- Email: [email protected]
Primate Early Life Stress Leads to Long-Term Mild Hippocampal Decreases in Corticosteroid Receptor Expression
BRIEF REPORTS
Primate Early Life Stress Leads to Long-Term Mild Hippocampal Decreases in Corticosteroid Receptor Expression Dimitrula Arabadzisz, Rochellys Diaz-Heijtz, Irene Knuesel, Elisabeth Weber, Sonia Pilloud, Andrea C. Dettling, Joram Feldon, Amanda J. Law, Paul J. Harrison, and Christopher R. Pryce Background: Expression of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) genes are moderately reduced in several brain regions in depression. These reductions could be partly due to early life stress (ELS), which predicts emotional disorders. Controlled primate studies are important to test whether ELS sufficient to induce long-term emotional changes also induces long-term altered MR and/or GR brain expression. Methods: In the common marmoset, ELS of daily 30 –120-min social isolation across month-1 resulted in some long-term changes in homeostasis and emotional behavior. In some of these same subjects, the aim of this study was to use marmoset-specific riboprobes to determine whether ELS produced long-term effects on brain MR and GR gene expression. Results: At adolescence, relative to control subjects, ELS marmosets exhibited mildly reduced messenger RNA signal for both MR (⫺15%, p ⫽ .05) and GR (⫺13%, p ⫽ .02) in hippocampus—primarily CA1-2— but not in prefrontal cortex, other cortical regions, or hypothalamus. Conclusions: In adolescent marmoset monkey brains, reduced hippocampal expression of MR and GR are consistent chronic-indicators of ELS. It is unlikely that these chronic, mild, specific reductions were acute-mediators of the observed long-term emotional effects of ELS. However, they do suggest involvement of hippocampal MR/GR in the neurodevelopmental effects of ELS. Key Words: Corticosteroid receptor, depression, early life stress, hippocampus, marker, neurodevelopment, neuropathology, primate
D
epression is predicted by prior early life stress (ELS), such as parent–infant/child neglect or abuse (1,2), but mediating mechanisms and processes are not wellunderstood. Acute stress responses to ELS, including of the hypothalamic-pituitary-adrenal axis, might trigger neurodevelopmental changes with long-term consequences (3). Repeated 3-hour maternal separation in rat pups led to increased mineralocorticoid receptor (MR) and decreased glucocorticoid receptor (GR) expression in hippocampus in adulthood (4). When adult human probands who committed depression-associated suicide were separated according to presence or absence of ELS, the ELS/suicide cohort exhibited reduced hippocampal GR expression relative to non-ELS/suicide and control cohorts (5). In depression with unknown early life history, there is reduced MR expression in hippocampus (6,7) and reduced GR expression in hippocampus, prefrontal cortex, and temporal cortex (6,8,9). Reflecting this, there is interest in both MR and GR as antidepressant targets (10). A study of brain MR and GR expression in a
From the Laboratory of Behavioral Neurobiology (DA, IK, EW, SP, ACD, JF, CRP), Swiss Federal Institute of Technology, Zürich, Switzerland; Department of Neuroscience (RD-H), Karolinska Institutet, Stockholm, Sweden; Department of Psychiatry (AJL, PJH), University of Oxford, Warneford Hospital, Oxford, United Kingdom; and the Clinical Brain Disorders Branch (AJL), National Institute of Mental Health, National Institute of Health, Bethesda, Maryland. Authors DA and RD-H contributed equally to this work. Address correspondence to Christopher R. Pryce, Ph.D., Clinic for Affective Disorders and General Psychiatry, Psychiatric University Hospital Zurich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland; E-mail: Christopher. [email protected]. Received Aug 3, 2009; revised Oct 16, 2009; accepted Dec 14, 2009.
0006-3223/$36.00 doi:10.1016/j.biopsych.2009.12.016
nonhuman primate ELS model with long-term altered homeostasis and emotionality is lacking. We have conducted a controlled in vivo–ex vivo study of effects of daily parental separationisolation in common marmosets otherwise reared normally in their family groups. As reported previously, these marmosets exhibited short-term stress hormone responses to the daily ELS (11) and long-term effects on homeostasis and emotional behavior, including increased basal urinary monoamine levels, reduced motivation for palatable reward, and increased sensitivity to negative feedback at reversal learning (12,13). Here we present findings on effects of ELS on brain MR and GR expression at adolescence and integrate these with in vivo findings.
Methods and Materials For a complete description of methods and materials see Supplement 1. Early deprivation (ED) comprised separation of infant from parent and isolation in a neutral environment for 30 –120 min/ day on postnatal Days 2–28. The control procedure (CON) was handling of the infant on the parent. Otherwise, all subjects remained with the family group and were studied longitudinally in terms of physiology and behavior. Subjects were killed at age 48 weeks. There were 11 ED subjects, 7 male and 4 female, from 10 breeding pairs, and 9 CON subjects, 5 male and 4 female, from 9 of the same breeding pairs. The brains were coded and analyzed blind. Each brain was serially sectioned on a cryostat in the coronal plane at 10 m. In situ hybridization of MR and GR messenger RNAs (mRNAs) was conducted with common marmoset-specific 35S-labeled riboprobes (14), according to Heijtz et al. (15). The MR/GR mRNA were each measured in hippocampus, cortex, and prefrontal cortex (PFC), and GR mRNA was measured in hypothalamus. For each receptor and brain region, all sections were included in a single in situ hybridization run; controls comprised concurrent hybridization with the respective 35S-UTP-labeled sense complementary RNA probe. Optical density values were quantified after BIOL PSYCHIATRY 2010;67:1106 –1109 © 2010 Society of Biological Psychiatry
D. Arabadzisz et al. background subtraction. A 14C step standard (GE Healthcare, Uppsala, Sweden) was included to calibrate optical density readings and convert measured values into kBq/g. Subjects’ mean optical densities were analyzed per region with analysis of variance with a within-subject factor of Area and between-subject factors of Manipulation and Gender; post hoc testing was conducted with the Bonferroni’s test. For brain regions/areas exhibiting significant Manipulation effects, correlations were analyzed both across and, if significant, within Manipulation: for basal cortisol titers at death/brain collection and for in vivo measures affected by ED, (i.e., physiology: increased basal urinary norepinephrine [13] and dopamine titers [12]; behavior: reduced social play [11], increased impulsivity [12], reduced motivation for palatable reward, reduced reversal learning [13]). Statistical testing was conducted with SPSS (SPSS Inc., Cary, North Carolina), with ␣ ⫽ .05.
Results Relative expression levels of MR mRNA were DG ⬎ CA14 ⬎ Cortex ⬎ PFC. In hippocampus (Figures 1A–1E), there was a significant main effect of Manipulation, indicating that
BIOL PSYCHIATRY 2010;67:1106 –1109 1107 MR mRNA levels were significantly reduced in ED relative to CON subjects [F (1,16) ⫽ 4.38, p ⫽ .05], with no effect of Gender (p ⬎ .1; Table S1 in Supplement 1). A-posteriori analysis of individual areas revealed a significant reduction in ED versus CON in CA2 [F (1,16) ⫽ 7.59, p ⫽ .01] (reduction in mean value ⫽ ⫺16%) and CA1 [F (1,16) ⫽ 8.14, p ⫽ .01] (⫺15%). In cortex (Table 1) there was no significant effect of Manipulation or Gender (p ⬎ .2). In PFC (Table 1) there was a significant Manipulation ⫻ Area interaction [F (3,21) ⫽ 4.47, p ⫽ .01], but in no single PFC area was there a significant effect of Manipulation (p values ⬎ .1). Relative expression levels of GR mRNA were DG ⬎ PFC ⬎ CA1-4 ⫽ Cortex ⫽ paraventricular nucleus. In hippocampus (Figures 1F–1J), there was a significant main effect of Manipulation, indicating that GR mRNA levels were significantly reduced in ED relative to CON subjects [F (1,16) ⫽ 6.80, p ⫽ .02], with no effect of Gender (p ⬎ .1; Table S1 in Supplement 1). There was a significant reduction in ED versus CON in CA2 [F (1,16) ⫽ 12.51, p ⫽ .003] (⫺12%) and in CA1 [F (1,16) ⫽ 11.36, p ⫽ .004] (⫺14%). In cortex, PFC, and hypothalamus (Table 1), there was no significant effect of Manipulation or Gender (p ⬎ .1).
Figure 1. Individual and mean optical density (OD) values for (A) mineralocorticoid receptor (MR) messenger RNA (mRNA) and (F) glucocorticoid receptor (GR) mRNA, in the dentate gyrus (DG) and Cornu ammonis subfields (CA) 4 –1, of control procedure (CON) (open circles, n ⫽ 9) versus early deprivation (ED) (filled circles, n ⫽ 11) adolescent common marmosets. The p values are for main effect of manipulation on the basis of a posteriori area-specific analysis of variance. Representative autoradiograms from coronal brain sections at the level of hippocampal formation showing MR mRNA signal in (B, C) a CON marmoset and (D, E) an ED marmoset and GR mRNA signal in (G, H) a CON marmoset and (I, J) an ED marmoset. The pseudocoloring indicates signal intensity from low (black/purple) to high (yellow/white). The scale bar in B denotes 2 mm. The arrow (with tail) indicates the estimated midregion of CA2, and the arrowhead indicates the estimated midregion of CA1.
www.sobp.org/journal
1108 BIOL PSYCHIATRY 2010;67:1106 –1109
D. Arabadzisz et al.
Table 1. MR and GR mRNA Levels (kBq/g, mean ⫾ SD) in CON and ED Marmosets MR Brain Region/Area
CON
ED
Cortex
n⫽9
n ⫽ 11
Parietal Insular Temporal Prefrontal Cortex Dorsal Medial Orbital Lateral
6.1 ⫾ .9 5.9 ⫾ .6 4.9 ⫾ .4 4.7 ⫾ .6 4.8 ⫾ .6 4.4 ⫾ .6 Par ⬎ Ins ⫽ Temp n⫽5
n⫽6
2.4 ⫾ .4 2.5 ⫾ .3 2.4 ⫾ .3 2.3 ⫾ .2 2.2 ⫾ .3 2.3 ⫾ .2 2.5 ⫾ .4 2.4 ⫾ .2 Dor ⬎ Orb; Lat ⬎ Orb
PVN
GR a
p
CON
ED
⬎.2
n⫽9
n ⫽ 11
.0001 ⬎ .5
.001
4.7 ⫾ 1.2 4.1 ⫾ 1.0 4.0 ⫾ .6 3.4 ⫾ .5 3.4 ⫾ .5 3.2 ⫾ .7 Par ⬎ Ins ⫽ Temp n⫽5
n⫽6
7.2 ⫾ .6 7.6 ⫾ .7 6.6 ⫾ .4 6.7 ⫾ .7 6.6 ⫾ .5 6.5 ⫾ .8 7.4 ⫾ .8 7.4 ⫾ .6 Dor ⫽ Lat ⬎ Med ⫽ Orb n⫽7
n ⫽ 11
4.3 ⫾ 1.1
3.8 ⫾ 1.1
pa ⬎.1
.0001 ⬎.6
.0001 ⬎.3
For logistical reasons, analysis of early deprivation (ED) effects on prefrontal cortex mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) expression was conducted with matched sections from only 5– 6 subjects/ manipulation group. Due to low expression levels in marmoset (14), MR messenger RNA (mRNA) was not quantified in paraventricular nucleus (PVN). CON, control procedure; Par, parietal; Ins, Insular; Temp, temporal; Dor, dorsal; Orb, orbital; Lat, lateral; Med, medial. a The p values are for main effect of manipulation within region and main effect of area within region.
Because ED induced similar magnitude of reductions in hippocampal MR and GR mRNA signals, there was no ED effect on MR/GR ratios in any area (e.g., CA1: CON ⫽ 2.3 ⫾ .4 [mean ⫾ SD], ED ⫽ 2.4 ⫾ .3; CA2: CON ⫽ 4.5 ⫾ .5, ED ⫽ 4.6 ⫾ .7) (p ⱖ .7). There was no significant effect of ED on cortisol titers (Figure S1 in Supplement 1). Turning to correlations with in vivo measures; for cortisol titers at death (n ⫽ 19, 8 CON, 11 ED), there were no significant correlations with CA-area MR or MR: GR mRNA (Supplement 1). For urinary monoamine titers (n ⫽ 11, 5 CON, 6 ED), there were significant inverse correlations across Manipulation of CA1-2 GR mRNA with NE titers (e.g., CA1: r ⫽ ⫺.68, p ⫽ .02) and DA titers (e.g., CA1: r ⫽ ⫺.64, p ⫽ .03) and of CA1 MR mRNA with NE titers (r ⫽.63, p ⫽ .04). The partial correlations (e.g., CA1 GR mRNA and urinary NE) (partial r ⫽ ⫺.50, p ⬎ .14) and groupspecific correlations (CON r ⫽ ⫺.65, p ⬎ .25, ED r ⫽ ⫺.32, p ⬎ .53) were inverse but did not attain significance. There was no significant across-Manipulation correlation of MR or GR mRNA in CA1/2 with any behavioral measure (p ⱖ .2).
Discussion This study demonstrates that ELS in primate infants leads in adolescence to mild reductions in MR and GR gene expression in hippocampus and not in PFC, cortex, or paraventricular nucleus. The ED and CON siblings had been studied in vivo, and several depression-relevant physiological and behavioral effects had been demonstrated. In adult human probands of unknown early life history, depression is associated with mild–moderate reductions in hippocampal MR (⫺20%/⫺30%) (6) and GR (⫺20%) (6,9) expression; depression-related suicide was associated with a moderate (⫺25%) reduction in hippocampal GR expression in ELS but not in non-ELS probands (5). The current findings suggest that reduced hippocampal MR/GR expressions are long-term markers of ELS that are mild but nonetheless consistent across individuals/families. This is www.sobp.org/journal
striking, given that ED marmosets otherwise experienced stressfree development and, unlike humans, do not self-reflect on life history (16). The mechanism underlying the reductions is unknown; increased gene-promoter methylation is suggested by recent human ELS evidence (5). The magnitude and specificity of the effects strongly suggest that reduced MR/GR expressions do not mediate the depression-relevant behavioral effects of ED demonstrated in these same subjects (11–13,17). It remains unclear whether the reductions in MR in hippocampus (6,7) and in GR in hippocampus, PFC, and temporal cortex (6,8,9) contribute to depression etiology. The inverse correlations of hippocampal MR/GR (decreased in ED) with urinary monoamine titers (increased across development in ED) are compromised by limited statistical power but are interesting, given that urinary monoamines are increased in humans who experienced ELS (18). The ED monkeys did not exhibit altered basal/early stress response cortisol titers, perhaps reflecting the unchanged hippocampal MR/GR ratio (19). The combination of reduced MR expression and unchanged basal cortisol suggests that the binding levels of high-affinity MR would be mildly but continuously reduced in hippocampus of ED marmosets (19). Although the long-term reductions are mild, this does not preclude that ELS induced short-term hyper-stimulation and/or downregulation of hippocampal MR/GR and that these events were important in modifying neurodevelopment and thereby long-term homeostasis and emotional function. We do not have data to test this hypothesis but note that neonate-infant marmoset brain expresses MR/GR (14) and that cortisol responses to daily ED occurred (11). That the primate hippocampus is particularly sensitive to long-term effects of ELS is evidenced by MR/GR here and by reduced expression of serotonin1A receptor and growthassociated protein-43 as reported previously for the same subjects (20). Therefore, in adolescent marmoset monkey brains, reduced hippocampal expression of MR and GR are consistent chronic-
BIOL PSYCHIATRY 2010;67:1106 –1109 1109
D. Arabadzisz et al. indicators of ELS. It is unlikely that these chronic, mild, specific reductions were acute-mediators of the observed long-term emotional effects of ELS. However, they do suggest involvement of hippocampal MR/GR in the neurodevelopmental effects of ELS. This research was funded by the Swiss Federal Institute of Technology (ETH) Zurich, the Swiss National Science Foundation (SNSF) National Center for Competence in Research (NCCR), Swiss Etiological Study of Adjustment and Mental Health, Grant 51A240 –104890, the NCCR Neural Plasticity and Repair, a SNSF Project Grant 31-67791.02, and the Wellcome Trust, United Kingdom. We are extremely grateful to Jean-Marc Fritschy for providing laboratory facilities; Corinne Sidler, Mary Walker, and Helen Gordon-Andrews for their expert technical assistance; and Sandor Vizi for helpful and stimulating discussion. Dr. Harrison reports having received, in the past 3 years, honoraria from AstraZeneca, Janssen Cilag, Merck, Novartis, Organon, Sanofi, Schering Plough, Servier, and Wyeth and an unrestricted educational grant from GlaxoSmithKline. Dr. Pryce has been an employee of Novartis. All other authors reported no biomedical financial interests or potential conflicts of interest. Supplementary material cited in this article is available online. 1. Kendler KS, Gardner CO, Prescott CA (2002): Toward a comprehensive development model for major depression in women. Am J Psychiatry 159:1133–1145. 2. Spatz Widom C, DuMont K, Czaja SJ (2007): A prospective investigation of major depressive disorder and comorbidity in abused and neglected children grown up. Arch Gen Psychiatry 64:49 –56. 3. Tarullo AR, Gunnar MR (2006): Child maltreatment and the developing HPA axis. Horm Behav 50:632– 639. 4. Ladd CO, Huot RL, Thrivikraman KV, Nemeroff CB, Plotsky PM (2004): Long-term adaptations in glucocorticoid receptor and mineralocorticoid receptor mRNA and negative feedback on the hypothalamo-pituitary-adrenal axis following neonatal maternal separation. Biol Psychiatry 55:367–375. 5. McGowan PO, Sasaki A, D’Alessio AC, Dymov S, Labonte B, Szyf M, et al. (2009): Epigenetic regulation of the glucocorticoid receptor in human brain associates with child abuse. Nat Neurosci 12:342–348. 6. Lopez JF, Chalmers DT, Little KY, Watson SJ (1998): Regulation of serotonin1A, glucocorticoid, annd mineralocorticoid receptor in rat and
7. 8.
9.
10. 11. 12. 13. 14.
15. 16. 17.
18.
19. 20.
human hippocampus: Implications for the neurobiology of depression. Biol Psychiatry 43:547–573. Xing G-Q, Russell S, Webster MJ, Post RM (2004): Decreased expression of mineralocorticoid receptor mRNA in the prefrontal cortex in schizophrenia and bipolar disorder. Int J Neuropsychopharmacol 7:143–153. Wang S-S, Kamphuis W, Huitinga I, Zhou J-N, Swaab D (2008): Gene expression analysis in the human hypothalamus in depression by laser microdissection and real-time PCR: The presence of multiple receptor imbalances. Mol Psychiatry 13:786 –799. Webster MJ, Knable MB, O’Grady J, Orthmann J, Weickert CS (2002): Regional specificity of brain glucocorticoid receptor mRNA alterations in subjects with schizophrenia and mood disorders. Mol Psychiatry 7:985–994. Schüle C, Baghai TC, Eser D, Rupprecht R (2009): Hypothalamic-pituitary-adrenocortical system dysregulation and new treatment strategies in depression. Expert Rev Neurother 9:1005–1019. Dettling AC, Feldon J, Pryce CR (2002): Repeated parental deprivation in the infant common marmoset (Callithrix jacchus, Primates) and analysis of its effects on early development. Biol Psychiatry 52:1037–1046. Pryce CR, Dettling AC, Feldon J (2004): Evidence for altered monoamine activity and emotional and cognitive disturbance in marmoset monkeys exposed to early life stress. Ann N Y Acad Sci 1032:245–249. Pryce CR, Dettling AC, Spengler M, Schnell CR, Feldon J (2004): Deprivation of parenting disrupts development of homeostatic and reward systems in marmoset monkey offspring. Biol Psychiatry 56:72–79. Pryce CR, Feldon J, Fuchs E, Knuesel I, Oertle T, Sengstag C, et al. (2005): Postnatal ontogeny of hippocampal expression of the mineralocorticoid and glucocorticoid receptors in the common marmoset. Eur J Neurosci 21:1521–1535. Heijtz RD, Alexeyenko A, Castellanos FX (2007): Calcyon mRNA expression in the frontal-striatal circuitry and its relationship to vesicular processes and ADHD. Behav Brain Funct 3:33. Craig AD (2009): How do you feel—now? The anterior insula and human awareness. Nat Rev Neurosci 10:59 –70. Dettling AC, Schnell CR, Maier C, Feldon J, Pryce CR (2007): Behavioral and physiological effects of an infant-neglect manipulation in a biparental, twinning primate: Impact is dependent on familial factors. Psychoneuroendocrinology 32:331–349. Pervanidou P, Chrousos GP (2007): Post-traumatic stress disorder in children and adolescents: From Sigmund Freud’s “trauma” to psychopathology and the (dys)metabolic syndrome. Horm Metab Res 39:413– 419. De Kloet ER, Vreugdenhil E, Oitzl MS, Joels M (1998): Brain corticosteroid receptor balance in health and disease. Endocr Rev 19:269 –301. Law AJ, Pei Q, Walker M, Gordon-Andrews H, Weickert CS, Feldon J, et al. (2009): Early parental deprivation in the marmoset monkey produces long-term changes in hippocampal expression of genes involved in synaptic plasticity and implicated in mood disorder. Neuropsychopharmacology 34:1381–1394.
www.sobp.org/journal
Primate Early Life Stress Leads to Long-Term Mild Hippocampal Decreases in Corticosteroid Receptor Expression Dimitrula Arabadzisz, Rochellys Diaz-Heijtz, Irene Knuesel, Elisabeth Weber, Sonia Pilloud, Andrea C. Dettling, Joram Feldon, Amanda J. Law, Paul J. Harrison, and Christopher R. Pryce Background: Expression of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) genes are moderately reduced in several brain regions in depression. These reductions could be partly due to early life stress (ELS), which predicts emotional disorders. Controlled primate studies are important to test whether ELS sufficient to induce long-term emotional changes also induces long-term altered MR and/or GR brain expression. Methods: In the common marmoset, ELS of daily 30 –120-min social isolation across month-1 resulted in some long-term changes in homeostasis and emotional behavior. In some of these same subjects, the aim of this study was to use marmoset-specific riboprobes to determine whether ELS produced long-term effects on brain MR and GR gene expression. Results: At adolescence, relative to control subjects, ELS marmosets exhibited mildly reduced messenger RNA signal for both MR (⫺15%, p ⫽ .05) and GR (⫺13%, p ⫽ .02) in hippocampus—primarily CA1-2— but not in prefrontal cortex, other cortical regions, or hypothalamus. Conclusions: In adolescent marmoset monkey brains, reduced hippocampal expression of MR and GR are consistent chronic-indicators of ELS. It is unlikely that these chronic, mild, specific reductions were acute-mediators of the observed long-term emotional effects of ELS. However, they do suggest involvement of hippocampal MR/GR in the neurodevelopmental effects of ELS. Key Words: Corticosteroid receptor, depression, early life stress, hippocampus, marker, neurodevelopment, neuropathology, primate
D
epression is predicted by prior early life stress (ELS), such as parent–infant/child neglect or abuse (1,2), but mediating mechanisms and processes are not wellunderstood. Acute stress responses to ELS, including of the hypothalamic-pituitary-adrenal axis, might trigger neurodevelopmental changes with long-term consequences (3). Repeated 3-hour maternal separation in rat pups led to increased mineralocorticoid receptor (MR) and decreased glucocorticoid receptor (GR) expression in hippocampus in adulthood (4). When adult human probands who committed depression-associated suicide were separated according to presence or absence of ELS, the ELS/suicide cohort exhibited reduced hippocampal GR expression relative to non-ELS/suicide and control cohorts (5). In depression with unknown early life history, there is reduced MR expression in hippocampus (6,7) and reduced GR expression in hippocampus, prefrontal cortex, and temporal cortex (6,8,9). Reflecting this, there is interest in both MR and GR as antidepressant targets (10). A study of brain MR and GR expression in a
From the Laboratory of Behavioral Neurobiology (DA, IK, EW, SP, ACD, JF, CRP), Swiss Federal Institute of Technology, Zürich, Switzerland; Department of Neuroscience (RD-H), Karolinska Institutet, Stockholm, Sweden; Department of Psychiatry (AJL, PJH), University of Oxford, Warneford Hospital, Oxford, United Kingdom; and the Clinical Brain Disorders Branch (AJL), National Institute of Mental Health, National Institute of Health, Bethesda, Maryland. Authors DA and RD-H contributed equally to this work. Address correspondence to Christopher R. Pryce, Ph.D., Clinic for Affective Disorders and General Psychiatry, Psychiatric University Hospital Zurich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland; E-mail: Christopher. [email protected]. Received Aug 3, 2009; revised Oct 16, 2009; accepted Dec 14, 2009.
0006-3223/$36.00 doi:10.1016/j.biopsych.2009.12.016
nonhuman primate ELS model with long-term altered homeostasis and emotionality is lacking. We have conducted a controlled in vivo–ex vivo study of effects of daily parental separationisolation in common marmosets otherwise reared normally in their family groups. As reported previously, these marmosets exhibited short-term stress hormone responses to the daily ELS (11) and long-term effects on homeostasis and emotional behavior, including increased basal urinary monoamine levels, reduced motivation for palatable reward, and increased sensitivity to negative feedback at reversal learning (12,13). Here we present findings on effects of ELS on brain MR and GR expression at adolescence and integrate these with in vivo findings.
Methods and Materials For a complete description of methods and materials see Supplement 1. Early deprivation (ED) comprised separation of infant from parent and isolation in a neutral environment for 30 –120 min/ day on postnatal Days 2–28. The control procedure (CON) was handling of the infant on the parent. Otherwise, all subjects remained with the family group and were studied longitudinally in terms of physiology and behavior. Subjects were killed at age 48 weeks. There were 11 ED subjects, 7 male and 4 female, from 10 breeding pairs, and 9 CON subjects, 5 male and 4 female, from 9 of the same breeding pairs. The brains were coded and analyzed blind. Each brain was serially sectioned on a cryostat in the coronal plane at 10 m. In situ hybridization of MR and GR messenger RNAs (mRNAs) was conducted with common marmoset-specific 35S-labeled riboprobes (14), according to Heijtz et al. (15). The MR/GR mRNA were each measured in hippocampus, cortex, and prefrontal cortex (PFC), and GR mRNA was measured in hypothalamus. For each receptor and brain region, all sections were included in a single in situ hybridization run; controls comprised concurrent hybridization with the respective 35S-UTP-labeled sense complementary RNA probe. Optical density values were quantified after BIOL PSYCHIATRY 2010;67:1106 –1109 © 2010 Society of Biological Psychiatry
D. Arabadzisz et al. background subtraction. A 14C step standard (GE Healthcare, Uppsala, Sweden) was included to calibrate optical density readings and convert measured values into kBq/g. Subjects’ mean optical densities were analyzed per region with analysis of variance with a within-subject factor of Area and between-subject factors of Manipulation and Gender; post hoc testing was conducted with the Bonferroni’s test. For brain regions/areas exhibiting significant Manipulation effects, correlations were analyzed both across and, if significant, within Manipulation: for basal cortisol titers at death/brain collection and for in vivo measures affected by ED, (i.e., physiology: increased basal urinary norepinephrine [13] and dopamine titers [12]; behavior: reduced social play [11], increased impulsivity [12], reduced motivation for palatable reward, reduced reversal learning [13]). Statistical testing was conducted with SPSS (SPSS Inc., Cary, North Carolina), with ␣ ⫽ .05.
Results Relative expression levels of MR mRNA were DG ⬎ CA14 ⬎ Cortex ⬎ PFC. In hippocampus (Figures 1A–1E), there was a significant main effect of Manipulation, indicating that
BIOL PSYCHIATRY 2010;67:1106 –1109 1107 MR mRNA levels were significantly reduced in ED relative to CON subjects [F (1,16) ⫽ 4.38, p ⫽ .05], with no effect of Gender (p ⬎ .1; Table S1 in Supplement 1). A-posteriori analysis of individual areas revealed a significant reduction in ED versus CON in CA2 [F (1,16) ⫽ 7.59, p ⫽ .01] (reduction in mean value ⫽ ⫺16%) and CA1 [F (1,16) ⫽ 8.14, p ⫽ .01] (⫺15%). In cortex (Table 1) there was no significant effect of Manipulation or Gender (p ⬎ .2). In PFC (Table 1) there was a significant Manipulation ⫻ Area interaction [F (3,21) ⫽ 4.47, p ⫽ .01], but in no single PFC area was there a significant effect of Manipulation (p values ⬎ .1). Relative expression levels of GR mRNA were DG ⬎ PFC ⬎ CA1-4 ⫽ Cortex ⫽ paraventricular nucleus. In hippocampus (Figures 1F–1J), there was a significant main effect of Manipulation, indicating that GR mRNA levels were significantly reduced in ED relative to CON subjects [F (1,16) ⫽ 6.80, p ⫽ .02], with no effect of Gender (p ⬎ .1; Table S1 in Supplement 1). There was a significant reduction in ED versus CON in CA2 [F (1,16) ⫽ 12.51, p ⫽ .003] (⫺12%) and in CA1 [F (1,16) ⫽ 11.36, p ⫽ .004] (⫺14%). In cortex, PFC, and hypothalamus (Table 1), there was no significant effect of Manipulation or Gender (p ⬎ .1).
Figure 1. Individual and mean optical density (OD) values for (A) mineralocorticoid receptor (MR) messenger RNA (mRNA) and (F) glucocorticoid receptor (GR) mRNA, in the dentate gyrus (DG) and Cornu ammonis subfields (CA) 4 –1, of control procedure (CON) (open circles, n ⫽ 9) versus early deprivation (ED) (filled circles, n ⫽ 11) adolescent common marmosets. The p values are for main effect of manipulation on the basis of a posteriori area-specific analysis of variance. Representative autoradiograms from coronal brain sections at the level of hippocampal formation showing MR mRNA signal in (B, C) a CON marmoset and (D, E) an ED marmoset and GR mRNA signal in (G, H) a CON marmoset and (I, J) an ED marmoset. The pseudocoloring indicates signal intensity from low (black/purple) to high (yellow/white). The scale bar in B denotes 2 mm. The arrow (with tail) indicates the estimated midregion of CA2, and the arrowhead indicates the estimated midregion of CA1.
www.sobp.org/journal
1108 BIOL PSYCHIATRY 2010;67:1106 –1109
D. Arabadzisz et al.
Table 1. MR and GR mRNA Levels (kBq/g, mean ⫾ SD) in CON and ED Marmosets MR Brain Region/Area
CON
ED
Cortex
n⫽9
n ⫽ 11
Parietal Insular Temporal Prefrontal Cortex Dorsal Medial Orbital Lateral
6.1 ⫾ .9 5.9 ⫾ .6 4.9 ⫾ .4 4.7 ⫾ .6 4.8 ⫾ .6 4.4 ⫾ .6 Par ⬎ Ins ⫽ Temp n⫽5
n⫽6
2.4 ⫾ .4 2.5 ⫾ .3 2.4 ⫾ .3 2.3 ⫾ .2 2.2 ⫾ .3 2.3 ⫾ .2 2.5 ⫾ .4 2.4 ⫾ .2 Dor ⬎ Orb; Lat ⬎ Orb
PVN
GR a
p
CON
ED
⬎.2
n⫽9
n ⫽ 11
.0001 ⬎ .5
.001
4.7 ⫾ 1.2 4.1 ⫾ 1.0 4.0 ⫾ .6 3.4 ⫾ .5 3.4 ⫾ .5 3.2 ⫾ .7 Par ⬎ Ins ⫽ Temp n⫽5
n⫽6
7.2 ⫾ .6 7.6 ⫾ .7 6.6 ⫾ .4 6.7 ⫾ .7 6.6 ⫾ .5 6.5 ⫾ .8 7.4 ⫾ .8 7.4 ⫾ .6 Dor ⫽ Lat ⬎ Med ⫽ Orb n⫽7
n ⫽ 11
4.3 ⫾ 1.1
3.8 ⫾ 1.1
pa ⬎.1
.0001 ⬎.6
.0001 ⬎.3
For logistical reasons, analysis of early deprivation (ED) effects on prefrontal cortex mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) expression was conducted with matched sections from only 5– 6 subjects/ manipulation group. Due to low expression levels in marmoset (14), MR messenger RNA (mRNA) was not quantified in paraventricular nucleus (PVN). CON, control procedure; Par, parietal; Ins, Insular; Temp, temporal; Dor, dorsal; Orb, orbital; Lat, lateral; Med, medial. a The p values are for main effect of manipulation within region and main effect of area within region.
Because ED induced similar magnitude of reductions in hippocampal MR and GR mRNA signals, there was no ED effect on MR/GR ratios in any area (e.g., CA1: CON ⫽ 2.3 ⫾ .4 [mean ⫾ SD], ED ⫽ 2.4 ⫾ .3; CA2: CON ⫽ 4.5 ⫾ .5, ED ⫽ 4.6 ⫾ .7) (p ⱖ .7). There was no significant effect of ED on cortisol titers (Figure S1 in Supplement 1). Turning to correlations with in vivo measures; for cortisol titers at death (n ⫽ 19, 8 CON, 11 ED), there were no significant correlations with CA-area MR or MR: GR mRNA (Supplement 1). For urinary monoamine titers (n ⫽ 11, 5 CON, 6 ED), there were significant inverse correlations across Manipulation of CA1-2 GR mRNA with NE titers (e.g., CA1: r ⫽ ⫺.68, p ⫽ .02) and DA titers (e.g., CA1: r ⫽ ⫺.64, p ⫽ .03) and of CA1 MR mRNA with NE titers (r ⫽.63, p ⫽ .04). The partial correlations (e.g., CA1 GR mRNA and urinary NE) (partial r ⫽ ⫺.50, p ⬎ .14) and groupspecific correlations (CON r ⫽ ⫺.65, p ⬎ .25, ED r ⫽ ⫺.32, p ⬎ .53) were inverse but did not attain significance. There was no significant across-Manipulation correlation of MR or GR mRNA in CA1/2 with any behavioral measure (p ⱖ .2).
Discussion This study demonstrates that ELS in primate infants leads in adolescence to mild reductions in MR and GR gene expression in hippocampus and not in PFC, cortex, or paraventricular nucleus. The ED and CON siblings had been studied in vivo, and several depression-relevant physiological and behavioral effects had been demonstrated. In adult human probands of unknown early life history, depression is associated with mild–moderate reductions in hippocampal MR (⫺20%/⫺30%) (6) and GR (⫺20%) (6,9) expression; depression-related suicide was associated with a moderate (⫺25%) reduction in hippocampal GR expression in ELS but not in non-ELS probands (5). The current findings suggest that reduced hippocampal MR/GR expressions are long-term markers of ELS that are mild but nonetheless consistent across individuals/families. This is www.sobp.org/journal
striking, given that ED marmosets otherwise experienced stressfree development and, unlike humans, do not self-reflect on life history (16). The mechanism underlying the reductions is unknown; increased gene-promoter methylation is suggested by recent human ELS evidence (5). The magnitude and specificity of the effects strongly suggest that reduced MR/GR expressions do not mediate the depression-relevant behavioral effects of ED demonstrated in these same subjects (11–13,17). It remains unclear whether the reductions in MR in hippocampus (6,7) and in GR in hippocampus, PFC, and temporal cortex (6,8,9) contribute to depression etiology. The inverse correlations of hippocampal MR/GR (decreased in ED) with urinary monoamine titers (increased across development in ED) are compromised by limited statistical power but are interesting, given that urinary monoamines are increased in humans who experienced ELS (18). The ED monkeys did not exhibit altered basal/early stress response cortisol titers, perhaps reflecting the unchanged hippocampal MR/GR ratio (19). The combination of reduced MR expression and unchanged basal cortisol suggests that the binding levels of high-affinity MR would be mildly but continuously reduced in hippocampus of ED marmosets (19). Although the long-term reductions are mild, this does not preclude that ELS induced short-term hyper-stimulation and/or downregulation of hippocampal MR/GR and that these events were important in modifying neurodevelopment and thereby long-term homeostasis and emotional function. We do not have data to test this hypothesis but note that neonate-infant marmoset brain expresses MR/GR (14) and that cortisol responses to daily ED occurred (11). That the primate hippocampus is particularly sensitive to long-term effects of ELS is evidenced by MR/GR here and by reduced expression of serotonin1A receptor and growthassociated protein-43 as reported previously for the same subjects (20). Therefore, in adolescent marmoset monkey brains, reduced hippocampal expression of MR and GR are consistent chronic-
BIOL PSYCHIATRY 2010;67:1106 –1109 1109
D. Arabadzisz et al. indicators of ELS. It is unlikely that these chronic, mild, specific reductions were acute-mediators of the observed long-term emotional effects of ELS. However, they do suggest involvement of hippocampal MR/GR in the neurodevelopmental effects of ELS. This research was funded by the Swiss Federal Institute of Technology (ETH) Zurich, the Swiss National Science Foundation (SNSF) National Center for Competence in Research (NCCR), Swiss Etiological Study of Adjustment and Mental Health, Grant 51A240 –104890, the NCCR Neural Plasticity and Repair, a SNSF Project Grant 31-67791.02, and the Wellcome Trust, United Kingdom. We are extremely grateful to Jean-Marc Fritschy for providing laboratory facilities; Corinne Sidler, Mary Walker, and Helen Gordon-Andrews for their expert technical assistance; and Sandor Vizi for helpful and stimulating discussion. Dr. Harrison reports having received, in the past 3 years, honoraria from AstraZeneca, Janssen Cilag, Merck, Novartis, Organon, Sanofi, Schering Plough, Servier, and Wyeth and an unrestricted educational grant from GlaxoSmithKline. Dr. Pryce has been an employee of Novartis. All other authors reported no biomedical financial interests or potential conflicts of interest. Supplementary material cited in this article is available online. 1. Kendler KS, Gardner CO, Prescott CA (2002): Toward a comprehensive development model for major depression in women. Am J Psychiatry 159:1133–1145. 2. Spatz Widom C, DuMont K, Czaja SJ (2007): A prospective investigation of major depressive disorder and comorbidity in abused and neglected children grown up. Arch Gen Psychiatry 64:49 –56. 3. Tarullo AR, Gunnar MR (2006): Child maltreatment and the developing HPA axis. Horm Behav 50:632– 639. 4. Ladd CO, Huot RL, Thrivikraman KV, Nemeroff CB, Plotsky PM (2004): Long-term adaptations in glucocorticoid receptor and mineralocorticoid receptor mRNA and negative feedback on the hypothalamo-pituitary-adrenal axis following neonatal maternal separation. Biol Psychiatry 55:367–375. 5. McGowan PO, Sasaki A, D’Alessio AC, Dymov S, Labonte B, Szyf M, et al. (2009): Epigenetic regulation of the glucocorticoid receptor in human brain associates with child abuse. Nat Neurosci 12:342–348. 6. Lopez JF, Chalmers DT, Little KY, Watson SJ (1998): Regulation of serotonin1A, glucocorticoid, annd mineralocorticoid receptor in rat and
7. 8.
9.
10. 11. 12. 13. 14.
15. 16. 17.
18.
19. 20.
human hippocampus: Implications for the neurobiology of depression. Biol Psychiatry 43:547–573. Xing G-Q, Russell S, Webster MJ, Post RM (2004): Decreased expression of mineralocorticoid receptor mRNA in the prefrontal cortex in schizophrenia and bipolar disorder. Int J Neuropsychopharmacol 7:143–153. Wang S-S, Kamphuis W, Huitinga I, Zhou J-N, Swaab D (2008): Gene expression analysis in the human hypothalamus in depression by laser microdissection and real-time PCR: The presence of multiple receptor imbalances. Mol Psychiatry 13:786 –799. Webster MJ, Knable MB, O’Grady J, Orthmann J, Weickert CS (2002): Regional specificity of brain glucocorticoid receptor mRNA alterations in subjects with schizophrenia and mood disorders. Mol Psychiatry 7:985–994. Schüle C, Baghai TC, Eser D, Rupprecht R (2009): Hypothalamic-pituitary-adrenocortical system dysregulation and new treatment strategies in depression. Expert Rev Neurother 9:1005–1019. Dettling AC, Feldon J, Pryce CR (2002): Repeated parental deprivation in the infant common marmoset (Callithrix jacchus, Primates) and analysis of its effects on early development. Biol Psychiatry 52:1037–1046. Pryce CR, Dettling AC, Feldon J (2004): Evidence for altered monoamine activity and emotional and cognitive disturbance in marmoset monkeys exposed to early life stress. Ann N Y Acad Sci 1032:245–249. Pryce CR, Dettling AC, Spengler M, Schnell CR, Feldon J (2004): Deprivation of parenting disrupts development of homeostatic and reward systems in marmoset monkey offspring. Biol Psychiatry 56:72–79. Pryce CR, Feldon J, Fuchs E, Knuesel I, Oertle T, Sengstag C, et al. (2005): Postnatal ontogeny of hippocampal expression of the mineralocorticoid and glucocorticoid receptors in the common marmoset. Eur J Neurosci 21:1521–1535. Heijtz RD, Alexeyenko A, Castellanos FX (2007): Calcyon mRNA expression in the frontal-striatal circuitry and its relationship to vesicular processes and ADHD. Behav Brain Funct 3:33. Craig AD (2009): How do you feel—now? The anterior insula and human awareness. Nat Rev Neurosci 10:59 –70. Dettling AC, Schnell CR, Maier C, Feldon J, Pryce CR (2007): Behavioral and physiological effects of an infant-neglect manipulation in a biparental, twinning primate: Impact is dependent on familial factors. Psychoneuroendocrinology 32:331–349. Pervanidou P, Chrousos GP (2007): Post-traumatic stress disorder in children and adolescents: From Sigmund Freud’s “trauma” to psychopathology and the (dys)metabolic syndrome. Horm Metab Res 39:413– 419. De Kloet ER, Vreugdenhil E, Oitzl MS, Joels M (1998): Brain corticosteroid receptor balance in health and disease. Endocr Rev 19:269 –301. Law AJ, Pei Q, Walker M, Gordon-Andrews H, Weickert CS, Feldon J, et al. (2009): Early parental deprivation in the marmoset monkey produces long-term changes in hippocampal expression of genes involved in synaptic plasticity and implicated in mood disorder. Neuropsychopharmacology 34:1381–1394.
www.sobp.org/journal