Motor hyperactivity caused by a deficit in dopaminergic neurons and the effects of endocrine disruptors: a study inspired by the physiological roles of PACAP in the brain

Abstract

Recent studies have revealed that the pituitary adenylate cyclase-activating polypeptide (PACAP) might act as a psychostimulant. Here we investigated the mechanisms underlying motor hyperactivity in patients with pervasive developmental disorders, such as autism, and attention-deficit hyperactivity disorder (ADHD). We studied the effects of intracisternal administration of 6-hydroxydopamine (6-OHDA) or endocrine disruptors (EDs) on spontaneous motor activity (SMA) and multiple gene expression in neonatal rats. Treatment with 6-OHDA caused significant hyperactivity during the dark phase in rats aged 4–5 weeks. Motor hyperactivities also were observed after treatment with endocrine disruptors, such as bisphenol A, nonylphenol, diethylhexyl phthalate and dibutyl phthalate, during both dark and light phases. Gene-expression profiles produced using cDNA macroarrays of 8-week-old rats with 6-OHDA lesions revealed the altered expression of several classes of gene, including the N-methyl-d-aspartate (NMDA) receptor 1, glutamate/aspartate transporter, γ-aminobutyric-acid transporter, dopamine transporter 1, D4 receptor, and peptidergic elements such as the galanin receptor, arginine vasopressin receptor, neuropeptide Y and tachykinin 2. The changes in gene expression caused by treatment with endocrine disruptors differed from those induced by 6-OHDA. These results suggest that the mechanisms underlying the induction of motor hyperactivity and/or compensatory changes in young adult rats might differ between 6-OHDA and endocrine disruptors.

Introduction

During the 1990s, we investigated the physiological profile of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rodent central nervous system and revealed that this peptide might have physiological functions as a neurotransmitter/modulator [1], [2], [3], [4], [5], [6], [7]. We also demonstrated that PACAP stimulates neurotransmitter release [3], [8]. Intracerebroventricular injection of PACAP in adult rats caused behavioural hyperactivity and counteracted reserpine-induced hypothermia [9], suggesting that PACAP might act as an endogenous psychostimulant. Interestingly, PACAP-knockout mice also showed behavioural hyperactivity [10]. The contradictory effects of PACAP on motor activity prompted us to study the mechanisms underlying hyperkinetic disorder.

The etiology of attention-deficit hyperactivity disorder (ADHD) and pervasive developmental disorders, including autism, is not yet clear. Patients with these disorders display motor hyperactivity, especially in childhood, which is sometimes referred to as ‘hyperkinetic disorder’. In 1976, Shaywitz et al. [11], [12] developed the first animal model of hyperkinetic disorder using lesions in dopamine (DA) neurons, which were caused by the intracisternal administration of 6-hydroxydopamine (6-OHDA) to rats during the neonatal period. The animals showed increased spontaneous motor activity (SMA) during the juvenile period, which was attenuated by psychostimulant drugs [11], [13], [14], [15]. Psychostimulants, such as methamphetamine or methylphenidate, are known to enhance extracellular DA concentration and can attenuate the motor hyperactivity in hyperkinetic disorders [16], [17]. The mechanisms underlying the different effects of psychostimulants on motor activity are not clear. Furthermore, it is not known whether the mesocorticolimbic DA system shows hypo- or hyperfunction in hyperkinetic disorders [18]. However, the inconsistent effects of psychostimulants suggest the involvement of DA neurons in hyperkinetic disorders.

Several recent studies have focused on the effects that environmental chemicals derived from industrial products have on living organisms. A number of these compounds show oestrogen-like activity, and can disrupt the endocrine system and reproduction [19]; such chemicals are known as ‘endocrine disruptors (EDs)’. Polychlorinated biphenyls (PCBs) have been shown to disrupt neuronal development [20], [21]. Mori et al. [22] detected dioxins, PCB, bisphenol A and heavy metals in the human umbilical cord and cord serum. EDs might therefore be transferred from the mother to the fetus via the placenta, and could have effects on brain function; for example, fetal exposure to bisphenol A has been reported to induce aggressive behaviour in mice [23], [24].

In the present study, we investigated the effects of neonatal treatment with EDs, including bisphenol A, nonylphenol, diethylhexylphthalate (DEHP) and dibutylphthalate (DBP), on SMA compared with the effects of neonatal 6-OHDA lesions in rats aged 4–5 weeks. Changes in gene expression might vary during this period as a consequence of alterations in the expression of early-response genes and as a response to hyperactivity itself. Therefore, gene expression also was examined in the brains of 8-week-old rats using a cDMA macroarray (BD Biosciences Clontech, Palo Alto, CA, USA). This method provides results similar to those obtained by reverse transcription-linked PCR [25], [26], [27], [28]. We focused our investigation on the striatum and the midbrain, as these regions contain major dopaminergic cell bodies and terminals.