Impaired hypothalamic-pituitary-adrenal axis and its feedback regulation in serotonin transporter knockout mice
Introduction
The serotonin (5-HT) transporter (SERT, 5-HTT) functions as a 5-HT reuptake site to take extracellular 5-HT back into the nerve terminals, resulting in termination of 5-HT receptor stimulation. Thus, the function of SERT is critical to control 5-HT activity, which plays an important role in regulation of emotions. SERT is not only a target for selective serotonin reuptake inhibitors (SSRIs), the most widely used antidepressants, but also may be a genetic component for the pathogenesis of affective disorders. Several polymorphisms and mutations have been found in the SERT promoter and coding regions (Murphy et al., 2004), such as a polymorphism in the 5-HT transporter-linked promoter region (5-HTTLPR) (Hu et al., 2005, Hu et al., 2006, Nakamura et al., 2000, Lesch et al., 1996). Studies have demonstrated that individuals carrying lower SERT expression genotypes are more sensitive to stress. Several recent studies revealed that the number of stressful life events correlates to the severity and number of episodes of major depression in the individuals carrying lower SERT expression genotypes (Caspi et al., 2003, Zalsman et al., 2006, Roy et al., 2007). These data raise the question as to why individuals with lower SERT expression genotypes are more vulnerable to stressful events. A recent study reported that psychological stress, such as public speech, significantly increases plasma ACTH and cortisol levels in the individuals with lower SERT expression genotypes (Jabbi et al., 2007). These data suggest that the sensitivity of HPA axis response to stress is increased in the individuals with lower SERT expression genotypes. Since increased activity of HPA axis is known to be involved in the pathophysiology of affective disorders, the increased sensitivity of HPA axis response to stress could be a trigger for the development of affective disorders in these individuals when they suffer stressful events. Therefore, studying the mechanisms by which the reduction in SERT function during development influences the sensitivity of HPA axis response to stress will provide significant insight for our understanding the etiology of affective disorders and thus, may lead to develop better approaches to prevent and treat affective disorders.
SERT knockout mice were generated by homologous recombination that replaced the second exon of SERT with a neo-cassette (Bengel et al., 1998). In these mice, SERT is constitutively absent (SERT(/() or reduced (SERT+/(). In several aspects, the SERT knockout mice, especially SERT+/( mice, are similar to humans with lower SERT expression genotypes (Li, 2006, Kalueff et al., 2007, Murphy and Lesch, 2008). For example, both SERT (+/() and humans with lower SERT expression genotypes have about a 50% reduction in SERT expression during early development relative to the SERT+/+ mice and individuals with high expression genotype of 5-HTTLPR, respectively. In addition, the SERT uptake rate is slightly reduced in both SERT+/( (vs. SERT+/+) mice and individuals with lower expression genotype of 5-HTTLPR (vs. high expression genotype) (Greenberg et al., 1999). Behavioral studies reveal that the SERT knockout mice are more anxious compared to SERT+/+ mice (Holmes et al., 2003d). Furthermore, SERT knockout mice (both SERT+/( and (/( mice) are more sensitive to stress. Mild stress, such as handling and saline injection, significantly increases ACTH secretion in SERT+/( and (/( mice (Li et al., 1999). These data suggest that disruption of SERT function early in life may alter the development of the HPA axis, resulting in increased sensitivity to stress.
To test the hypothesis that the HPA axis and its feedback regulation are altered in SERT knockout mice, in the present study, we characterized the components of the HPA axis and its feedback regulation in SERT knockout mice. The HPA axis is a major system responsible for maintaining homeostasis in response to stress. Corticotrophin-releasing factor (CRF) in the paraventricular nucleus (PVN) of the hypothalamus is the initial point of the HPA axis-stress system. Stress activates the CRF neurons through activation of numerous neurotransmitters and consequently increases the CRF secretion through the medial eminence to the anterior pituitary. The CRF then activates CRF type 1 receptors (CRF R1) in the anterior pituitary and stimulates ACTH release into the circulation. The ACTH activates the secretor cells in the zona fasciculata of the adrenal cortex to release glucocorticoid, corticosterone (in rodent) or cortisol (in human). The glucocorticoids trigger gene transcription and alter protein synthesis through activation of mineralocorticoid (MR) and glucocorticoid receptors (GR). Activation of MR and GR also produces a negative feedback regulation for the HPA axis, which serves as a major control of the HPA axis. GR is expressed in the hypothalamus, pituitary, adrenal cortex and also in the hippocampus, as an upstream control of the HPA axis. On the other hand, MR is mainly expressed in the hippocampus and is responsible for controlling physiological and mild stress-related variation of the HPA axis (Pace and Spencer, 2005). In the present study, the function and expression of components of the HPA axis, such as CRF and CRF R1, and its feedback regulation, such as GR, were examined in the hypothalamus, pituitary and adrenal gland of SERT+/+, +/( and (/( mice under basal (non-stressed) and stressed conditions.
Section snippets
Animals
SERT knockout mice were purchased from Taconic Farm Inc. (Hudson, NY) and bred in the University of Texas Medical Branch animal facility. In SERT knockout mice, the second exon of the SERT gene was replaced by a neo-cassette (Bengel et al., 1998). The mice were backcrossed on a congenital C57/BL6 genetic background for 14 generations. SERT+/( mice were used for mating to minimize potential effects of differences in maternal care behaviors. The genotypes of the mice were identified using a PCR
Alterations in CRF mRNA in the PVN
Using in situ hybridization, we detected high density of CRF mRNA in the PVN of of mice (Fig. 1A). The CRF mRNA level in the PVN of SERT+/( and (/( mice was significantly reduced in comparison to SERT+/+ mice under non-stressed (basal) conditions (Fig. 1B; One-Way ANOVA: F(2,8) = 14.57, P < 0.01). The reductions of CRF mRNA in SERT+/( and (/( were 30% and 43%, respectively. To confirm the results, we further conduced a quantitative RT-PCR in the hypothalamus of SERT+/( and (/( mice. Consistently,
Discussion
In the present studies, we determined the function and expression of several components of the HPA axis and its negative feedback regulation in SERT knockout mice under basal and stressed conditions. As summarized in Table 2, our results demonstrated that the HPA axis in SERT+/( and (/( mice is impaired. The overall basal activity of the HPA axis in the SERT knockout mice was reduced, as demonstrated by the decreased CRF expression (Fig. 1) and low basal plasma corticosterone concentrations (Li
Role of funding source
Funding for this study was partly provided by a NARSAD Young Investigator Award and USPHS MH72938 to Qian Li; The NARSAD and NIMH had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Conflict of interest
All authors reported no biomedical financial interests or potential conflicts of interest.
Acknowledgments
The authors thank Mary Caraway and Yan Liu for their important technical assistance with the experiments. The authors also thank Drs. Kathryn Cunningham and Brid Nic Dhonnchadha in the University of Texas Medical Branch, and Drs. Nancy A. Muma and Dania Rossi in the University of Kansas, who kindly provided feedback and proof-reading of the manuscript. The studies were partly supported by a NARSAD Young Investigator Award and USPHS MH72938 to Qian Li.
References (53)
- et al.
Regulation of pituitary corticotropin releasing hormone receptors
Peptides
(2001) - et al.
Abnormal behavioral phenotypes of serotonin transporter knockout mice: parallels with human anxiety and depression
Biol. Psychiatry
(2003) - et al.
Behavioral profile of wild mice in the elevated plus-maze test for anxiety
Physiol. Behav.
(2000) - et al.
Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-compulsive disorder
Am. J. Hum. Genet.
(2006) - et al.
The developing use of heterozygous mutant mouse models in brain monoamine transporter research
Trends Pharmacol. Sci.
(2007) - et al.
Molecular manipulations as tools for enhancing our understanding of 5-HT neurotransmission
Trends Pharmacol. Sci.
(1999) - et al.
Late-life depressive symptoms are associated with both hyperactivity and hypoactivity of the hypothalamo-pituitary-adrenal axis
Am. J. Geriatr. Psychiatry
(2007) - et al.
Regulation of the developing hypothalamic-pituitary-adrenal axis in corticotropin releasing hormone receptor 1-deficient mice
Neuroscience
(2003) - et al.
Maternal programming of steroid receptor expression and phenotype through DNA methylation in the rat
Front Neuroendocrinol.
(2005) - et al.
Differential regulation of hypothalamic pituitary corticotropin releasing hormone receptors during development of adjuvant-induced arthritis in the rat
J. Endocrinol.
(1997)