurons and produced neurotoxic effects in these structures. Because large structures of the brain (the brain stem, cortex, hypothalamus and the residual parts of the brain) were modestly affected (from –13% to –33%) or not changed (the cerebellum) by 6-OHDA, the marked effect of the neurotoxin on the small structures located

close to the ventricle could have been masked when serotonin level was measured in the whole brain [12, 15]. Accordingly, a decrease in the 5-HT level in some structures of the brain (the hippocampus, hypothalamus, pons-medulla) after 6-OHDA treatment was observed by Reader and Gauthier, who used a higher dose of the neurotoxin (200 μg, icv) [10]. In summary, this report is the first showing the effect of small doses of 6-OHDA on the level of monoaminergic neurotransmitters in different rat brain regions. The results of our experiment indicate that an effective and relatively selectively induced lesion of noradrenergic innervation of the hypothalamus may be obtained after icv injection of 6-OHDA. Small doses of 6-OHDA (25 or 50 μg per ventricle) could damage the noradrenergic system in that brain structure without significant interference with the DA system and with a modest effect on 5-HT neurons. Injection of 6-OHDA by the icv route seems to be a more effective and safer method for the production of selective lesions affecting NA hypothalamic neurons than direct intrastructural administration of the neurotoxin, which can cause mechanical damage to this region.

Acknowledgments:

This study was supported by grant no. N N405 304836 from the Ministry of Science and Higher Education (Warszawa, Poland) and by statutory funds from the Institute of Pharmacology, Polish Academy of Sciences (Kraków, Poland). We also thank Justyna Staryñska for her excellent technical assistance.

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Received:

October 6, 2010; in the revised form: October 25, 2010.