SYNAPSE 00:00–00 (2014)
Asymmetrical Expression of BDNF and NTRK3 Genes in Frontoparietal Cortex of Stress-Resilient Rats in an Animal Model of Depression SARA FARHANG,1,2 JALEH BARAR,2 ALI FAKHARI,1 MEHRAN MESGARIABBASI,3 SAJJAD KHANI,2 YADOLLAH OMIDI,2 AND ALIREZA FARNAM1* 1 Clinical Psychiatry Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 2 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran 3 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
KEY WORDS
depression; lateralization; gene expression; BDNF; NTRK3; chronic mild stress
ABSTRACT The current study is based on the “approach–withdrawal” theory of emotional regulation and lateralization of brain function in rodents, which has little been studied. The aim was to indentify asymmetry in hemispheric genes expression during depression. Depressive-like symptoms were induced in rats using chronic mild stress protocol. The sucrose consumption test was performed to identify the anhedonic and stress-resilient rats. After decapitation, RNA was extracted from frontotemporal cortex of both hemispheres of anhedonic and stress-resilient rats. The pattern of gene expression in these samples was compared with controls by real-time polymerase chain reaction. A linear mixed model analysis of variance was fitted to the data to estimate the effect of rat line. From the total of 30 rats in the experimental group, five rats were identified to be anhedonic and five were stress-resilient, according to the result of sucrose-consumption test. BDNF and NTRK-3 were expressed at significantly lower levels in the right hemisphere of anhedonic rats compared with stress-resilient rats. No significant difference was found between left hemispheres. Hemispheric asymmetry in the level of gene expression was only observed for the BDNF gene in stressresilient rats, upregulated in right hemisphere compared with the left. Expression of NTRK3, HTR2A, COMT, and SERT was not lateralized. There was no significant asymmetry between hemispheres of anhedonic rats. This study supports the evidence for the role of genes responsible for neural plasticity in pathophysiology of depression, emphasizing probable hemispheric asymmetry at level of gene expression. Synapse C 2014 Wiley Periodicals, Inc. 00:000–000, 2014. V
INTRODUCTION Major depressive disorder is common (Roca et al., 2009), costly (Gibson et al., 2010), and recurrent (Curry, 2011), whereas not all of the patients’ response is the same for the therapy regiments (Little, 2009). Heterogeneity of clinical manifestations and a very wide range of response to different classes of medications indicate a vast need for further investigations of the etiological factors. There is a consensus that a biopsychosocial model is needed to explain its etiology (i.e., existence of a vulnerability activated by stressful life events). However, despite several studies in this area, results are not yet conclusive. Results obtained from different investigations searching for the culprit genes for depression are usually Ó 2014 WILEY PERIODICALS, INC.
complex (Sadock et al., 2009). Identification of genes for a specific endophenotype seems to be the most logical solution. Depression is characterized by a change in mood, including dysphoria and anhedonia (inability to experience pleasure) as well as several behavioral symptoms. The condition has intimate, but not yet defined,
Contract grant sponsor: Clinical Psychiatry Research Center, Tabriz University of Medical Sciences. *Correspondence to: Dr. Alireza Farnam, Razi Hospital, Elgoli Road, Tabriz, East Azerbaijan, Iran. E-mail:
[email protected] Received 11 February 2014; Accepted 16 April 2014 DOI: 10.1002/syn.21746 Published online 19 April 2014 in Wiley Online Library (wileyonlinelibrary. com).
2
S. FARHANG ET AL.
relation to emotional processing. Davidson (Davidson et al., 1990; Schaffer et al., 1983) proposed a model for emotional processing linked to asymmetrical frontal activity during emotional states. He hypothesized that frontal asymmetry is related to the motivational system connected to certain stimuli, with the left prefrontal cortex (PFC) involved in a system facilitating “approach behavior” and the right PFC involved in a system facilitating “withdrawal behavior.” This is an aspect of the so called “lateralization” of brain function that has been well described in humans in various behaviors and cognitive functions. There is a growing body of evidence for this phenomenon among non-human animals (Vallortigara et al., 2011). The underlying mechanism is still largely unknown. Several studies have been conducted based on Davidson (Davidson et al., 1990) ever since, mostly on the human brain. They have demonstrated the presence of interhemispheric imbalance in depression with regard to function and activity (Hecht, 2010; Koek et al., 1999; Maeda et al., 2000) and response to negative and positive stimulus (Harmon-Jones et al., 2002; Herrington et al., 2010), indicating a hyperactive right hemisphere and a relatively hypoactive left hemisphere. Hemispheric asymmetries have also been reported in treatment response of patients with depression. For instance, it has been shown that transcranial magnetic stimulation had a superior response when administered to the left hemisphere of patients with major depressive disorder (Triggs et al., 2010). Interestingly, asymmetry across cortical regions could be distinguished between resilient and sensitive-to-stressor children, where greater left hemisphere activity was a characteristic of those who were resilient to stressor (Curtis and Cicchetti, 2007). Evolving animal models of mental disorders offer the advantage of replicating structured conditions. Chronic mild stress (CMS) is a valid model of depression in rodents (Willner, 1997), modeling environmental factors connected to depression in humans as chronic unexpected stressors (Willner, 2005). These stressors also decrease consumption of a palatable sweet solution, which is assumed to represent a decreased responsiveness to reward that is comparable with anhedonia in humans (as the cardinal symptom of melancholic depression) (Willner et al., 1987). But this effect does not take place in all of the animals, and some animals remain resilient to stressors. So the CMS model replicates our understanding of the biopsychosocial model, indicating a biological predisposition to developing depression as well. To date, there are several studies on hemispheric differences in humans, but there is very little research about lateralization in rodents. Most of such studies in the field of gene expression have focused only on a specific functional region of the brain. The design of this study was based on the described Synapse
asymmetry in hemispheric activation during depression. The first hypothesis was that expression of genes in the PFC region might be also following this asymmetric pattern in anhedonic rats and such a lateralization might not exist in stress-resilient rats. In the next step, lateralization of the pattern of gene expression was also probed in anhedonic and stressresilient rats separately. MATERIALS AND METHODS Ethics The study was conducted in accordance with the European Communities Council Directive of November 1986 and has been approved by the ethical Committee at Tabriz University of Medical Sciences. Efforts were made to minimize animal pain and discomfort. Animals Male Wistar rats (3 months of age) were purchased from Pasteur Institute of Tehran, Iran, and brought into the laboratory 1 week before starting the experiment. Mean animal weight was 203.8 g (range: 141– 257 g). Thirty rats were randomly chosen to enter the CMS model, and 10 rats served as controls. Room temperature was set at 21 C, and the rats were housed individually unless grouping was applied during the protocol. A 12-h light/dark cycle was maintained except when a specific condition was needed in the course of stressor schedule. Food and water were freely available except as part of the deprivations during the stressor schedule. Control animals had no contact with the stressed animals and were undisrupted except for a water and food deprivation for 18 h prior to sucrose consumption test. Chronic mild stress protocol The experimental group of the current study was exposed to a variety of ecologically relevant stressors over 9 weeks. The stressor procedure was performed according to an optimized procedure of CMS protocol (Jayatissa et al., 2009) to achieve depressive-like symptoms. This protocol consists of mild unpredictable stressors, which are: intermittent illumination, stroboscopic light, grouping, food or water deprivation, soiled cage, and 45 cage tilting. Grouping indicates housing a rat in pairs with different partners while an individual rat alternately becomes a resident or an intruder. All the stressors lasted for 14 h. Sucrose consumption test The sucrose consumption test was administered to measure any change in preference of rats for consuming a palatable solution. Animals were first trained to consume a 1.5% sucrose solution for 5 weeks. In this period, the sucrose consumption test was performed
3
ASYMMETRICAL GENE EXPRESSION IN DEPRESSION
centrifuged at 12,000g at 4 C for 10 min, yielding a total RNA pellet that was washed with 75% ethanol twice. The air-dried samples were dissolved in diethylpyrocarbonate-treated water, and the quality and quantity of the extracted RNA was measured by Nanodrop1000 spectrophotometer (NanoDrop, Wilmington, DE). Reverse transcription reaction and cDNA preparation
Fig. 1. Amounts of sucrose consumption in rats subjected to 9 weeks of chronic mild stress. A 25% decrease in sucrose intake within-subject defined rats to be anhedonic and a
