Gestational manganese intoxication and anxiolytic-like effects of diazepam and the 5-HT

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Gestational manganese intoxication and anxiolytic-like effects of diazepam

and the 5-HT _1A receptor agonist 8-OH-DPAT in male Wistar rats

Adam Kwieciñski, Przemys³aw Nowak

Department of Pharmacology, Medical University of Silesia, H. Jordana 38, PL 41-808 Zabrze, Poland Correspondence: Przemys³aw Nowak, e-mail: pnowak@sum.edu.pl

Abstract:

In the present study, the effects of prenatal manganese (Mn) intoxication on the anxiolytic-like effects of diazepam and the 5-HT_1A receptor agonist R-(+)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT) were examined. Wistar dams were exposed to MnCl₂•4H₂O at 5,000 ppm in the drinking water for the duration of pregnancy. On the day of parturition, Mn was discontinued as an additive in the drinking water. Control rats were derived from dams that consumed tap water and had no exposure to Mn. Male offspring were tested at the age of 12 weeks. The anxiolytic-like effect was assessed in an elevated plus maze device and with the Vogel conflict test. The benzodiazepine anxiolytic diazepam (5 mg/kg,ip) increased the percentage of time spent in open arms in control rats (in comparison to saline treatment) (p < 0.05); no such effect was seen in Mn-exposed rats. Conversely, the serotoninergic 5-HT_1Aagonist 8-OH-DPAT (0.3 mg/kg,ip) increased the percentage of time spent in open arms in both experimental groups. In the Vogel drinking test, an anxiolytic-like effect was also observed in both test groups (in controls this was of borderline significance). In contrast, 8-OH-DPAT did not evoke an anxiolytic-like action in control or in Mn-exposed rats in the anticonflict test. In conclusion, findings indicate that gestational Mn exposure attenuated benzodiazepine-mediated anxiolytic-like effects but not those of the 5-HT_1Arecep- tor agonist 8-OH-DPAT.

Key words:

manganese, gestational, exposure, anxiety, diazepam, 8-OH-DPAT, rats

Abbreviations: CNS – central nervous system, GABA – g -aminobutyric acid, Mn – manganese, 8-OH-DPAT – R-(+)-8- hydroxy-dipropylaminotetralin

Introduction

There is a large body of literature on the association between occupational manganese (Mn) intoxication

and neurodegenerative, respiratory, and reproductive effects in humans. The majority of cases have been documented in miners [34]. Mn exposure through welding fumes has been reported to cause a parkin- sonian syndrome, sometimes described as welding fume-related parkinsonism and often misdiagnosed as Parkinson’s disease because of similarities in neurological features [25]. In addition, adverse mood effects and other psychological disturbances of overex- posure to Mn have been described [6].

Pharmacological Reports 2009, 61, 10611068 ISSN 1734-1140

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shown that blood levels of Mn are also increased in patients with impaired liver function [35]. Neonates also accumulate more Mn than adults due to enhanced Mn absorption, an incomplete blood-brain barrier, and little to no biliary excretion until weaning [13]. In- deed, the brain Mn concentration is higher in neonatal vs. adult rats [43], suggesting an increased requirement for Mn during development. However, the higher levels of Mn in neonates may increase the risk of neurotoxicity. Accordingly, Sahni et al. [39] observed that Mn intoxication occurred in young children.

Recent studies have revealed that exposure to envi- ronmental Mn essentially affects a variety of neurotransmitter systems in animals and causes a wide range of long-lasting adverse effects, particularly in the developing brain [1, 18, 23, 44]. In previous studies from our laboratory, we examined the effect of prenatal Mn intoxication on the reactivity of dopamine (DA), serotonin (5-HT) and muscarinic receptors to specific agonists and antagonists applied to adult rat offspring. We demonstrated that prenatal Mn increased the reactivity of DA D₁, DA D₃, and muscarinic receptors but not of 5-HT receptors in adult rats [9]. In a subsequent experiment, we showed that Mn reduced L-3,4-dihydroxyphenylacetic acid (DO- PAC) release in amphetamine-treated rats and diminished DA and 5-HT turnover in the striatum [10]. Fur- thermore, in the same paper we showed that Mn levels in the brain and liver of mothers pre-exposed to Mn at 10,000 ppm for 21 days was significantly increased. Levels of Mn in offspring tissues studied (one day old rats) were 20 times lower than in their mothers. Only some non-significant increases in Mn levels were observed in the brain and liver. This demonstrated that the neurotoxic effects of Mn may occur despite the lack of its accumulation in the neural tissue. In summary, these studies confirm earlier reports on the harmful effects of Mn on nigrostriatal neurotransmission and may, at least in part, explain the neurological symptoms observed in humans.

There are also few recent reports regarding Mn- induced neuropsychological disturbances in humans, including increased anxiety, nervousness, irritability, emotional disturbance, and aggression [5, 6]. Other authors undertaking this issue pointed out that the emotional and other nonspecific neurobehavioral

(CNS) development and mood disturbances in laboratory animals. Conversely, similar to other heavy metals [30], exposure to Mn could alter the vulnerability to anxiolytic-like effects of benzodiazepines, ethanol and other similarly acting agents (results in hyper- or hyposensitivity). Indirect proofs were supplied by Erikson et al. [16] who demonstrated decreased brain tissue levels ofg-aminobutyric acid (GABA) as well as decreased [³H]-GABA uptake in synaptosomes in Mn-exposed rats. Others found that Mn intoxication results in an increase in extracellular GABA concen- trationsvia altered expression of transport and receptor proteins [1].

Taking the above into consideration, the aim of this study was to determine whether gestational Mn intoxication modifies the behavioral responses to diazepam (benzodiazepine receptor agonist) and the 5-HT_1Are- ceptor agonist R-(+)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT) in male rat offspring.

Materials and Methods

Chemicals

MnCl₂•4H₂O was purchased from POCH (Gliwice, Poland), diazepam was from Jelfa (Jelenia Góra, Po- land), and 8-OH-DPAT (HBr) was obtained from Sigma Chemicals (St. Louis, MO, USA).

Animals and treatment

The study was initiated in pregnant Wistar rats weigh- ing 200–220 g at the start of the experiment. Dams were housed in temperature-controlled facilities under a 12 h light : 12 h inverse dark cycle and fed standard rat chow (Altromin-1324, Lage, Germany). From day 1 of pregnancy, when vaginal plugs were found, pregnant rats were housed singly. One group received tap water for drinking, while the second group received tap water containing 5,000 ppm MnCl₂ as drinking water. On the day of parturition, Mn was discontinued. Control and Mn-exposed litters were reared sepa- rately, and weaning occurred on the 21st day after birth. At 12 weeks, experimental testing was initiated.

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The local Bioethical Committee for Animals, Medical University of Silesia approved the experiment (per- mission no 19/06 issued on 01.03.2006). Animals were treated in accordance with the Institutional Ani- mal Care Committee and the principles and guidelines described in the NIH booklet Care and Use of Labora- tory Animals. The number of control and Mn- intoxicated rats in each experiment was 10 per group.

In the present study, diazepam and 8-OH-DPAT were used as pharmacological tools to assess the anxiolytic-like effects in control and Mn-exposed rats.

We chose these agents, because the evidence base for pharmacologic treatment of anxiety disorders is cur- rently greatest for the serotonin 5-HT_1Aagonists (buspirone and gepirone) and benzodiazepines [21, 47].

The doses of each anxiolytic were based on literature reports [20, 26, 28, 38, 46].

Experimental procedures in the elevated plus maze

The elevated plus maze for rats consisted of wood painted black with two opposing open arms (50 × 10 cm) without legs and two opposing open arms of the same dimension but with 40 cm high walls. The arms were attached to a central square (10 cm²) shaped as a plus sign. The entire device was placed 50 cm above the floor. A lamp (20 W bulbs) was placed on the sides above the apparatus, and experiments took place in this dimmed light (100 lux, measured on the central platform). Anxiety-like behavior was measured ac- cording to Pellow et al. [36].

Rats from both test groups were administered either NaCl (0.9%, 1 ml/kg, ip), diazepam (5 mg/kg, ip), or 8-OH-DPAT (0.3 mg/kg, ip). Immediately after injection, rats were returned to their home cage for 60 min before the 5-min plus maze test. Each rat was placed in the central square of the plus maze, facing an enclosed arm. An arm entry was defined when all four paws entered an arm. The following scores were recorded: number of entries into open and closed arms and time spent in both open and enclosed arms.

Scores were presented as open/total time in open arms and absolute open arm entries as anxiety indexes. The absolute number of entries into enclosed arms re- flected general motor activity.

During testing, the observer remained seated qui- etly, approximately one meter from the maze. The maze was cleaned between tests with a mild disinfec- tant (70% ethanol).

Experimental procedures in the Vogel conflict drinking test

A modification of the method of Vogel et al. [45], described below, was employed. Male Wistar rats were water-restricted for three consecutive 24 h periods. At the end of the first and second 24 h period, rats were placed in a test chamber containing a drinking bottle, and they were allowed to drink freely for 15 min. At the end of the third 24-h period, control and Mn- intoxicated rats were injected with 0.9% NaCl (1 ml/kg, ip), diazepam (5 mg/kg, ip), or 8-OH-DPAT (0.3 mg/kg,ip). One hour later, the rats were placed in the test chamber and allowed to drink for 30 s. Imme- diately afterwards, drinking attempts were punished with an electric shock between the grid floor and the drinking spout (0.5 mA; 250 ms) triggered every 20th lick.

Data analysis

Group differences were assessed by a two-way analysis of variance (ANOVA) and the post-ANOVA test of Newman-Keuls. A p value < 0.05 was taken as the level of significant difference.

Results

Elevated plus maze

The benzodiazepine anxiolytic diazepam (5 mg/kg, ip) increased the percentage of time spent in open arms in control rats (vs. saline controls) (two-way ANOVA, groups [F = 2.561, p < 0.118]; substances [F = 18.273, p < 0.0001]; both factors [F = 1.360, p <

0.252]; there was no effect in Mn-exposed rats (Fig. 1, lower panel). Diazepam did not modify the number of entries into open arms in control and Mn-exposed rats (groups [F = 0.071, p < 0.786]; substances [F = 0.729, p < 0.398]; both factors [F = 2.727, p < 0.107]). Con- versely, the number of entries into closed arms was reduced after diazepam in both experimental groups (Fig. 1, upper panel) (groups [F = 2.756, p < 0.105];

substances [F = 51.754, p < 0.0001]; both factors [F = 0.544, p < 0.465]).

Basal anxiety, assessed as the time spent in open arms after saline treatment, was greater in Mn-

Manganese intoxication and anxiety

Adam Kwieciñski and Przemys³aw Nowak

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(control and Mn-exposed) (two-way ANOVA, groups [F = 0.0002, p < 0.987]; substances [F = 24.788, p <

0.0001]; both factors [F = 2.148, p < 0.151]) (Fig. 2, lower panel). 8-OH-DPAT enhanced the number of entries into open arms in Mn-exposed rats; there was no effect in the control group (groups [F = 0.737, p <

0.390]; substances [F = 17.472, p < 0.0001]; both factors [F = 6.639, p < 0.014]) (Fig. 2, upper panel).

Conversely, 8-OH-DPAT increased the number of entries into closed arms in control but not in Mn- intoxicated animals (groups [F = 2.520, p < 0.121];

Vogel conflict drinking test

Diazepam (5 mg/kg, ip) produced an anxiolytic-like effect in the Vogel conflict test in both tested groups, increasing the number of electric shocks in Mn- exposed rats (2.00 ± 0.71 saline vs. 6.10 ± 0.17 shocks) (Fig. 3, upper panel). A similar trend was obtained in control groups (2.70 ± 1.16vs. 6.50 ± 2.59) (two-way ANOVA, groups [F = 0.127, p < 0.728];

Fig. 1. Percentage of time spent in open arms and the number of entries into open and closed arms of the elevated plus maze after saline (1 ml/kg, ip) or diazepam (5 mg/kg) treatment of adult rats that were prenatally exposed to Mn (n = 10). * p < 0.05; control (1) vs. control + diazepam (3)

Fig. 3. Numbers of shocks received by prenatal Mn-exposed rats after ip saline (1 ml/kg), diazepam (5 mg/kg) or 8-OH-DPAT (0.3 mg/kg) (n =10). * p < 0.05; Mn (2) vs. Mn + diazepam (4) Fig. 2. Percentage of time spent in open arms and the number of en-

tries into open and closed arms of the elevated plus maze after saline (1 ml/kg, ip) or 8-OH-DPAT (0.3 mg/kg, ip) treatment of adult rats prenatally exposed to Mn (n =10). * p < 0.05; control (1) vs. control + 8-OH-DPAT (3); Mn 5,000 ppm (2) vs. Mn 5,000 ppm + 8-OH-DPAT (4); # p < 0.05; control (1) vs. Mn 5,000 ppm (2)

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In contrast, 8-OH-DPAT (0.3 mg/kg, ip) failed to evoke an anxiolytic-like action in either control or Mn-exposed rats in the Vogel conflict drinking test.

The number of electric shocks control rats received after salinevs. 8-OH-DPAT injection was 2.20 ± 0.66 vs. 2.50 ± 1.12, respectively. In Mn-intoxicated rats, the number of shocks was 2.60 ± 0.86 (saline)vs. 3.10

± 1.05 (8-OH-DPAT) (Fig. 3, lower panel) (two-way ANOVA, groups [F = 0.287, p < 0.595]; substances [F

= 0.183, p < 0.670]; both factors [F = 0.01, p < 0.915]).

Discussion

In a long series of studies published over a period of 10 years, we have shown that gestational Mn intoxication, similar to that with other heavy metals, such as cadmium, lead, and mercury, has a prominent influence on a variety of brain functions in animals (e.g., cognitive, behavioral and motor abnormalities) result- ing from impaired dopaminergic and cholinergic neurotransmission [8–12, 15, 22, 29–33, 40–42]. Some recent reports demonstrated that an excess of Mn may disturb GABA-ergic neurotransmission in the brains of laboratory animals [1, 16]. Since GABA is deeply involved in the anxiety behavior, it may suggest that prenatal Mn may have long-lasting implications on the emotional behavior in rats and, consequently, alter the susceptibility of the offspring to drug administration. It is worth knowing that neurotoxins and other chemicals, such as methylmercury, lead, antiepileptic drugs and ethanol, administered during the brain development period affect the vulnerability to different kinds of drugs applied in adulthood [3, 4, 19, 27].

The present study showed that prenatal Mn perma- nently alters the susceptibility to the anxiolytic-like effects of diazepam (benzodiazepine) but not to those of 8-OH-DPAT (5-HT_1Aagonist) in rats. We demonstrated that Mn increased basal anxiety (after saline), as shown in the elevated plus maze test. This contrasts with findings by Ponzoni et al. [37], but it is in agree- ment with clinical observations [6]. Diazepam pre- treatment increased the percentage of time spent in open arms in control rats, and there was no effect in Mn-exposed rats. It is notable that diazepam simulta- neously suppressed locomotor activity in both test

groups, as a decrease in the number of entries into closed arms was recorded. Despite that action, an anxiolytic-like effect was still observed in control animals. The above suggests that there is diminished sensitivity to the diazepam anxiolytic-like effect in Mn- treated rats. Conversely, in the Vogel conflict test, a significant increase in the number of electric shocks was received by Mn-exposed rats (i.e., an anticonflict effect). A similar trend was obtained in control groups. To the best of our knowledge, there are no literature data on this topic. In a recently published study, we found that prenatal lead acetate (250 ppm) intoxication diminished sensitivity to the diazepam anxiolytic-like effect in rats [30]. These data contrast with the present findings, but one must recognize that Mn and lead are different xenobiotics, each with a dis- tinct neurotoxic mechanism.

In the present study, we further demonstrated that 8-OH-DPAT produced an increase in the percentage of time spent by control and Mn-intoxicated rats in the open arms. 8-OH-DPAT also enhanced the number of entries into open arms (regarded as the second index of anxiety-like behavior) in both tested groups (although non-significantly in controls). It must be added that 8-OH-DPAT likewise increased the number of entries into closed arms (p < 0.05 in control rats), suggesting that the behavioral effect observed in control rats was due, at least in part, to an increase in exploratory activity. To summarize, gestational Mn exposure did not affect the anxiolytic-like effects mediated by a 5-HT_1Areceptor agonist.

Astonishingly, in the Vogel drinking anticonflict test, 8-OH-DPAT did not have an effect. On the other hand, one must recognize that 5-HT_1Areceptors are presynaptic receptors (on the soma and dendrites of 5-HT neurons of midbrain raphe nuclei) and inhibitory auto- or postsynaptic receptors on 5-HT neurons in forebrain regions [24]. The respective roles of (pre- and postsynaptic) 5-HT_1Areceptors in the control of behavior in the Vogel conflict test and other para- digms is a complex issue to which an answer depends upon the model (type of anxiety), serotoninergic tone, gender, level of stress of the drug under study as well as its dose and the site of administration. It is likely that the dose of 8-OH-DPAT used in the elevated plus maze was adequate for testing but too high to properly respond to a conflict situation (Vogel conflict) [26].

The molecular mechanism involved in the diazepam-mediated anxiolytic-like abnormalities of Mn- intoxicated rats remains difficult to define. As men-

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[16]. In addition, Mn intoxication results in an in- crease in extracellular GABA concentrations [1]. This is in line with the work of Drapeau and Nachshen [14], who found that the addition of Mn to K-rich so- lutions stimulated the release of the neurotransmitters 5-hydroxytryptamine and GABA but not of acetyl- choline from striatal synaptosomes. Others demonstrated an increase in the ratio of GABA to glutamate, indicating enhanced inhibitory transmission in the brain of rats after maternal exposure to a diet supple- mented with excess levels of Mn [18]. Xiao et al. [48]

provided electrophysiological evidence that another heavy metal (Pb²⁺) inhibited action potential-dependent GABA release by inhibiting presynaptic voltage- gated calcium channels. Braga et al. [7] also showed that Pb²⁺increases the frequency of GABA- and glutamate-mediated miniature postsynaptic currents (MPSCs) recorded by the patch-clamp technique with cultured hippocampal neurons. Given that synaptic activity is a key mechanism for the establishment of stable synaptic connections early in development, it is possible that, by interfering with spontaneous transmitter release, heavy metals evoke long-lasting effects on neuronal maturation and plasticity. Since GABA and serotonin are particularly involved in the CNS anxiety effects of diazepam and 8-OH-DPAT, behavioral consequences of Mn exposure during the gestational period could be related to changes in the development of these neuronal phenotypes.

In conclusion, this is the first report demonstrating that gestational Mn intoxication diminishes the anxiolytic-like effects of benzodiazepine (diazepam) without having an effect on the 5-HT_1A-mediated anxiety behavior. Accordingly, prenatal Mn exposure has a great impact on the GABA-ergic system in rats.

Acknowledgment:

This study was supported by grant 2 PO5D 066 27 from the Ministry of Education and Science, Warszawa, Poland.

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Gestational manganese intoxication and anxiolytic-like effects of diazepam and the 5-HT

Gestational manganese intoxication and anxiolytic-like effects of diazepam

and the 5-HT 1A receptor agonist 8-OH-DPAT in male Wistar rats

and the 5-HT _1A receptor agonist 8-OH-DPAT in male Wistar rats