Research ReportDopamine receptor gene expression in human amygdaloid nuclei: Elevated D4 receptor mRNA in major depression
Introduction
The dopaminergic system plays an important role in the regulation of motor, cognitive, and emotional functions. Disturbances of the dopaminergic system have been strongly implicated in several neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, and depressive disorders. Though most research on depressive disorders has focused on serotonin and norepinephrine, there is a vast array of compelling clinical and laboratory animal evidence of a disruption of dopaminergic neuronal activity in depression (Dunlop and Nemeroff, 2007, Kapur and Mann, 1992, Roy et al., 1992, Swerdlow and Koob, 1987). For example, several drugs (neuroleptics, reserpine, α-methyl-p-tyrosine) that decrease dopaminergic transmission are known to precipitate depressive episodes (Charney, 1998, Willner, 1983a). In addition, low CSF concentrations of the dopamine (DA) metabolite, homovanillic acid (HVA; see Willner, 1983b) and reduced venoarterial plasma concentrations of HVA have been reported in depression (Lambert et al., 2000). Consistent associations between DA receptor polymorphisms and depression have not been found (Dunlop and Nemeroff, 2007), although a recent metanalysis of 12 studies demonstrated a significant association of a specific repeat polymorphism of the D4 dopamine receptor (DRD4.2) with depression (López León et al., 2005). Results of neuroimaging studies of D2 receptor binding in subjects with major depressive disorder (MDD) have not been consistent and are reviewed by Dunlop and Nemeroff (2007). A recent functional magnetic resonance imaging study demonstrated that subjects with MDD have a greater behavioral response to the rewarding effects of amphetamine (Tremblay et al., 2005). In fact, the severity of depression correlates with the magnitude of reward experienced by administration of amphetamine, a drug that increases the synaptic availability of DA. This latter finding was used to argue that there is reduced synaptic DA in depression, leading to postsynaptic supersensitivity including supersensitivity to psychostimulants. The interpretation of postsynaptic supersensitivity is likely to be an oversimplification of the pathological process in depression, because numerous studies demonstrate that enhancement of D2-like receptor sensitivity is an important action of antidepressant drug action (Gershon et al., 2007). Despite our lack of a thorough understanding of DA pathobiology in depression, both preclinical and clinical findings suggest that brain dopaminergic activity is reduced in depression.
A region of the brain that is richly innervated by DA and that has considerable relevance to depression is the amygdala. The amygdala is involved in many emotion-laden behaviors and stress-related responses associated with depression (Ramel et al., 2007, Whalen et al., 2002). In fact, functional abnormalities in the amygdala correlate with the severity of MDD, i.e. resting cerebral blood flow and metabolism correlate positively with ratings of depression severity. In addition, antidepressant treatment reduces amygdala metabolism towards normal in MDD patients (Drevets, 2003). Patients with MDD show a greater activation of the amygdala in response to sad facial expressions, an effect that is attenuated by antidepressant treatment (Fu et al., 2004, Surguladze et al., 2005). Abnormal amygdala volume has also been demonstrated in MDD subjects, but consistent changes have not been observed across studies (Drevets, 2003), possibly because of unexpected familial and perhaps genetic influences on amygdala size (Munn et al., 2007). Low glial cell density has been observed in postmortem amygdala from MDD subjects (Bowley et al., 2002, Hamidi et al., 2004), as has been observed in the prefrontal cortex (Rajkowska et al., 1999), suggesting that reduced glia support to neuronal functions contribute to amygdala pathology in depression. Because of the important role of the amygdala in emotion processing and the putative role of DA in depression pathology, we previously investigated DA indices in postmortem amygdala from subjects with MDD. We found an elevation of D2 DA receptors and a reduction of DA transporters in the amygdala of subjects with MDD as compared to psychiatrically normal control subjects (Klimek et al., 2002). Remy et al. (2005) observed lower [11C]RTI-32 binding (an in vivo marker of DA and norepinephrine transporters) in the amygdala in depressed Parkinson's patients relative to non-depressed Parkinson's patients, also implicating an association of a reduced number of DA transporters (or reduced DA innervation) in the amygdala with depression. Interestingly, administration of a DA neurotoxin that produces DA depletion results in upregulation of D2 DA receptors (Angulo et al., 1991) and a reduction in DA transporters in the rat brain (Gordon et al., 1996). Hence, postmortem receptor findings in MDD subjects suggest that amygdala pathology in depression may be associated with deficient DA transmission in the amygdala.
Five distinct subtypes of DA receptors mediate the actions of DA. D1 and D5 receptors belong to the D1 subfamily, and D2, D3, and D4 represent the D2 subfamily; each receptor subtype having a distinct pharmacological profile. DA receptors have been extensively characterized with regard to the relative distributions of receptor proteins and mRNAs in a number of regions of the brain (Hall et al., 1996, Kessler et al., 1993, Meador-Woodruff et al., 1996, Smiley et al., 1994). However, few studies have concentrated on gene expression of DA receptors in the subregions of the human amygdala, and gene expression levels of amygdala DA receptors in depression have not been investigated. The present study aimed to clarify the quantitative distribution of mRNA of the five subtypes of DA receptors in subnuclei of the human amygdala. The present study also sought to determine whether D2 receptor upregulation in depression observed previously (Klimek et al., 2002) may be related to an increase in D2 receptor gene expression. In previous work, D2 receptor binding was highest in the basal amygdaloid nucleus, a region where an elevation of D2 receptor binding and a decrease in DAT binding was observed in MDD subjects as compared to normal control subjects. Hence, the basal nucleus was the focus of the present investigation of DA receptor gene expression changes in depression. The basal nucleus is part of the basolateral complex and is classically considered a place of formation of conditioned and unconditioned stimulus associations in fear conditioning, and plays a major role in regulating memory consolidation, particularly emotionally charged memories (McIntyre et al., 2003). The basal nucleus also has reciprocal connections with medial and orbital frontal cortex, areas where reduced activity has been observed in MDD (Drevets, 2007). DA is known to enhance memory retention in the basolateral amygdala (Lalumiere et al., 2004, LaLumiere et al., 2005) and putative altered dopaminergic input to the amygdala could contribute to the link between disrupted processing of emotionally charged stimuli and increased amygdaloid neural response that has been observed in MDD subjects (Surguladze et al., 2005). The basal nucleus also communicates directly with the major output nucleus of the amygdala, the central nucleus (Knapska et al., 2007), which projects to brainstem nuclei such as the locus coeruleus also known to be dysregulated in MDD (Ordway, 2007).
Section snippets
DA receptor mRNA distribution
Initial experiments determined the quantitative distribution of the 5 subtypes (D1–D5) of DA receptor mRNA in five amygdaloid subnuclei (Fig. 1), using tissue from seven normal control subjects. All PCR reactions were validated for the specificity of primers by melting curve analysis and agarose gel electrophoresis. DNA fragments with the expected size were confirmed by agarose gel electrophoresis of PCR reactions for the five DA receptors. In order to obtain copy numbers of the 5 DA receptor
Discussion
This study details for the first time the quantitative expression pattern of mRNAs for the 5 DA receptors in human amygdala subnuclei, using quantitative real-time RT-PCR. We confirm previous identifications of DA receptor gene expression in the amygdala, demonstrate the predominance of D1, D2 and D4 mRNAs in the amygdala, and clarify the relative abundance of the different receptor mRNAs in the various amygdaloid subnuclei. In addition, we report elevated levels of D4 DA receptor mRNA in the
Human subjects
Human brain tissue was collected at autopsy at the Cuyahoga County Coroner's Office in Cleveland, Ohio, in accordance with an approved Institutional Review Board Protocol, as described previously (Karolewicz et al., 2005). For the study of receptor mRNA distribution, discarded brain tissues from 7 subjects (5 males, 2 females) were collected at autopsy and detailed medication and psychiatric histories were unavailable. These subjects died as result of cardiovascular disease (4),
Acknowledgments
The excellent assistance of the Cuyahoga County Coroner's Office, Cleveland, OH is greatly appreciated. This research was supported by the National Institute of Mental Health (MH63187, MH46692, MH02031, MH67996) and by the National Center for Research Resources (RR17701).
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