Differential gene expression in a rat model of depression based on persistent differences in exploratory activity

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Abstract

Affective disorders are often accompanied by changes in motivation and anxiety. We investigated the genome-wide gene expression patterns in an animal model of depression that separates Wistar rats belonging into clusters of persistently high anxiety/low motivation to explore and low anxiety/high motivation to explore (low explorers and high explorers, LE and HE, respectively), in three brain regions previously implicated in mood disorders (raphe, hippocampus and the frontal cortex). Several serotonin-, GABA-, and glutamatergic genes were differentially expressed in LE- and HE-rats. The analysis of Gene Ontology biological process terms associated with the differentially regulated genes identified a significant overrepresentation of genes involved in the neuron development, morphogenesis, and differentiation; the most enriched pathways from the Kyoto Encyclopedia of Genes and Genomes were the Wnt signalling, MAPK signalling, long-term potentiation, and long-term depression pathways. These findings corroborate some expression data from other models of depression, and suggest additional targets.

Introduction

In recent years, animals bred or preselected for individual variability are proving to be increasingly useful in modelling aspects of human psychopathology, particularly anxiety-related disorders and addiction (Piazza et al., 1989, Landgraf and Wigger, 2002, Kabbaj, 2006, White et al., 2007, Pawlak et al., 2008). Differences in behavioural traits are accompanied by differences in monoaminergic neurotransmission and sensitivity to pharmacological manipulations (Piazza et al., 1989, Hooks et al., 1992, Rouge-Pont et al., 1998).

Behaviour of animals in unfamiliar environments is always a function of their natural curiosity and fear of the unknown (Harro, 1993); alterations in these motivational processes—low motivation and high anxiety—are regarded as core features of depression. The exploration box test that measures novelty-related behaviour in rats typically yields a distribution that strongly deviates from normality and facilitates separation of rats into groups of high motivation to explore/low neophobia and low motivation to explore/high neophobia (HE and LE, respectively), with relatively few animals occupying the middle ground. These differences in spontaneous exploratory activity levels are stable over time, persist with repeated testing and predict activity in other behavioural tests (Mällo et al., 2007). Compared to HE-animals, LE-rats are less active and more anxious in the elevated plus-maze and display passive coping strategies in the forced swimming test. In the fear conditioning test, LE-animals acquire a more persistent association between neutral and stressful stimuli (Mällo et al., 2007). The LE-rats also develop behavioural sensitization to repeated amphetamine treatment more readily (Alttoa et al., 2007). The HE- and LE-animals spend a similar amount of time in active social interaction, thus do not differ in their social anxiety (Mällo et al., 2007). The differences in novelty-related behaviour in HE- and LE-rats cannot be explained by the variations in general locomotor activity, as in the elevated plus-maze the number of entries into the closed arm of the maze is similar in HE- and LE-rats, and there are no differences in their activity in a novel home-cage like environment (Mällo et al., 2007).

The distinct behavioural profiles of HE- and LE-rats are also reflected in the differences in neurochemistry. We have shown previously that the HE-animals have higher basal and stimulated extracellular dopamine levels in the striatum but not in the nucleus accumbens (Mällo et al., 2007), and a higher proportion of dopamine-D2 receptors in the functional high-affinity state (Alttoa et al., 2009). There are also differences between high and low exploring groups in the properties of the serotonergic system in the prefrontal cortex and hippocampus (Mällo et al., 2008) that may underlie their anxiety-related behaviours. Specifically, although serotonin release in the prefrontal cortex and dentate gyrus in baseline conditions is similar in HE- and LE-rats, the number of serotonin transporter binding sites in the prefrontal cortex is higher in the LE-animals. Thus, after blocking the serotonin transporters in the prefrontal cortex by a local infusion of citalopram, the serotonin release in that brain region is significantly increased in the LE group (Mällo et al., 2008). The drug-free, amphetamine-stimulated and amphetamine-sensitized behaviour of the HE- and LE-rats is differentially regulated by noradrenaline, as revealed after a partial noradrenergic denervation of locus coeruleus projections with a selective neurotoxin DSP-4 (Alttoa et al., 2005, Alttoa et al., 2007). Furthermore, the two groups have distinct cerebral metabolic activity in areas that are involved in defensive behaviours and cognitive processing of sensory stimuli (Matrov et al., 2007). Thus, the LE-rats demonstrate a behavioural profile that is characterized by low motivation to explore, high anxiety, high vulnerability to stress and cognitive rigidity—the core symptoms of depression, and thus, resembles a ‘depressed’ phenotype. Differences in monoaminergic neurotransmission between LE- and HE-rats are also compatible with the notion of LE-rats being an animal model of depression.

In order to investigate the genetic differences between the ‘healthy’ and the ‘depressed’, and identify novel mechanisms that contribute to or underlie the observed phenotype, genome-wide microarrays are the method of choice. Thus, the aim of the current study was to identify the genes and molecular pathways that contribute to the differences between HE- and LE-rats by examining the gene expression profiles in three brain regions most consistently implicated in the pathogenesis of depression and anxiety (the raphe, the hippocampus and the frontal cortex) using Illumina RatRef-12 BeadChips.

Section snippets

Animals

Male Wistar rats weighing 275–472 g (Scanbur BK AB, Sweden) were housed four per cage in standard polypropylene cages in a light controlled room (12-h light/dark cycle; lights on at 7:00 a.m.) maintained at 22 °C. Food and water were available ad libitum. All behavioural experiments were carried out between 13:00 and 19:00. All experiments were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Ethics Committee of the University of

Individual differences in spontaneous exploratory activity

On the second exposure to the exploration box, which was the basis for the differentiation between high exploring (HE) and low exploring (LE) animals, none of the LE-rats emerged from the small chamber of the exploration box, while the mean number of exploratory events of the twelve randomly selected HE-animals was 216 (SEM 6.78). Thus, the HE-rats were significantly more active than the LE-animals in their spontaneous novelty-related behaviour in the exploration box (Mann–Whitney U test, p < 

Discussion

In the present study, we report gene expression data from three brain regions of rats with persistently high or low exploratory activity. The focus on the raphe nuclei, the hippocampus and the frontal cortex, comprising the ascending serotonergic pathway, was determined by their role in the pathogenesis and pathophysiology of anxiety and depression.

Web resources

The URLs for resources presented herein are as follows:

Role of the funding source

This study was supported by the European Commission Framework 6 Integrated Project NEWMOOD (LSHMCT-2004-503474) and the Estonian Ministry of Education and Science Project No. 0180027. The funding sources were not involved in the data analysis and the preparation of the manuscript.

Contributors

Aet Alttoa performed the behavioural experiments, RNA extractions, KEGG and GO analyses, and contributed to the first draft of the paper. Kadri Kõiv performed the behavioural experiments and RNA extractions. Timothy A. Hinsley performed the microarray data normalisation, conceived the bioinformatical analyses, and performed the REEF analysis. Andrew Brass supervised the bioinformatical analyses. Jaanus Harro oversaw the project, and data collection and analysis, and contributed to the first

Conflict of interests

None for any author.

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