Multiple evidence of a segmental defect in the anterior forebrain of an animal model of hyperactivity and attention deficit

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Abstract

Molecular biology and microscope imaging techniques were used to map putative neural substrates of hyperactivity and attention deficit in an animal model, the juvenile prehypertensive male spontaneously hypertensive rat (SHR). We have studied in anterior forebrain sections of SHR and Wistar-Kyoto Normotensive (WKY) controls the spatial distribution of neural markers such as: (i) dopamine (DA) D-1 and D-2 receptor families by radioligand binding studies; (ii) the Ca2+/calmodulin-dependent protein kinase II (CaMKII); and (iii) the transcription regulators of gene expression (TFs) c-FOS and JUN-B by Immunocytochemistry (ICC). Microcomputer-assisted high-resolution image analysis showed in the SHR a higher density of DA D-1 receptors and a lower density of D-3 autoreceptors paralleled by a reduced number of elements positive for CaMKII and TFs in a restricted segment of the anterior forebrain that included the most rostral portions of the caudate-putamen, pole and shell of the nucleus accumbens and olfactory tubercle. The differential rostro-caudal distribution of D-1 receptors and D-3 autoreceptors is discussed in the light of current hypotheses of DA mesocorticolimbic system functioning. In addition, the segmental defect was partially reversed by subchronic treatment with a DA re-uptake blocker, Methylphenidate (MPH; 3 mg/kg) and by environmental stimulation during the fifth and sixth postnatal week. The findings are consistent with the role of genetic determinants and environmental factors in the phenotypic expression of hyperactivity and attention deficit.

Introduction

The frontal and posterior parietal cortices are thought to be the neural sites responsible for attentive processes pertaining to the motivational/emotional and the visuo-spatial domain, respectively [1], [2].

The major pathology of attentive processes is represented by Attention-Deficit Hyperactivity Disorder in children (ADHD; see Ref. [3] for a review). ADHD is a neurodevelopmental disorder characterized by attentional problems, hyperkinesis, restlessness, and disturbances in timing. It affects mostly male children [3] with a high genetic component as demonstrated by family, twin and adoption studies, and the allelic polymorphism of the dopamine (DA) D-4 receptor gene and the DA transporter protein (DAT) [4].

The most widely studied model system for ADHD is represented by the spontaneously hypertensive rat (SHR) [5]. Some of the behavioral problems of ADHD children and SHR have been linked to dysfunction of the mesocorticolimbic DA system that innervates the nucleus accumbens, and the prefrontal cortex (see Ref. [6] for a review) and is a major component of a multiple system involved in attention and reinforcement mechanisms [7], [8]. DA is known to bind to a heterogeneous class of synaptic and extra-synaptic receptors of the D-1 and D-2 subfamilies [9], thus increasing the signal-to-noise ratio [10].

We have focused upon the most anterior portion of the forebrain including structures such as the nucleus accumbens (ACB), that constitutes an anatomical and physiological unit, which has been divided into three subterritories: the “rostral pole”, the “core”, and the “shell” [11]. ACB is the target of a wide glutamate innervation from prefrontal cortex, midline intralaminar thalamic nuclei, hippocampus, basal amygdala nuclei [12], [13], as well as an extensive DA projection from the ventral tegmental area (VTA), serotonin fibers from raphĕ nuclei and histamine fibers from the posterior hypothalamus. In turn, ACB sends efferent to all mesencephalic DA sources [14]. The “core” projects to the prefrontal cortex, while the shell and rostral pole have a wide output to the lateral hypothalamus, basal amygdala and hippocampus [14].

The Ca2+/calmodulin-dependent protein kinase II (CaMKII; see Ref. [15] for a review) is a member of the serine/threonine kinase family that constitutes the main protein of the postsynaptic density, but is present also at the presynaptic terminal [16]. CaMKII is a multifunctional protein that acts as one of the major molecular devices in signal transduction, on which converge several signals derived from the cross-talk between different second messenger systems.

Transcription factors, such as c-FOS and JUN-B, are members of a numerous and heterogeneous gene superfamily, also referred to as immediate early genes (IEG; [17]). They code for proteins, which mediate many of the long-term neuronal responses to trans-synaptic signals [18]. In fact, they dimerize and enter the nucleus where they bind to specific DNA binding sites, such as the AP-1 site [17] for FOS and JUN-B and the SP-1 site for ZIF/268.

Therefore, using the D-1 and D-2 dopamine receptor types, CaMKII, and the transcription factors c-FOS and JUN-B as markers of neuronal activity, the aim of this study was to investigate their spatial distribution in the antero-posterior plane in the juvenile SHR and Wistar-Kyoto Normotensive (WKY) controls.

Section snippets

Animals

Juvenile (4-week-old) male SHRs and WKY controls (Charles-River, Italy) were housed in standard cages during a 2-week period for acclimatization and handling. All other parameters fulfilled the requirements of the “Guide for the Care and Use of Laboratory Animals” by the National Research Council, implemented by EU rules.

Procedures

Rats were either given no injection (basal conditions) or given a daily intraperitoneal injection of vehicle (vehicle control) or methylphenidate-HCl (3 mg/kg) for 14 days. Rats

Dopamine receptor autoradiography

A higher density of DA D-1 binding sites and a lower density of D-3 autoreceptors was demonstrated in the SHR, by saturation analysis (Fig. 1) for the D-1 sites by 3H-SCH23390 and by competition analysis with 3H-raclopride as ligand and 7-OH-DPAT as nonlabeled displacer for the D-3 sites, respectively (see Table 1 for a summary of the results). This was assessed in a pool of sections from anterior but not posterior portions of different forebrain structures. A significantly higher level of D-1

Discussion

The multiple high resolution light microscope imaging probe analysis used in this series of studies revealed the existence of a segmental defect in the anterior forebrain of a genetic model of hyperactivity and attention deficit, i.e. the juvenile prehypertensive SHR rats. This defect comprises the most rostral portion of the dorsal and ventral striatum and the olfactory tubercle.

This has been demonstrated by multiple neuronal markers, namely: (1) an increased density of dopamine D-1 [25] and

Acknowledgements

This project has been supported by EU HCM grant (1994–96) and by Telethon-Italy grant E.513 (1997-98).

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