Invited Symposium: What Can Genetic Models Tell Us About Attention-Deficit Hyperactivity Disorder (ADHD)?
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Introduction
This presentation discusses an investigation of the neural networks of behavioral excitability and attention deficits of two genetic models, Naples-High Excitability (NHE) rats and Naples Low-Excitability (NLE) rats, with random-bred (NRB) rats as controls (Cerbone, Pellicano, & Sadile, 1993b). NHE rats are hyperexcitable and show attention deficits in behavioral paradigms. The Naples High-Excitability and Low-Excitability rat lines were thus named for their respective behavior on spatial novelty tasks, such as a Lát maze, a hexagonal tunnel maze and an asymmetric radial arm maze. These rats' reactivity to novelty cannot be attributed to general motor hyperactivity as baseline motor activity between strains is not significantly different (Cerbone, Patacchioli, & Sadile, 1993a). Therefore, the NLE and NHE strains may be used as animal models of important features of attention deficit hyperactivity disorder (ADHD) in children (Swanson et al., 1998).
The studies reported here analyze the regional neuroanatomical profile from functional imaging with cytochrome oxidase (C.O.) quantitative histochemistry (Papa et al., 1998; Gonzalez-Lima & Cada, 1998). C.O. has been demonstrated to reflect long-lasting changes in tissue metabolic capacity induced by neuronal activity and learning and memory processes (Poremba, Jones & Gonzalez-Lima, 1998ab). Cytochrome oxidase (C.O.) is an integral transmembrane protein of the inner eukaryotic mitochondrial membrane. It acts as a terminal enzyme in the electron transport chain which catalyses transfer of electrons from its reduced substrate, ferrocytochrome c, to molecular oxygen to form water. C.O. is coupled to the process of oxidative phosphorylation, which is responsible for the generation of ATP. This energy device is then used for active ion pumping to maintain the resting membrane potential, fast axoplasmic transport, and synthesis of macromolecules and neurotransmitters. The maintenance of ion balance constitutes the major energy-consuming function of neurons. In fact, increased neuronal activity promotes heightened cellular respiration in order to generate more ATP for the accelerated activity of Na/K transporting ATPase (Wikstrom, Krab, & Saraste, 1981).
Wong-Riley (1989) postulated that the level of neuronal C.O. activity should correlate positively with the functional activity level of neurons. Much evidence shows a correlation between C.O. activity and functional activity of various neural regions under normal conditions. There are adaptive adjustments of C.O. activity in response to experimentally induced changes in neuronal functional activity (Gonzalez-Lima & Jones, 1994; Wong-Riley, 1989; Wong-Riley et al., 1993; Gonzalez-Lima, 1992).
In the Naples lines, quantitative C.O. histochemistry was carried out under comparable conditions to monitor long-lasting changes in neural networks. Baseline (unstimulated) conditions were chosen because hyperactive rodent models as well as ADHD children show attentional deficits at low motivational levels (van der Meere, Vreeling, & Sergeant, 1992).
Materials and Methods
ANIMALS.
Male rats of the NHE, NLE and random-bred controls (NRB) were from our animal colony in Naples, Italy. The experiments were carried out on adult (six months old) animals. Rats were housed in groups of two in standard cages and put under standard conditions on a 12:12 light-dark cycle. The whole brain was removed and frozen in cold isopentane and stored at -80 °C. The brains were air-freight shipped in dry ice to Austin, Texas, for C.O. quantitative histochemical analysis.
QUANTITATIVE C.O. HISTOCHEMISTRY.
Brains were taken from 15 naive rats selectively bred for activity level on maze tasks (Sprague-Dawley derived). After decapitation, brains were removed without perfusion or fixation and frozen in isopentane. The brains were sectioned with a cryostat at 40 µm sections. The sections were thaw-mounted to slides and kept frozen until staining. The staining procedure first required a series of chemical exposures: 0.1M phosphate buffer with 10% sucrose (4 changes, 5 min. each), a preincubation with Tris buffer (10 min. each), and a 0.1M phosphate buffer rinse. Then the slides were incubated for 60 minutes with DAB (preceded by bubbling oxygen into DAB for 5 min.) at 37°C while stirring. The slides then sat in buffered formalin (10%) with 10% sucrose for 30 min before a series of dehydration baths in 30, 50, 70, 90, 95 (two baths) and 100 (three baths) percent ethanol, with 5 min in each bath. The slides then went through a series of three xylene baths of 5 min each before being permounted and coverslipped. (For details, see Gonzalez-Lima and Cada, 1998.)
C.O. reactivity was measured in optical density (OD) units using an image-processing system: high-gain camera, Targa image capture board, computer running JAVA software (Jandel Scientific), Sony color monitor and DC-powered illuminator. C.O. activity was measured spectrophotometrically at 37°C, pH 7, using calibration standards with brain tissue homogenates, and transformation of OD into C.O. activity units (Cada et al., 1995). Three data points were taken in each region of interest for each of the three sections selected per brain region. A vertical OD reading was taken through the layers of the hippocampus giving average sample readings for those layers.
Results
Cytochrome Oxidase Activity in Naples NHE, NLE and NRB Rats. The significant (p below 0.05) results are summarized in three parts, corresponding to regions of the hippocampal formation (Part 1), cerebral cortex (Part 2), and subcortical nuclei (Part 3).
(1) In the hippocampal formation, the outer granular cell layer of the dentate showed significant differences between the NLE and NHE groups. In fact, this was the only region showing a significant difference between these groups. This difference was mainly due to the outer granular cell layer of the dentate showing a localized decrease in C.O. activity in NLE. In addition, the entorhinal cortex (outer layers) showed an increased C.O. activity in NHE vs. NRB.
(2) In the cerebral cortex, the medial frontal cortex showed an increased C.O. activity in NLE vs. NRB. The posterior parietal cortex (outer and middle layers) showed a decreased C.O. activity in NHE. The perirhinal cortex (in dorsal, middle and ventral parts) showed decreased C.O. activities in NHE vs. NRB. The dorsal perirhinal cortex also showed a decreased C.O. activity in NLE vs. NRB. Note that all differences in the cerebral cortex are in comparison with the NRB group. In NLE, the increase in C.O. activity was only in medial frontal cortex, a region implicated in depressive behavior.
(3) In subcortical nuclei, most regions showed no differences, including nucleus accumbens and caudate-putamen. However, the cortical amygdala showed a decreased C.O. activity in NHE vs. NRB, corresponding to a region whose dysfunction is traditionally linked to increased emotionality.
These results suggest that the neural networks for behavioral excitability are mediated by neural systems differentiating the NHE and NLE rats from other hyperactive rats. The NHE rats are not hyperactive under basal (nonincentive) conditions lacking novelty such as running wheel activity. Instead, NHE and NLE rats are hyperreactive and hyporeactive to spatial novelty, respectively. These results show that quantitative C.O. histochemistry can successfully discriminate the neural regions mediating different behaviors in genetic animal models.
Discussion and Conclusion
The major conclusion of these experiments with the Naples lines is that they differ in metabolic capacity in neural regions thought to be involved in the processing of limbic and spatial information. Interestingly, of all the 71 regions analyzed (including sensory and motor regions) only prefrontal and limbic regions showed C.O. metabolic differences. Hippocampal metabolic differences between the NLE and NHE were found in the granular cell layer of the outer blade of the dentate gyrus. This was the only region with a significant difference between the experimental strains (NLE vs. NHE) and supports the conclusion that these strains differ in hippocampal function (Cerbone et al., 1993). Granule cells of the dentate gyrus show postnatal cell division in rats, and are particularly vulnerable to manipulations such as X-irradiation in infancy (Altman, 1986). Indeed, granule cell hypoplasia in the X-irradiated infant rat leads to hyperreactivity and learning deficits which have been hypothesized to simulate deficits found in ADHD children (Diaz-Granados et al., 1994).
Other regions may interact with the hippocampal granular cells to modify neural network operations in the Naples rats. For example, greater C.O. activity of entorhinal cortex was found in the high behavioral reactivity NHE group as compared to NRB controls. Entorhinal influences on hippocampal function may be related to hyperreactivity (Cerbone et al., 1993). Genetic selection in NLE rats led to greater metabolic capacity in the medial frontal cortex correlated to low behavioral reactivity. The medial frontal cortex has been related to depressive behavior in 2-deoxyglucose autoradiographic studies (Caldecott-Hazard & Weissman, 1992).
In addition, lower metabolism in the posterior parietal cortex, the perirhinal cortex, and the cortical amygdala of both NHE and NLE suggests a potential learning impairment because these regions form a network related to the differentiation of associative effects of conditioned stimuli in rats (Gonzalez-Lima & Scheich, 1986; McIntosh & Gonzalez-Lima, 1994).
As the ADHD syndrome in children has been described with variants showing prevailing attention or activity deficits (Barkley, DuPaul, & McMurray, 1991; Goodyear & Hynd, 1992), similarly, animal models can reproduce and feature the main aspects of the clinical heterogeneity. In fact, NLE and NHE rat lines show altered non-selective attention, as measured by the duration of rearing episodes, but NHE rats are hyperreactive and NLE rats are hyporeactive. Therefore, the NHE might model the ADD-plus variants of ADHD (Aspide et al., 1997). These data support the hypothesis that NLE/NHE rats may be an appropriate model for studying genetically altered limbic and cortical regions related to impaired emotional processing. Altogether, the results support the involvement of limbic-cortical networks in the forebrain in attentive processes and impulsiveness.
ACKNOWLEDGEMENTS. This research has been supported by EU Human Capital and Mobility contract ERBCHRXCT930303, by Telethon-Italy grant #E513, by NIH grant NS37755 and by NSF grant IBN9222075. The assistance of D. Hu , K. Nixon, and J. Shumake is gratefully acknowledged.
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