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Endocrinology. Aug 2009; 150(8): 3709–3716.
Published online Apr 2, 2009. doi:  10.1210/en.2008-1721
PMCID: PMC2717884

Influence of Sex and Corticotropin-Releasing Factor Pathways as Determinants in Serotonin Sensitivity

Abstract

Stress sensitivity and sex are predictive factors in affective disorder susceptibility. Serotonin (5-HT) pathway recruitment by corticotropin-releasing factor (CRF) during stress is necessary in adaptive coping behaviors, but sex differences in such responses have not been investigated. Using selective 5-HT reuptake inhibitor (SSRI) administration to acutely elevate 5-HT in a genetic model of stress sensitivity, we examined behavioral and physiological responses in male and female stress-sensitive CRF receptor-2-deficient (R2KO) mice. Chronic SSRI treatment was used to confirm outcomes were specific to acute 5-HT elevation and not antidepressant efficacy. We hypothesized that R2KO mice would show a greater sensitivity to acute changes in 5-HT and that, because females typically are more stress sensitive, R2KO females would be the most responsive. Our results supported this hypothesis because females of both genotypes and R2KO males showed a greater sensitivity to an acute 10 mg/kg dose of citalopram in a tail suspension test, displaying decreased immobile time and increased latency to immobility. Furthermore, acute citalopram promoted significant anxiogenic-like effects that were specific to R2KO females in the elevated plus maze and light-dark box tests. Chronic citalopram did not produce these behavioral changes, supporting specificity to acute 5-HT modulation. Mechanistically, females had decreased hippocampal 5-HT transporter (SERT) levels, whereas R2KO mice showed reduced SERT in the prefrontal cortex, supporting a possible intersection of sex and genotype where R2KO females would have the lowest SERT to be blocked by the SSRI. This sensitivity to 5-HT-mediated anxiety in females may underlie a heightened vulnerability to stress-related affective disorders.

Affective disorders such as depression and anxiety comprise a complex pattern of neurocircuitry dysregulation (1,2,3). Females present with depression at twice the frequency of males (3,4,5,6,7), suggesting an important sex-dependant susceptibility to disease risk factors, including exposure to stress. Although a link between stress sensitivity and disease susceptibility has been observed in clinical settings, the full implications of sex differences have not yet been examined. Furthermore, use of intact, healthy organisms in research has not provided the necessary assessment of the maladaptive stress responses that likely contribute to disease onset.

The recruitment of the serotonin (5-HT) system provides a mechanism by which stress alters neurotransmitter signaling critical in mood regulation and in the prevention of affective disorders (8,9,10,11,12,13). One mediator in the interaction between stress and 5-HT is the neuropeptide corticotropin-releasing factor (CRF), which is induced by stress exposure and acts on its two receptors, CRF receptor-1 (CRFR1) and -2 (CRFR2) (14,15,16,17). In the raphe, stress exposure results in increased gene expression related to 5-HT production (18) and alterations in excitatory and inhibitory drives on 5-HT producing raphe neurons (19,20,21,22,23,24,25). Additionally, sex hormones have been implicated as modulators of 5-HT transmission in raphe projection regions (26,27). Despite the known complex modulation of 5-HT by stress, sex differences in these midbrain dorsal and median raphe nuclei producing the main ascending 5-HT projections have not been determined.

To examine the contribution of stress pathway dysregulation and sex on behavioral responses to acute increases in 5-HT, we have used a mouse model of stress sensitivity deficient for CRFR2 [CRFR2 knockout (R2KO)]. R2KO mice display maladaptive physiological and behavioral responses to stress perturbations (28,29) and fail to produce adaptive and homeostatic responses in the raphe after stress (30). We hypothesize that because CRF modulates 5-HT pathways, mice with a genetic disruption in CRF signaling will show a greater sensitivity to changes in local 5-HT in behavioral and physiological outcomes after acute citalopram treatment. Chronic citalopram treatment was assessed as a control to ascertain the specificity of these effects to acute elevations in 5-HT rather than antidepressant efficacy. Because intrinsic differences in the 5-HT system may contribute to altered responses to citalopram, sex and genotypic comparisons for 5-HT transporter (SERT) levels were conducted in brain regions where stress-mediated signaling interact and modulate 5-HT pathways, including the dorsal and ventral hippocampus, prefrontal cortex, and amygdala.

Materials and Methods

Animals

All mice used were generated in-house from CRFR2 heterozygous breeding on a mixed C57BL/6:129J background as previously described (28). Male and female wild-type (WT) and CRFR2-deficient (R2KO) littermates (9–12 wk, genotyped at 3 wk) were group housed under a 12-h light, 12-h dark cycle (lights on at 0700 h), with food and water ad libitum. All studies were in accordance with experimental protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee, and all procedures were conducted in accordance with institutional guidelines.

Citalopram administration

Acute

To examine the physiological and behavioral effects of acute increases in 5-HT, WT and R2KO male (n = 13–14) and female (n = 18) mice were treated with 10 mg/kg of the selective 5-HT reuptake inhibitor (SSRI) citalopram (Anawa, Zurich, Switzerland) dissolved in saline and administered via ip injection (100 μl) or saline vehicle control (100 μl). Citalopram was administered at 10 mg/kg as a moderate dose shown to produce behavioral responses in mice (31,32,33). Behavioral measures were examined 30 min after administration of citalopram or vehicle (34).

Chronic

To distinguish between antidepressant efficacy and effects of acute elevations in 5-HT in these studies, a separate cohort of WT and R2KO male (n = 17–18) and female (n = 19–24) mice were exposed to chronic citalopram dissolved in their drinking water for 4 wk as previously described (35). For controls in chronic administration groups, male and female WT and R2KO littermates were given water without drug. Body weights were taken at weekly intervals, and water consumption of mice was measured and water volumes refreshed with new drug solutions at 3-d intervals (see supplemental Table 1). Citalopram levels in drinking water were adjusted based on recent consumption and weight data to ensure continued delivery of a physiologically relevant dosage of 10 mg/kg. In behavioral testing, responses were measured in mice after 4 wk citalopram (35). Mice continued to receive citalopram during behavioral testing periods.

Behavioral responses after citalopram administration

Behavioral stress responses were examined in male and female mice 30 min after acute citalopram administration. A separate cohort of mice was examined during chronic citalopram treatment. Testing was completed in a designated room within the mouse home environment. Behavioral tests were separated by 5–7 d and occurred in the following order: elevated plus maze, tail suspension test, and light-dark box. At the end of each test, female mice were vaginally lavaged with warm saline to attain cellular samples for determining estrous cycle stage. All studies were conducted by an investigator blinded to genotype and treatment groups.

Tail suspension test

Testing was performed as previously described (28). Briefly, the 6-min behavioral test was performed between 1100 and 1300 h. Immobility was hand scored by an experimenter blinded to experimental details. Mice were excluded from statistical analysis if they climbed their tails during the test. Data analysis was aided by the use of AnyMaze software (Stoelting, Wood Dale, IL) to measure time spent immobile and latency to first bout of immobility.

Elevated plus maze

Testing was conducted as previously described (36). Briefly, the 5-min test occurred during the light cycle between 1100 and 1300 h. Light intensity in open arms was 6 lux. Analysis used AnyMaze software to measure open and closed arm time and entries and total distance traveled. Mice were excluded from statistical analysis if they fell off the open arms of the maze.

Light-dark box

Testing was performed as previously described (28). Briefly, light intensity was 5 lux in the dark compartment and 300 lux in the light compartment. Test duration was 10 min and occurred 2 h into the dark cycle. AnyMaze software was used to measure transitions between light and dark sides of the test and time spent in the light side.

Estrous cycle

To control for peak estrogen levels during the time of testing, estrous cycle stages were examined by vaginally lavaging each female with warm saline immediately after completion of each test. The smear was air dried on a slide and hematoxylin stained. Estrous cycle stages were grouped according to hormone levels into proestrus, estrus, and diestrus categories.

Hypothalamic-pituitary-adrenal (HPA) axis assessment

To examine sex and genotypic effects of acute citalopram administration on the physiological HPA axis stress response, corticosterone levels were examined after a 15-min restraint stress in a separate cohort of male and female mice. Testing was administered between 0900–1100 h by placing mice in a 50-ml conical tube. In acute treatment groups, restraint onset began 30 min after administration of citalopram to male (n = 7–11) and female (n = 7–10) WT and R2KO mice. Tail blood samples (~15 μl per sample) were taken at onset and completion of restraint (0 and 15 min, respectively). For female mice, vaginal lavage saline samples were also collected immediately after the last blood sample collection for estrous cycle determination. To examine the HPA axis stress recovery phase, additional samples were collected at 15 min and 75 min after restraint end. Chronic citalopram male (n = 9–10) and female (n = 8–12) WT and R2KO mice were exposed to the same restraint and blood collection procedure. Corticosterone was determined by RIA (MP Biomedicals, Orangeburg, NY) using 3 μl plasma with a variance coefficient R2 = 0.9985.

SERT autoradiography

To determine whether sex differences in response to acute citalopram were related to transporter levels, SERT binding was analyzed in male and female mice using [3H]citalopram autoradiography. Fresh-frozen brain sections (30 μm) were selected from experimentally naive male and female WT and R2KO mice (n = 6–8). Sections were atlas matched (37) and immersed in incubation buffer (4 C, 10 min) containing 50 mm Tris (pH 7.6), 120 mm NaCl, and 5 mm KCl. Sections were then placed in incubation buffer containing [3H]citalopram at room temperature (0.7 nm; Perkin-Elmer, Boston, MA) for 1 h. Slides were washed in incubation buffer and rapidly dipped in cold deionized H2O, dried under a cold airstream, and apposed to Kodak Hyperfilm (Eastman Kodak, Rochester, NY) for 21 d. Images were normalized to background and analyzed by a predetermined region of interest tool in the basolateral amygdala, prefrontal cortex, and ventral and dorsal hippocampus. OD measurements of matched slides were conducted using IPlabs software (BD Biosciences/Scanalytics, Rockville, MD) and averaged across two consecutive sections for each animal. Analyses were conducted by an investigator blinded to sex and genotype.

Statistical analyses

Examination of sex differences in citalopram effects on behavioral tests and physiological measurements used multifactor ANOVA with factors for sex, genotype, and drug. Additional analysis of main effects by genotype was accomplished using ANOVA with factors for sex and drug treatment separated by WT and R2KO. For further distinction of differential effects of citalopram in males and females, data were analyzed by multifactor ANOVA, separated by sex, with factors for genotype and drug treatment. HPA measurements were analyzed using repeated-measures ANOVA over time with the same factors as above. Assessing estrous cycle effects used an ANOVA model for cycle effects in a behavioral measurement from each test (immobility time, open arm time, light-dark transitions). All acute and chronic groups were analyzed separately, and an additional test was run to assess acute-chronic differences where treatment duration was added as a factor to the tests described above. Analysis was carried out with JMP statistical software (SAS, Cary, NC).

Results

Behavioral tests

Tail suspension test

Analysis of male and female vehicle-treated controls by ANOVA revealed that R2KO mice displayed a main effect for basal elevation in immobile time compared with WT controls [F(1, 62) = 2.6; P < 0.05; Fig. 1A1A].]. Post hoc testing within sexes showed that this effect was significant in males [F(1, 15) = 2.3; P < 0.05]. In measuring latency to become immobile within vehicle-treated mice, a main effect of genotype was again observed [F(1, 62) = 2.6; P < 0.05; Fig. 1B1B]] because R2KO mice displayed a decreased latency to become immobile compared with WT.

Figure 1
Enhanced sensitivity in females and CRFR2-deficient mice after acute citalopram administration in the tail suspension test. Citalopram (CIT) treatment decreased immobile time (A) in male R2KO and female mice relative to vehicle-treated mice (VEH) and ...

Acute citalopram administration resulted in main effects of drug for decreased immobility time [F(1, 60) = 5.3; P < 0.01; Fig. 1A1A]] and increased latency to first become immobile [F(1, 60) = 3.8; P < 0.01; Fig. 1B1B].]. Acute citalopram reduced time spent immobile for male and female R2KO mice [F(1, 16) = 3.7 and 3.1, respectively; P < 0.01] as well as in WT females [F(1, 11) = 2.5; P < 0.05] but not in WT male mice [F(1, 15) = 1.3; P = 0.20].

Chronic citalopram did not alter time spent immobile [F(1, 66) = 0.11; P = 0.91] or latency to become immobile [F(1, 66) = 0.12; P = 0.91; see Table 11]] in male or female WT or R2KO mice.

Table 1
Behavioral effects of chronic citalopram administration

Elevated plus maze

In the elevated plus maze, analysis by ANOVA revealed a main effect of drug for acute citalopram to decrease open arm time [F(1, 62) = 7.9; P < 0.01; Fig. 2A2A].]. Testing within sexes revealed this effect to be specific to female mice [F(1, 23) = 10.7; P < 0.01]. Male WT and R2KO groups displayed no change in open arm time after citalopram injection [F(1, 20) = 0.9; P = 0.36]. Further analysis showed a main effect of sex where female mice displayed fewer open arm entries compared with males [F(1, 62) = 6.5; P < 0.01; Fig. 2B2B],], although this measure was not affected by citalopram. Females displayed a citalopram-mediated decrease in total number of entries in the maze [F(1, 23) = 5.7; P < 0.05; Fig. 2C2C]] and elevated distance traveled in the maze when compared with males [F(1, 62) = 9.2; P < 0.01; Fig. 2D2D].

Figure 2
Female mice display increased anxiety-like behavior after acute citalopram in the elevated plus maze. A, Administration of citalopram (CIT) produced decreased time in open arms relative to vehicle-treated controls (VEH) in WT and R2KO females but not ...

No effects of chronic citalopram administration were observed in male or female WT or R2KO mice (Table 11).

Light-dark box

Acute citalopram produced a sex-specific drug effect on light side time [drug × sex interaction, F(1, 68) = 2.1; P < 0. 05], where drug administration reduced time spent in the light side for females but not for males (Fig. 3A3A).). This effect was significant in R2KO female mice [F(1, 15) = 2.7; P < 0.05]. Acute citalopram administration also caused a reduction in light-dark transitions across all groups [F(1, 68) = 2.6; P < 0.01; Fig. 3B3B].]. This effect was significant only in female R2KO mice [F(1, 15) = 2.5; P < 0.05]. In vehicle-treated mice, there was a main effect of sex for females to show increased transitions between dark and light sides [F(1, 68) = 2.2; P < 0.05] and decreased time spent in the light side of the apparatus [F(1, 68) = 2.7; P < 0.01; Fig. 3A3A].

Figure 3
Female R2KO mice display enhanced sensitivity to acute citalopram in the light-dark box test. In R2KO females, citalopram (CIT) produced a reduction in time spent in the light side (A) and in the number of light-dark transitions (B) relative to wild-type ...

Chronic citalopram administration resulted in a drug by genotype interaction effect in male mice [F(1, 33) = 2.1; P < 0. 05; see Table 11],], where only WT male mice displayed increased light side time after citalopram.

Estrous cycle

Estrous cycle analysis after behavior and physiological tests revealed no significant differences among acute or chronic treatment groups (supplemental Table 2).

HPA stress axis

In groups administered citalopram acutely before restraint stress, a main effect was observed where the HPA response was significantly elevated in both male and female mice [F(1, 81) = 25.1; P < 0.0001; Fig. 44,, A and B, respectively]. Vehicle-treated mice displayed a significant sex effect where female mice displayed an elevated corticosterone response across multiple testing time points compared with males [F(1, 81) = 37.1; P < 0.0001]. In male mice, there was a trend for a genotype-specific effect of citalopram [drug × genotype interaction F(1, 27) = 2.0; P = 0.06], where citalopram-treated R2KO mice displayed greater corticosterone levels at 30 min compared with WT males administered drug. In females, acute citalopram significantly increased basal (0 min) corticosterone levels in R2KO mice compared with WT mice [F(3, 59) = 6.7; P = 0.0001]. Acute citalopram delayed HPA axis recovery 90 min after stress in females [F(1, 54) = 2.5; P < 0.05] and males [F(1, 27) = 3.3; P < 0.01]. Post hoc testing of the 90-min time point revealed that although all citalopram-treated groups showed apparent corticosterone elevation, this effect was significant in R2KO males and females [F(1, 11) = 2.0–2.4; P < 0.05].

Figure 4
HPA activation after acute citalopram is enhanced in female and R2KO mice. Administration of citalopram (CIT) produced enhanced corticosterone (CORT) levels in males (A) and females (B) relative to vehicle-treated mice (VEH). Female R2KO mice displayed ...

Chronic administration of citalopram produced no significant effects on HPA axis stress response in male or female WT or R2KO mice (supplemental Table 3). Although an apparent reduction in corticosterone was seen among female mice at the 0-min time point, this did not reach significance [F(3, 40) = 3.0; P = 0.09].

SERT autoradiography

To examine whether sex and genotypic differences in 5-HT reuptake in limbic and frontal areas might contribute to observed behavioral responses to acute citalopram, SERT levels were compared between male and female WT and R2KO mice in the amygdala, hippocampus, and prefrontal cortex. In the hippocampus, an interaction effect between sex and genotype was observed in dorsal dentate gyrus [DG; F(1, 24) = 2.2; P < 0.05; Fig. 5A5A],], where lower SERT levels relative to males were observed in female WT mice but not in R2KO females. A main effect of sex was also detected in the dorsal and ventral DG, where males displayed elevated SERT compared with females [F(1, 11) = 3.8 and 2.1, respectively; P < 0.01; Fig. 5B5B].]. A genotypic effect for reduced SERT was detected in the dorsal DG of R2KO males compared with WT male controls [F(1, 11) = 2.1; P < 0.05]. An interaction between sex and genotype was also detected in dorsal area CA3 [F(1, 24) = 2.6; P < 0.05; Fig. 5E5E],], where female mice, but not males, displayed lower SERT levels. Males also displayed significantly elevated SERT compared with females in dorsal DG [F(1, 11) = 3.9; P < 0.01], and a trend was detected where R2KO males showed reduced SERT compared with WT males in dorsal DG [F(1, 11) = 2.0; P = 0.06]. No effects of sex or genotype were observed in the CA1 region of the dorsal or ventral hippocampus.

Figure 5
Female and R2KO mice display reduced SERT levels in hippocampus and prefrontal cortex. SERT levels were examined by autoradiography in the DG (A and D), area CA1 (B and E), and area CA3 (C and F) in the dorsal and ventral hippocampus (dHpc and vHpc, respectively), ...

A significant main effect of genotype was observed in the prefrontal cortex [F(1, 23) = 2.1; P < 0.05] with a decrease in SERT in R2KO mice (Fig. 5G5G).). Additionally, a significant interaction between sex and genotype was observed in the basolateral amygdala [F(1, 23) = 2.1; P < 0.05, see Fig. 5H5H],], where females displayed decreased SERT in WT mice but not in R2KO groups.

Discussion

Examination of behavioral and physiological responses to acute SSRI exposure in a model of stress dysregulation may be a critical step toward understanding the neurobiology of affective disorders and improving therapeutic efficacy. The inability to effectively recruit and maintain the homeostatic changes in 5-HT pathways mediated by CRF may be a factor in precipitating stress-induced disease onset (1,29,38,39,40). Furthermore, sex differences in affective disorder presentation necessitate the inclusion of females in these studies. Male and female stress-sensitive R2KO were examined for behavioral and physiological responses to acute or chronic citalopram treatment. In these studies, multiple behavioral paradigms were used to explore the interaction of stress pathway dysregulation and 5-HT in active and passive behavioral coping responses (41,42,43,44,45). Although acute testing required repeated SSRI administration that complicates interpretation from single tests, consistent effects across tests indicated a more robust support of 5-HT sensitivity.

In the tail suspension test, the predicted genotypic differences were detected where R2KO mice showed both increased immobile time and reduced latency to immobility compared with WT littermates. Acute citalopram administration at a dose of 10 mg/kg resulted in significant reductions in immobility in females of both genotypes. Interestingly, reduced immobility in males was only detected in R2KO mice, which may suggest that males are less sensitive to behavioral effects of acute 5-HT manipulation compared with female mice, which display significant drug effects across genotypes. The observed increase in latency to immobility and decrease in immobile time support our hypothesis that a stress-sensitive phenotype correlates with increased responsivity to acute SSRI administration. We hypothesize that both basal sex differences and a dysregulation of upstream stress pathways likely produces compensatory changes in the 5-HT system that may be responsible for the observed increased sensitivity to acute SSRI administration (38,41). As predicted, chronic citalopram administration did not produce these behavioral effects, supporting their dependence on acute increases in 5-HT and not antidepressant efficacy (46,47).

In an elevated plus maze, females displayed increased sensitivity to the anxiogenic effects of acute citalopram as exhibited by significant reductions in open arm time and entries after treatment. Interestingly, these outcomes were specific to females because male mice showed no significant behavioral changes in this test after acute citalopram. Similar anxiogenic responses to acute citalopram were observed in the light-dark box where female R2KO mice showed decreased time in the light side of the box. Chronic citalopram failed to exhibit effects on these anxiety measures for either sex or genotype in the elevated plus maze. Acute antidepressant administration has previously been associated with anxiogenic behavioral effects (47,48,49,50,51), and a subclass of patients also report increased anxiety during initial SSRI treatment (52,53). Interestingly, chronic citalopram produced opposing results in the light-dark box, with anxiolytic-like effects that were restricted to WT males, hypothesized to be the least stress-sensitive group in these studies. Such a finding may be dependent in part on the nature of the light-dark box paradigm where testing occurs during the dark cycle when recent water consumption may have resulted in a greater intake of drug before testing. Additionally, inspection of acute and chronic vehicle groups revealed an apparent reduction in light-dark transitions and light side time in chronic mice compared with acute vehicle groups, indicating that injection alone may induce a behavioral state more sensitive to increased 5-HT. The profile of acute responses across behavioral stress tests clearly delineates two additive factors in stress sensitivity: 1) sex and 2) CRF receptor dysregulation. Gonadal hormones may have an influence on synaptic plasticity and neurotransmission that mediates these sex differences. In females, differences in responsivity may arise from rapid actions between estrogen receptors and glutamatergic signaling in limbic brain regions (54), particularly within regions receiving stress-mediated 5-HT inputs. This enhanced excitatory input could be exacerbated by increased activity of raphe neurons in R2KO animals, ultimately resulting in modified responses in behavioral stress measurements (30).

SERT levels in male and female WT and R2KO mice present a possible contributing factor to increased SSRI sensitivity observed in behavioral tests. Autoradiographic analysis revealed a main effect of sex in the dorsal and ventral hippocampus where females had overall lower SERT levels. Reduced female SERT levels may indicate an altered ability to accommodate fluctuations in 5-HT levels after SSRI treatment, potentially sensitizing the 5-HT system and further enhancing drug effects. This finding, coupled with enhanced changes in behavioral stress measures within females, may contribute to the anxiety-like responses observed in our behavioral studies, supporting previous findings showing a link between serotonergic pathways and anxiety (55,56,57,58). Analysis of SERT in prefrontal cortex revealed a genotypic effect where both male and female R2KO mice showed significantly reduced SERT. Together, the decreased SERT among female mice in the hippocampus and in R2KO mice in the prefrontal cortex may provide a mechanism whereby female R2KO mice show the greatest sensitivity and behavioral responses to acute SSRI. Serotonergic influence on the cortex, including changes in SERT levels has been described as a point of genetic vulnerability in affective disorders (59,60,61,62). Although SERT is among multiple targets that modulates 5-HT activity, we have also recently reported increased expression of the rate-limiting synthesis enzyme for 5-HT, tryptophan hydroxylase-2, in the raphe of R2KO mice, further supporting a genetic basis for the increased sensitivity to acute citalopram in these mice (30).

The HPA stress axis is a physiological indicator of organismal stress state and is subject to sex-specific regulation (63). In our examination of corticosterone levels after an acute restraint stress, we detected the predicted sex differences where females displayed significantly elevated corticosterone compared with male mice. Acute citalopram treatment before the start of restraint increased baseline corticosterone for both sexes. Within females, this effect was significantly elevated in R2KO mice, again supporting the increased stress sensitivity of this group. In males, citalopram further increased corticosterone levels throughout the HPA time course. Both males and females treated with citalopram showed a main effect of drug to delay stress recovery, suggesting that acute pharmacological increases in 5-HT may override glucocorticoid-mediated negative feedback in the HPA stress axis. Although chronic citalopram failed to induce significant drug effects, trends in data suggesting a reduced HPA response after chronic SSRI exposure may indicate a potential blunting of the HPA and suggest a potential measure for therapeutic effects of chronic SSRI (53,64).

Overall, these studies have examined the hypothesis that a dysregulation of stress pathways produces alterations in 5-HT-mediated behavioral and physiological responses during stress. Furthermore, comparisons identifying the intersection between genotype and sex in these outcomes provided support for this hypothesis because the most stress-sensitive group, R2KO females, showed a consistent and significant response to acute citalopram treatment in behavioral tests. Sex and genotype effects on SERT levels that were brain region specific likely contribute to the complexity of affective disorder susceptibility and to the challenges in developing effective treatment strategies. We hypothesize that stress susceptibility may be produced by dysfunction of the stress-5-HT system arising from both basal sex differences as well as genetic dysregulation of the CRF system, leaving certain subpopulations at especially high risk of developing stress-induced affective disorders. These findings suggest that an increased sensitivity to acute SSRI administration may be a predictive marker of an underlying vulnerability to disease.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Y. Xiong and L. Levy for their technical support.

Footnotes

This study was supported by grants from the National Institutes of Health, MH 79754 (to J.G.M.) and MH 73030 (to T.L.B.).

Current address for K.A.S.: University of Colorado, Department of Integrative Physiology, 354 UCB, Boulder, Colorado 80309.

Disclosure Summary: The authors have nothing to disclose.

First Published Online April 2, 2009

For editorial see page 3440

Abbreviations: CRF, Corticotropin-releasing factor; CRFR1, CRF receptor-1; DG, dentate gyrus; 5-HT, serotonin; R2KO, CRFR2 knockout; SERT, 5-HT transporter; SSRI, selective 5-HT reuptake inhibitor; WT, wild type.

References

  • Bale TL 2006 Stress sensitivity and the development of affective disorders. Horm Behav 50:529–533 [PubMed]
  • Gorman JM 2006 Gender differences in depression and response to psychotropic medication. Gend Med 3:93–109 [PubMed]
  • Kessler RC, McGonagle KA, Zhao S, Nelson CB, Hughes M, Eshleman S, Wittchen HU, Kendler KS 1994 Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States. Results from the National Comorbidity Survey. Arch Gen Psychiatry 51:8–19 [PubMed]
  • Heninger GR 1997 Serotonin, sex, and psychiatric illness. Proc Natl Acad Sci USA 94:4823–4824 [PMC free article] [PubMed]
  • Kessler RC, McGonagle KA, Nelson CB, Hughes M, Swartz M, Blazer DG 1994 Sex and depression in the National Comorbidity Survey. II. Cohort effects. J Affect Disord 30:15–26 [PubMed]
  • Weissman MM, Bland RC, Canino GJ, Faravelli C, Greenwald S, Hwu HG, Joyce PR, Karam EG, Lee CK, Lellouch J, Lépine JP, Newman SC, Rubio-Stipec M, Wells JE, Wickramaratne PJ, Wittchen H, Yeh EK 1996 Cross-national epidemiology of major depression and bipolar disorder. JAMA 276:293–299 [PubMed]
  • Becker JB, Monteggia LM, Perrot-Sinal TS, Romeo RD, Taylor JR, Yehuda R, Bale TL 2007 Stress and disease: is being female a predisposing factor? J Neurosci 27:11851–11855 [PubMed]
  • Lopez JF, Vasquez DM, Chalmers DT, Watson SJ 1997 Regulation of 5-HT receptors and the hypothalamic-pituitary-adrenal axis. Implications for the neurobiology of suicide. Ann NY Acad Sci 29:106–134 [PubMed]
  • Nemeroff CB, Vale WW 2005 The neurobiology of depression: inroads to treatment and new drug discovery. J Clin Psychiatry 66(Suppl 7):5–13 [PubMed]
  • Strock M 2002 Depression. Bethesda, MD: National Institute of Mental Health; 1–25
  • Vergne DE, Nemeroff CB 2006 The interaction of serotonin transporter gene polymorphisms and early adverse life events on vulnerability for major depression. Curr Psychiatry Rep 8:452–457 [PubMed]
  • Mann JJ, McBride PA, Brown RP, Linnoila M, Leon AC, DeMeo M, Mieczkowski T, Myers JE, Stanley M 1992 Relationship between central and peripheral serotonin indexes in depressed and suicidal psychiatric inpatients. Arch Gen Psychiatry 49:442–446 [PubMed]
  • Mann JJ, McBride PA, Anderson GM, Mieczkowski TA 1992 Platelet and whole blood serotonin content in depressed inpatients: correlations with acute and life-time psychopathology. Biol Psychiatry 32:243–257 [PubMed]
  • Carrasco GA, Van de Kar LD 2003 Neuroendocrine pharmacology of stress. Eur J Pharmacol 463:235–272 [PubMed]
  • Lee HS, Kim MA, Valentino RJ, Waterhouse BD 2003 Glutamatergic afferent projections to the dorsal raphe nucleus of the rat. Brain Res 963:57–71 [PubMed]
  • Shekhar A, Truitt W, Rainnie D, Sajdyk T 2005 Role of stress, corticotropin releasing factor (CRF) and amygdala plasticity in chronic anxiety. Stress 8:209–219 [PubMed]
  • Berretta S 2005 Cortico-amygdala circuits: role in the conditioned stress response. Stress 8:221–232 [PubMed]
  • Chamas FM, Underwood MD, Arango V, Serova L, Kassir SA, Mann JJ, Sabban EL 2004 Immobilization stress elevates tryptophan hydroxylase mRNA and protein in the rat raphe nuclei. Biol Psychiatry 55:278–283 [PubMed]
  • Daugherty WP, Corley KC, Phan TH, Boadle-Biber MC 2001 Further studies on the activation of rat median raphe serotonergic neurons by inescapable sound stress. Brain Res 923:103–111 [PubMed]
  • Keeney A, Jessop DS, Harbuz MS, Marsden CA, Hogg S, Blackburn-Munro RE 2006 Differential effects of acute and chronic social defeat stress on hypothalamic-pituitary-adrenal axis function and hippocampal serotonin release in mice. J Neuroendocrinol 18:330–338 [PubMed]
  • Kirby LG, Pernar L, Valentino RJ, Beck SG 2003 Distinguishing characteristics of serotonin and non-serotonin-containing cells in the dorsal raphe nucleus: electrophysiological and immunohistochemical studies. Neuroscience 116: 669–683 [PMC free article] [PubMed]
  • Kirby LG, Rice KC, Valentino RJ 2000 Effects of corticotropin-releasing factor on neuronal activity in the serotonergic dorsal raphe nucleus. Neuropsychopharmacology 22:148–162 [PubMed]
  • Roche M, Commons KG, Peoples A, Valentino RJ 2003 Circutry underlying regulation of the serotonergic system by swim stress. J Neurosci 23:970–977 [PubMed]
  • Tan H, Zhong P, Yan Z 2004 Corticotropin-releasing factor and acute stress prolongs serotonergic regulation of GABA transmission in prefrontal cortical pyramidal neurons. J Neurosci 24:5000–5008 [PubMed]
  • Valentino RJ, Liouterman L, Van Bockstaele EJ 2001 Evidence for regional heterogeneity in corticotropin-releasing factor interactions in the dorsal raphe nucleus. J Comp Neurol 435:450–463 [PubMed]
  • Joffe H, Cohen LS 1998 Estrogen, serotonin, and mood disturbance: where is the therapeutic bridge? Biol Psychiatry 44:798–811 [PubMed]
  • Fink G, Sumner BE, Rosie R, Grace O, Quinn JP 1996 Estrogen control of central neurotransmission: effect on mood, mental state, and memory. Cell Mol Neurobiol 16:325–344 [PubMed]
  • Bale TL, Contarino A, Smith GW, Chan R, Gold LH, Sawchenko PE, Koob GF, Vale WW, Lee KF 2000 Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat Genet 24:410–414 [PubMed]
  • Bale TL, Vale WW 2003 Increased depression-like behaviors in corticotropin-releasing factor receptor-2-deficient mice: sexually dichotomous responses. J Neurosci 23:5295–5301 [PubMed]
  • McEuen JG, Beck SG, Bale TL 2008 Failure to mount adaptive responses to stress results in dysregulation and cell death in the midbrain raphe. J Neurosci 28:8169–8177 [PMC free article] [PubMed]
  • Weber M, Talmon S, Schulze I, Boeddinghaus C, Gross G, Schoemaker H, Wicke KM 2008 Running wheel activity is sensitive to acute treatment with selective inhibitors for either serotonin or norepinephrine reuptake. Psychopharmacology (Berl) 203:753–762 [PubMed]
  • Fish EW, Faccidomo S, Gupta S, Miczek KA 2004 Anxiolytic-like effects of escitalopram, citalopram, and R-citalopram in maternally separated mouse pups. J Pharmacol Exp Ther 308:474–480 [PubMed]
  • Egashira N, Matsuda T, Koushi E, Higashihara F, Mishima K, Chidori S, Hasebe N, Iwasaki K, Nishimura R, Oishi R, Fujiwara M 2008 Δ(9)-Tetrahydrocannabinol prolongs the immobility time in the mouse forced swim test: involvement of cannabinoid CB1 receptor and serotonergic system. Eur J Pharmacol 589:117–121 [PubMed]
  • Chenu F, Guiard BP, Bourin M, Gardier AM 2006 Antidepressant-like activity of selective serotonin reuptake inhibitors combined with a NK1 receptor antagonist in the mouse forced swimming test. Behav Brain Res 172:256–263 [PubMed]
  • Dulawa SC, Holick KA, Gundersen B, Hen R 2004 Effects of chronic fluoxetine in animal models of anxiety and depression. Neuropsychopharmacology 29:1321–1330 [PubMed]
  • Teegarden SL, Bale TL 2007 Decreases in dietary preference produce increased emotionality and risk for dietary relapse. Biol Psychiatry 61:1021–1029 [PubMed]
  • Paxinos G, Franklin KBJ 2003 The mouse brain in stereotactic coordinates. Compact 2nd ed. New York: Elsevier/Academic Press
  • Bale TL, Picetti R, Contarino A, Koob GF, Vale WW, Lee KF 2002 Mice deficient for both corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 have an impaired stress response and display sexually dichotomous anxiety-like behavior. J Neurosci 22:193–199 [PubMed]
  • Linthorst AC, Peñalva RG, Flachskamm C, Holsboer F, Reul JM 2002 Forced swim stress activates rat hippocampal serotonergic neurotransmission involving a corticotropin-releasing hormone receptor-dependent mechanism. Eur J Neurosci 16:2441–2452 [PubMed]
  • Dranovsky A, Hen R 2006 Hippocampal neurogenesis: regulation by stress and antidepressants. Biol Psychiatry 59:1136–1143 [PubMed]
  • Mueller BR, Bale TL 2007 Early prenatal stress impact on coping strategies and learning performance is sex dependent. Physiol Behav 91:55–65 [PubMed]
  • McEwen BS 2003 Mood disorders and allostatic load. Biol Psychiatry 54:200–207 [PubMed]
  • Mallo T, Koiv K, Koppel I, Raudkivi K, Uustare A, Rinken A, Timmusk T, Harro J 2008 Regulation of extracellular serotonin levels and brain-derived neurotrophic factor in rats with high and low exploratory activity. Brain Res 1194:110–117 [PMC free article] [PubMed]
  • Adamec R, Head D, Blundell J, Burton P, Berton O 2006 Lasting anxiogenic effects of feline predator stress in mice: sex differences in vulnerability to stress and predicting severity of anxiogenic response from the stress experience. Physiol Behav 88:12–29 [PubMed]
  • Mineur YS, Belzung C, Crusio WE 2006 Effects of unpredictable chronic mild stress on anxiety and depression-like behavior in mice. Behav Brain Res 175:43–50 [PubMed]
  • Kelliher P, Kelly JP, Leonard BE, Sánchez C 2003 Effects of acute and chronic administration of selective monoamine re-uptake inhibitors in the rat forced swim test. Psychoneuroendocrinology 28:332–347 [PubMed]
  • Griebel G, Moreau JL, Jenck F, Misslin R, Martin JR 1994 Acute and chronic treatment with 5-HT reuptake inhibitors differentially modulate emotional responses in anxiety models in rodents. Psychopharmacology (Berl) 113:463–470 [PubMed]
  • Burghardt NS, Sullivan GM, McEwen BS, Gorman JM, LeDoux JE 2004 The selective serotonin reuptake inhibitor citalopram increases fear after acute treatment but reduces fear with chronic treatment: a comparison with tianeptine. Biol Psychiatry 55:1171–1178 [PubMed]
  • Dazzi L, Seu E, Cherchi G, Biggio G 2005 Chronic administration of the SSRI fluvoxamine markedly and selectively reduces the sensitivity of cortical serotonergic neurons to footshock stress. Eur Neuropsychopharmacol 15:283–290 [PubMed]
  • Gerdelat-Mas A, Loubinoux I, Tombari D, Rascol O, Chollet F, Simonetta-Moreau M 2005 Chronic administration of selective serotonin reuptake inhibitor (SSRI) paroxetine modulates human motor cortex excitability in healthy subjects. Neuroimage 27:314–322 [PubMed]
  • Hesketh S, Jessop DS, Hogg S, Harbuz MS 2005 Differential actions of acute and chronic citalopram on the rodent hypothalamic-pituitary-adrenal axis response to acute restraint stress. J Endocrinol 185:373–382 [PubMed]
  • den Boer JA, Westenberg HG, Kamerbeek WD, Verhoeven WM, Kahn RS 1987 Effect of serotonin uptake inhibitors in anxiety disorders; a double-blind comparison of clomipramine and fluvoxamine. Int Clin Psychopharmacol 2:21–32 [PubMed]
  • Grillon C, Levenson J, Pine DS 2007 A single dose of the selective serotonin reuptake inhibitor citalopram exacerbates anxiety in humans: a fear-potentiated startle study. Neuropsychopharmacology 32:115–331 [PubMed]
  • Micevych PE, Mermelstein PG 2008 Membrane estrogen receptors acting through metabotropic glutamate receptors: an emerging mechanism of estrogen action in brain. Mol Neurobiol 38:66–77 [PMC free article] [PubMed]
  • Durand M, Berton O, Aguerre S, Edno L, Combourieu I, Mormède P, Chaouloff F 1999 Effects of repeated fluoxetine on anxiety-related behaviours, central serotonergic systems, and the corticotropic axis in SHR and WKY rats. Neuropharmacology 38:893–907 [PubMed]
  • Ichise M, Vines DC, Gura T, Anderson GM, Suomi SJ, Higley JD, Innis RB 2006 Effects of early life stress on [11C]DASB positron emission tomography imaging of serotonin transporters in adolescent peer- and mother-reared rhesus monkeys. J Neurosci 26:4638–4643 [PubMed]
  • Spindelegger C, Lanzenberger R, Wadsak W, Mien LK, Stein P, Mitterhauser M, Moser U, Holik A, Pezawas L, Kletter K, Kasper S 25 March 2008 Influence of escitalopram treatment on 5-HT1A receptor binding in limbic regions in patients with anxiety disorders. Mol Psychiatry 10.1038/mp.2008.35 [PubMed]
  • Romeo RD, McCarthy JB, Wang A, Milner TA, McEwen BS 2005 Sex differences in hippocampal estradiol-induced N-methyl-d-aspartic acid binding and ultrastructural localization of estrogen receptor-α. Neuroendocrinology 81:391–399 [PubMed]
  • Hariri AR, Holmes A 2006 Genetics of emotional regulation: the role of the serotonin transporter in neural function. Trends Cogn Neurosci 10:182–191 [PubMed]
  • Jennings KA, Loder MK, Sheward WJ, Pei Q, Deacon RM, Benson MA, Olverman HJ, Hastie ND, Harmar AJ, Shen S, Sharp T 2006 Increased expression of the 5-HT transporter confers a low-anxiety phenotype linked to decreased 5-HT transmission. J Neurosci 26:8955–8964 [PubMed]
  • Lira A, Zhou M, Castanon N, Ansorge MS, Gordon JA, Francis JH, Bradley-Moore M, Lira J, Underwood MD, Arango V, Kung HF, Hofer MA, Hen R, Gingrich JA 2003 Altered depression-related behaviors and functional changes in the dorsal raphe nucleus of serotonin transporter-deficient mice. Biol Psychiatry 54:960–971 [PubMed]
  • Parsey RV, Hastings RS, Oquendo MA, Huang YY, Simpson N, Arcement J, Huang Y, Ogden RT, Van Heertum RL, Arango V, Mann JJ 2006 Lower serotonin transporter binding potential in the human brain during major depressive episodes. Am J Psychiatry 163:52–58 [PubMed]
  • Handa RJ, Burgess LH, Kerr JE, O'Keefe JA 1994 Gonadal steroid hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis. Horm Behav 28:464–476 [PubMed]
  • Finley PR 1994 Selective serotonin reuptake inhibitors: pharmacologic profiles and potential therapeutic distinctions. Ann Pharmacother 28:1359–1369 [PubMed]
  • Paxinos G, Watson KB 2001 The mouse brain in stereotaxic coordinates. 2nd ed. New York: Elsevier

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