Article. Brain-Derived Neurotrophic Factor Signaling and Subgenual Anterior Cingulate Cortex Dysfunction in Major Depressive Disorder

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1 Article Brain-Derived Neurotrohic Factor Signaling and Subgenual Anterior Cingulate Cortex Dysfunction in Major Deressive Disorder Adam Tri, M.D., Ph.D. Hyunjung Oh, M.S. Jean-Philie Guilloux, Ph.D. Keri Martinowich, Ph.D. David A. Lewis, M.D. Etienne Sibille, Ph.D. Objective: The subgenual anterior cingulate cortex is imlicated in the athology and treatment resonse of major deressive disorder. Low levels of brain-derived neurotrohic factor (BDNF) and reduced markers for GABA function, including in the amygdala, are reorted in major deression, but their contribution to subgenual anterior cingulate cortex dysfunction is not known. Method: Using olymerase chain reaction, we first assessed the degree to which BDNF controls mrna exression (defined as BDNF deendency) of 15 genes relating to GABA and neuroetide functions in the cingulate cortex of mice with reduced BDNF function (BDNF-heterozygous [Bdnf +/2 ] mice and BDNF exon-iv knockout [Bdnf KIV ] mice). Gene exression was then quantified in the subgenual anterior cingulate cortex of 51 ostmortem subjects with major deressive disorder and comarison subjects (total subjects, N=102; 49% were women) and comared with revious amygdala results. Results: Based on the results in Bdnf +/2 and Bdnf KIV mice, genes were sorted into high, intermediate, and no BDNF deendency sets. In ostmortem human subjects with major deression, BDNF recetor (TRKB) exression, but not BDNF, was reduced. Postmortem deressed subjects exhibited down-regulation in genes with high and intermediate BDNF deendency, including markers of dendritic targeting interneurons (SST, NPY, and CORT) and a GABA synthesizing enzyme (GAD2). Changes extended to BDNF-indeendent genes (PVALB and GAD1). Changes were greater in men (otentially because of low baseline exression in women), dislayed notable differences from rior amygdala results, and were not exlained by demograhic or clinical factors other than sex. Conclusions: These arallel human/ mouse analyses rovide direct (low TRKB) and indirect (low exression of BDNFdeendent genes) evidence in suort of decreased BDNF signaling in the subgenual anterior cingulate cortex in individuals with major deressive disorder, imlicate dendritic targeting GABA neurons and GABA synthesis, and, together, suggest a common BDNF-/GABA-related athology in major deression with sex- and brain region-secific features. (Am J Psychiatry 2012; 169: ) Major deressive disorder is a debilitating disorder of low affect and altered mood regulation that affects aroximately 17% of the oulation at some oint in life, resulting in serious ersonal, social, and economic burdens (1). The revalence of major deressive disorder is two times higher in women than in men. Female atients with the disorder tend to have higher symtom numbers, a more severe tye of deression, and greater risk of recurring eisodes comared with male atients, but the underlying biological vulnerabilities have not been characterized (2). Changes in the structure, function, and coordinated activity of several brain regions may underlie imaired mood regulation in deression (3). Increased metabolic activity in one of these regions, the subgenual anterior cingulate cortex, has been consistently reorted in the induction of the deressive state, and subgenual anterior cingulate cortex metabolism is reversed by harmacological treatment (4) and dee brain stimulation (5). Low neurotrohic suort in limbic brain regions has been roosed as a unifying hyothesis for the reduced density or cell numbers in the frontal cortex (6) and amygdala (7) and the reduced hiocamal volume observed in individuals with major deression (8). Rodent studies have demonstrated that various antideressant treatments increase brain-derived neurotrohic factor (BDNF ) exression (9), and BDNF infusion into the hiocamus is sufficient to roduce an antideressantlike effect (10). Desite abundant animal studies suorting the close relationshi between BDNF and deression, direct evidence in humans is limited to reorts of low circulating eriheral BDNF levels, which are normalized by antideressant treatment (11), and studies demonstrating This article is discussed in an Editorial by Dr. Kerman (. 1137) 1194 aj.sychiatryonline.org Am J Psychiatry 169:11, November 2012

2 TRIPP, OH, GUILLOUX, ET AL. reduced ro-bdnf and BDNF levels in the ostmortem amygdala of deressed female subjects (12) and in the hiocamal tissue in deressed atients (13, 14). Additionally, studies have reorted that individuals who die by suicide exhibit low hiocamal and midbrain BDNF levels (15), reduced activity-deendent BDNF exression by hyermethylation of romoter/exon IV of the BDNF gene (16), and, in carriers of the BDNF Met allele, increased risk for violent suicide (17, 18), together roviding additional evidence that BDNF has a role in the sychoathology of major deression. In arallel, human imaging and basic science studies have suggested excitation/inhibition imairment in individuals with major deression that is otentially mediated by decreased GABA content (19). We recently reorted down-regulation of several GABA-related genes in the dorsolateral refrontal cortex (20), subgenual anterior cingulate cortex (21), and amygdala (12) in atients with major deression, otentially affecting somatostatinositive dendritic targeting interneurons. We further demonstrated that a set of amygdala-related gene changes (affecting the TAC1, CORT, NPY, SST, RGS4, and SNAP25 genes [Table 1]) correlate with reduced BDNF exression in deressed atients and in mice with reduced BDNF function, hence identifying a attern of reduced BDNFdeendent gene exression in major deression (12) and roviding suorting evidence for a link between the neurotrohic (9) and GABA (19) hyotheses imlicated in deression. In the resent study, we investigated molecular evidence for a low BDNF and reduced GABA function athway in the subgenual anterior cingulate cortex in individuals with major deressive disorder. We first tested the degree of BDNF deendency on gene exression of a set of GABAand BDNF-related genes in the cingulate cortex of mice with reduced BDNF function. Our choice of genes was determined by our revious study of the amygdala in deressed ostmortem subjects (12) to enable comarative analyses across the two studies. Based on this information, we assessed changes in three sets of genes, with high, intermediate, or no BDNF deendency, in human subgenual anterior cingulate cortex using ostmortem brain samles from a large cohort of subjects with major deressive disorder and matched comarison subjects. Exloratory analyses were erformed on utative sex differences in exression atterns, given the greater female vulnerability and the revious findings of more robust somatostatin down-regulation in women with major deression (21). Method Human Postmortem Subjects After consent from the next of kin, brain samles were obtained during autosies erformed at the Allegheny County Medical Examiner s Office (Pittsburgh) using rocedures aroved by the University of Pittsburgh Institutional Review Board and the Committee for Oversight of Research Involving the Dead. Consensus DSM-IV diagnoses were made by an indeendent committee of exerienced clinical research scientists using information from clinical records, toxicology results, and standardized sychological autosies (22). Fifty-one airs of subjects were analyzed. Each air consisted of one subject with major deressive disorder and a comarison subject matched for sex; grou means for age, ostmortem interval, and brain H were nearly identical (see Table S1 in the data sulement accomanying the online edition of this article). Subgenual anterior cingulate cortex samles containing all six cortical layers were harvested from coronal sections as described elsewhere (23). Mice BDNF-heterozygous (Bdnf +/2 ) mice (3 4 months old) were bred on a mixed S129/Sv3C57BL/6 genetic background (24). BDNF exon-iv knockout (Bdnf KIV ) mice were crossed on C57BL/ 6 as described elsewhere (25). All mice were maintained under standard conditions (i.e., in a 12/12-hour light-dark cycle, in 22 C [SD=1], and with food and water ad libitum). Brains were raidly removed and flash frozen on dry ice. The left and right cingulate cortices were microunctured using 0.5-mm diameter unches (23) and stored in TRIzol (Invitrogen, Carlsbad, Calif.) at 80 C. All animal care and treatment was in accordance with the Guide for Care and Use of Laboratory Animals established by the National Institutes of Health. Real-Time Quantitative Polymerase Chain Reaction (PCR) Total RNA was isolated from TRIzol homogenates of the subgenual anterior cingulate cortex in all 51 airs of ostmortem subjects (major deression and comarison subjects) and of the cingulate cortex in rodents. The samles were urified using RNeasy sin columns (Valencia, Calif.), and RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Walbronn, Germany). To generate cdna, 1 mg total RNA was mixed with oligo-dt rimers and SuerScrit II reverse transcritase (Invitrogen, Carlsbad, Calif.) er the manufacturer s rotocol. PCR roducts were amlified in quadrulets on a Mastercycler real-time PCR machine (Eendorf, Hamburg, Germany) using universal PCR conditions as described elsewhere (21). Results were calculated as the geometric mean of threshold cycles normalized to three validated internal controls (actin, glyceraldehyde-3-hoshate dehydrogenase, and cyclohilin G). Gene Selection We assessed mrna exression of several genes that we reviously demonstrated to be affected in the amygdala in individuals with major deression and that dislay various levels of BDNF deendency, based on exression levels in the amygdala in the same strains of mice with reduced BDNF functions (Bdnf +/2 and Bdnf KIV ) used in the resent study (24, 25). Our set includes genes related to BDNF signaling (BDNF and TRKB), BDNF-deendent genes (CORT, NPY, SST, VGF, TAC1, SNAP25, and RGS4), and GABA-associated genes (GAD1, GAD2, GABRA1, SLC6A1, CALB2, PVALB, SST, NPY, and CORT) (Table 1). Protein Isolation and BDNF Measurements Following RNA extraction, acetone reciitation of roteins was carried out from the TRIzol samles, and Western blot analysis was erformed as described elsewhere (23). Dual signals were detected using the Odyssey Infrared Imaging System (LI- COR Biosciences, Lincoln, Neb.), and BDNF signal ratios to actin were calculated. Samles were rocessed in matched airs on Am J Psychiatry 169:11, November 2012 aj.sychiatryonline.org 1195

3 BDNF AND SUBGENUAL ANTERIOR CINGULATE CORTEX DYSFUNCTION IN DEPRESSION TABLE 1. Assessment of Brain-Derived Neurotrohic Factor (BDNF) Deendency on Target Gene Exression in Bdnf +/2 and Bdnf KIV Mice a Gene Code Gene Name Bdnf +/2 Mice Combined Male-Female Male Female Bdnf-IX Brain-derived growth factor Trkb (Ntrk2) Tyrosine kinase recetor High BDNF deendency Cort Cortistatin Vg f Neurotrohic growth factor inducible Sst Somatostatin Tac1 Protachykinin E Ny Neuroetide Y Intermediate BDNF deendency Sna25 Synatosomal-associated rotein Gad2 (Gad65) Glutamate decarboxylase Low or no BDNF deendency Gad1 Glutamate decarboxylase (Gad67) Pvalb Parvalbumin Rgs4 Regulator of G rotein signaling Slc6a1 GABA transorter Calb2 Calretinin Gabra1 GABA-A recetor, alha a Mice heterozygous for a constitutive deletion of the Bdnf gene are identified as Bdnf +/2 (N=13 [seven were male, and six were female]; comarison mice: N=13 [seven were male, and six were female]), and mice with a targeted disrution of exon IV are identified as Bdnf KIV (N=12 [seven were male, and five were female]; comarison mice: N=12 [seven were male, and five were female]). b Data indicate average log ratio of the exerimental grou relative to the comarison grou. Significant at,0.05. Significant after correction for multile testing. the same gel, and results were relicated for a total of three different Western blots. Test assays were run with 5 50 mg of total rotein using the following antibodies: mouse antihuman BDNF (R&D Systems, Minneaolis), antihuman TrkB (sc-8316; Santacruz Biotechnology, Santa Cruz, Calif.), and antiactin (A2228; Sigma-Aldrich, St. Louis). We also measured BDNF using an enzyme-linked immunosorbent assay (ELISA) kit (Promega, Madison, Wisc.). Statistical Analysis Differences in diagnosis-deendent gene exression were determined by analysis of covariance (ANCOVA) using SPSS (SPSS, Chicago). To determine which covariates to include in the genesecific models, each nominal factor was tested as the main factor using analysis of variance (ANOVA), scale covariates were tested using Pearson s correlation, and reeated measures were corrected using the modified Holm-Bonferroni method (see Table S1 in the online data sulement). Samles were analyzed for the resence of antideressant medication at the time of death. Given the small number of observations (N=9), benzodiazeines were not formally analyzed. No other sychotroic medications were resent at the time of death. ANCOVA models, including significant cofactors, were then alied to the combined and sexsecific analyses. For the rodent analyses, ANOVA models with sex as a cofactor were alied to the combined analysis, and unaired two-tailed t tests were erformed in the searate male and female analyses. Values were then adjusted across genes for multile testing using a second modified Holm-Bonferroni test (i.e., false discovery rate-corrected). Uncorrected values for diagnosis effects are listed in Table 1. Results BDNF-Deendent Gene Exression in the Cingulate Cortex in Mice With Altered BDNF Function Since BDNF exression and function vary significantly deending on brain regions, sex, and cellular activity, we first tested the role of BDNF in regulating the exression of the genes of interest in the cingulate cortex of mice that were heterozygous for a constitutive deletion of the Bdnf gene (Bdnf +/2 ) or that had a targeted disrution of exon IV (Bdnf KIV ), with the latter mutation resulting in blockade of activity-deendent BDNF rotein exression (25). Bdnf mrna levels were reduced in Bdnf +/2 mice (Table 1). When the grous were searated by sex, this decrease reached statistical significance only in male mice, otentially reflecting reduced analytical ower in smaller grous. Bdnf mrna levels were also reduced in Bdnf KIV mice, in both the combined and sex-secific grous (Table 1). These latter results reveal that constitutive and activity-deendent functions of BDNF are not only reduced in the cingulate cortex in Bdnf KIV mice, which is consistent with an indeendent study of the same Bdnf KIV mouse strain reorting activity-deendent Bdnf KIV romoter-driven transcrition abolishment in the cortex (26), but they are also significantly reduced in basal transcrition by romoters I, II, III, VI, and 1196 aj.sychiatryonline.org Am J Psychiatry 169:11, November 2012

4 TRIPP, OH, GUILLOUX, ET AL. Bdnf KIV Mice Combined Male-Female Male Female E E E , E IXa. The levels of Trkb, the main recetor of Bdnf, werenot affected in the two mouse models. Using the Bdnf +/2 and Bdnf KIV mouse models, we next investigated the degree of BDNF regulation on target genes (defined as BDNF deendency) within the cingulate cortex. The quantitative PCR (qpcr) analysis revealed robust and significant decreases in gene transcrit levels for Cort, Vg f, Sst, Tac1, and Ny exression (false discovery rate-corrected significance in the combined male-female grou in at least one strain of mice) but less robust effects on Sna25 and Gad2 exression (uncorrected significance in the combined male-female grou in at least one strain of mice) and little or no effect of decreased BDNF function on Gad1, Pvalb, Rgs4, Gat1, and Gabra1 exression (no difference in the combined male-female grou and uncorrected significance in the searate sex grous in one strain of mice or no change observed at all). Combined, these findings rovided us with three sets of genes with a gradient of BDNF deendency in the cingulate cortex. Results of this analysis are summarized in Table 1. BDNF-Deendent Gene Exression Changes in the Subgenual Anterior Cingulate Cortex in Postmortem Subjects With Deression To evaluate subgenual anterior cingulate cortex BDNF function in the ostmortem brain of subjects with major deression and comarison subjects, we used qpcr to measure the mrna exression levels of BDNF, of the BDNF recetor TRKB, and of the three sets of genes with variable BDNF deendency described above. The qpcr measures were first evaluated for the effect of relevant cofactors to include in the main ANCOVA models for each gene, as described earlier. None of the investigated cofactors dislayed consistent effect. The cofactor analyses and ANCOVA inclusions are summarized in Table S2 in the online data sulement. Results from the ANCOVA analyses for the effects of major deression are summarized in Table 2. Although BDNF mrna levels were not changed, mrna levels of the BDNF recetor TRKB were significantly reduced by aroximately 30% after correction for multile testing. In view of the negative BDNF mrna finding and the comlex regulation of BDNF mrna at the rotein level, we attemted to measure extracts using quantitative Western blot tests and ELISA. However, the ro- and mature forms of BDNF were below detection levels in the subgenual anterior cingulate cortex samles using both aroaches. Similarly, the signal-to-background ratio for TrkB rotein levels was low in the samles and recluded robust quantification. Reduced exression was identified for CORT ( 33%), SNAP25 ( 38%), VGF ( 35%), SST ( 34%), NPY ( 37%), GAD1 ( 25%), GAD2 ( 28%), and PVALB ( 33%). Reduced exression was also observed for TAC1 ( 25%) but only at nominal uncorrected significance. The RGS4, GABA transorter 1/SLC6A1, calretinin/calb2, and GABRA1 genes were unchanged. When the grous were segregated by sex, BDNF levels remained unchanged, and TRKB was similarly reduced in both male ( 29%) and female ( 32%) deressed subjects Am J Psychiatry 169:11, November 2012 aj.sychiatryonline.org 1197

5 BDNF AND SUBGENUAL ANTERIOR CINGULATE CORTEX DYSFUNCTION IN DEPRESSION TABLE 2. Alterations in Proximal Brain-Derived Neurotrohic Factor (BDNF) Signaling Machinery and Distal BDNF- Deendent Genes in the Subgenual Anterior Cingulate Cortex of Postmortem Subjects With Major Deressive Disorder Combined Male-Female Male Female Gene Ratio a Ratio a Ratio a BDNF TRKB High BDNF deendency CORT E E-03 VGF E SST b TAC NPY E Intermediate BDNF deendency SNAP E GAD2 (GAD65) E Low or no BDNF deendency GAD1 (GAD67) E E PVALB (PV) RGS SLC6A1 (GAT1) CALB2 (calretinin) GABRA a Data indicate average log ratio of the exerimental grou relative to the comarison grou. b Data for SST quantitative olymerase chain reaction were reanalyzed from a revious study (21). Significant at,0.05. Significant after correction for multile testing. comared with the resective comarison subjects, although results were less robust and only showed nominal uncorrected significance. Overall, male deressed subjects exhibited more robust decreased exression across the gene anels than female deressed subjects comared with the resective comarison subjects. Out of the eight genes dislaying false discovery rate-corrected significance in the combined male-female grou, six remained significant at false discovery rate-corrected values and two at uncorrected values in the male cohort, comared with two and three genes, resectively, in the same categories in the female cohort. Out of the four genes showing no effect in the combined grou, calretinin/calb2 was decreased in the male cohort at false discovery rate-corrected significance. When summarized by BDNF-deendent categories (Table 2), these results suggest a robust decrease in BDNF signaling in the subgenual anterior cingulate cortex in deressed subjects, with changes related to GABA functions also including genes with moderate or low BDNF deendency. BDNF-Deendent mrna Sex Differences in Comarison Postmortem Subjects To investigate otential sources of the discreancy in altered major deression-related gene exression between male and female subjects, we comared exression differences between male and female comarison subjects (i.e., individuals without sychiatric diagnoses). We found no sex differences in BDNF or TRKB mrna exression, but several genes within the investigated anel, including CORT, NPY, CALB2, and TAC1, showed reduced baseline exression in women (Figure 1). This suggests that levels of exression in the female comarison subjects may already be closer to the low exression levels observed in deressed subjects. Discussion Seeking molecular evidence in suort of a low BDNF and reduced GABA function athway in the subgenual anterior cingulate cortex in individuals with major deression, we first relied on two strains of mice with reduced BDNF function to determine the extent to which our genes of interest deend on BDNF for exression in the cingulate cortex (Table 1). Translating this information to human subjects with major deression and measuring exression levels in their subgenual anterior cingulate cortex, we observed no changes in BDNF itself, reduced exression of TRKB, the main recetor through which BDNF signals, and reduced mrna levels of several genes for which exression deends on BDNF (Table 2). Among these BDNF-deendent and deression-affected genes, the reduced exression of several markers of GABA-ergic interneurons that secifically target the dendritic comartment of yramidal neurons (SST, NPY, and CORT) suggests the resence of a reduced dendritic inhibition henotye in 1198 aj.sychiatryonline.org Am J Psychiatry 169:11, November 2012

6 TRIPP, OH, GUILLOUX, ET AL. FIGURE 1. Significant Sex Differences in Gene Exression in Postmortem Comarison Subjects a 250 * * * * Relative mrna Exression (%) Female Male Female Male Female Male Female Male CORT NPY TAC1 CALB2 a Comarisons are between male and female comarison subjects without sychiatric diagnoses. SST mrna exression was lower in female subjects at a level that fell short of significance (,0.1). Black bars indicate mean values, and red bars indicate the coefficients of variation. The asterisk indicates statistical significance (,0.05). individuals with major deression, downstream from low BDNF signaling. GABA-related changes also extended to genes with modest or no evidence for BDNF deendency (based on mouse studies [also see Table 1 and Table 2]), suggesting that additional factors lead to reduced GABA function in deression. Overall, results were more robust in men, which is contrary to our revious observations in the amygdala, in which deressed women exhibited reduced BDNF levels (but not TRKB levels) and greater BDNFdeendent gene changes comared with men. Together, these results suggest a core BDNF-/GABA-related athology in major deression that affects markers of interneurons targeting yramidal cell dendrites and that dislays sexand brain region-secific features. With the excetion of sex and, to a lesser extent, age, none of the clinical, demograhic, and technical arameters had any consistent detectable effects on gene exression in our relatively large cohort of human subjects. Death by suicide has been associated with reduced BDNF exression, but differences observed in the resent study aeared to be more robust in subjects who did not die by suicide, comared with the resective comarison subjects (see Table S3 in the online data sulement). When the grous were segregated by sex, we observed similar changes in TRKB exression and overall lower statistical significance of changes in deression-related gene down-regulation in female subjects, although not systematically (Table 2). Notably, the exression levels of three BDNF-deendent genes (CORT, NPY, and TAC1) were already lower in female comarison subjects relative to male comarison subjects (Figure 1). Desite lower baseline levels, exression changes for CORT, TAC1, and NPY still dislayed greater or equal statistical significance and effect size in deressed female subjects comared with deressed male subjects. Thus, desite a less robust rofile of molecular changes, the low female baseline exression for some genes may result in a greater roensity to reach the threshold of low athohysiological function (Table 2). Finally, the overall male-female similarities in gene changes downstream from low BDNF in the two mouse strains (Table 1) and the reduction or absence of changes in human female subjects for genes Am J Psychiatry 169:11, November 2012 aj.sychiatryonline.org 1199

7 BDNF AND SUBGENUAL ANTERIOR CINGULATE CORTEX DYSFUNCTION IN DEPRESSION FIGURE 2. BDNF/TRKB and Associated GABA Marker Dysregulation in the Subgenual Anterior Cingulate Cortex and Amygdala in Postmortem Subjects With Major Deressive Disorder a BDNF and recetor BDNF deendency and major deression effect Major deression sex differences BDNF deendent Genes BDNF TRKB VGF CORT SST TAC1 NPY SNAP25 GAD2 GAD1 PVALB RGS4 CALB2 GABRA1 SLC6A1 Male Female Major deression effect: sgacc Decreased Amygdala No Change a Reduced BDNF signaling in major deressive disorder is suggested by the findings of low BDNF in the amygdala and low TrkB in the subgenual anterior cingulate cortex. Analyses of mice with reduced BDNF functions suggest that co-occurring GABA-related gene changes are artly downstream from low BDNF signaling, but the exact nature and extent of downstream gene changes are moderated by brain region- and sex-secific factors. Comarison findings in the amygdala are taken from a revious study (12). SgACC5Subgenual anterior cingulate cortex. with intermediate and low BDNF deendency (SNAP25, GAD1/GAD2 [Table 2]) suggest the resence of additional sex-secific moderating factors in human subjects. This is consistent with our revious study of the amygdala in deressed male (23) and female (12) subjects, in which BDNF and BDNF-deendent genes were robustly affected in women but not in men. Together, these results suort the concet that sex differences in the vulnerability to and the exression of major deression may not result from different athohysiological mechanisms but rather from moderating biological factors acting on a core henotye imlicating a utative reduction in GABA function and dendritic targeting interneuron vulnerability. SST, NPY, and CORT are three neuroetide coding genes with overlaing atterns of exression in mice that are found in aroximately 20% of interneurons and that have the functional characteristic of roviding GABAmediated inhibition to distal dendrites of yramidal neurons. Moreover, TAC1, the fourth neuroetide coding gene that is similarly down-regulated in the subgenual anterior cingulate cortex and amygdala in individuals with major deression (Figure 2), encodes for substance P, a gene roduct with utative antideressant activity (27), which in the cortex mainly activates SST-ositive cells through NK1R recetor binding (28). Since all four genes are deendent on BDNF for their exression (Table 1), low BDNF signaling may orchestrate a synergy between decreased TAC1, SST, NPY, andcort exression, leading to reduced inhibition onto the dendritic trees of targeted yramidal neurons. VGF and SNAP25, two genes involved in synatic function and reviously imlicated in major mental illnesses, were also found to be BDNF deendent and down-regulated in the subgenual anterior cingulate cortex in subjects with major deression, suggesting that a broader BDNF-deendent module may be affected. Finally, exression of GAD1, a gene encoding an enzyme that roduces GABA, and exression of PV, a gene encoding a marker for fast siking GABA interneurons targeting the cell body and axon initial segment, were also down-regulated. For PV and GAD1, the mechanism aears to be indeendent of reduced BDNF function (Table 1, Table 2). Notably, PV levels were not affected in other brain regions (the dorsolateral refrontal cortex [e.g., 20] and the amygdala [e.g., 12]), and calretinin (CALB2), a marker for a third interneuron subset, dislayed reduced baseline exression and no deressionrelated changes in the subgenual anterior cingulate cortex but was decreased in the amygdala in deressed female subjects (Figure 2). Together, these findings ut forward critical observations of the athology of major deression, which may relate to three consecutive biological scales: 1) molecular function, manifested by altered BDNF-/TrkB- and GABA-associated gene function; 2) cellular microcircuitry, in which findings aear to be clustered by function (i.e., dendritic inhibition); and 3) circuit moderators, in which sex-related factors and brain regions are relevant modulators to gene exression in major deression. Hence, reduced GABA-mediated inhibition of incoming information in yramidal dendrites may reresent a utative microcircuitry-level henotye underlying the increased activation of the subgenual anterior cingulate cortex and amygdala that is frequently reorted in studies of atients with major deression (29, 30). In turn, restoring dendritic inhibitory function may reduce yramidal cell activation and excitatory tone and contribute to the reduction in activation of the subgenual anterior cingulate cortex with ositive treatment resonse to theraeutic intervention (e.g., dee brain stimulation, antideressants) (4, 5). Some of the limitations of these results are inherent to investigation of heterogeneous cohorts and of ostmortem brain samles. Large numbers of clinical, demograhic, and technical arameters have to be taken into consideration, and results are mostly correlative and cannot rovide 1200 aj.sychiatryonline.org Am J Psychiatry 169:11, November 2012

8 TRIPP, OH, GUILLOUX, ET AL. insight into develomental rocesses in major deression. Our relatively large cohort size allowed us to rule out major effects of utative confounds (details are summarized in Table S2 in the online data sulement), but the results will need to be confirmed in indeendent cohorts. The causal link between reduced BDNF signaling and altered gene exression was inferred from analyses of rodents with genetically induced reduction in BDNF function, but secies differences may exist, and thus the different labels of gene-secific BDNF deendency may vary. The fact that regional variations in BDNF deendency were also observed in rodents suggests that asects of the human gene regulation atterns are conserved across secies. This latter observation suorts the need for further studies of rodent models with more refined genetic maniulations affecting secific interneuron oulations, for instance, to assess the effect on reduced dendritic inhibition on the local microcircuitry in the amygdala and cingulate cortex and downstream behavioral henotyes. Indeed, it is evident that the comlexity of the utative BDNF-mediated cellular and signaling henotye observed in human major deression is not fully relicated in currently available genetic rodent models, and thus caution should be alied when interreting rodent behavioral oututs downstream from broad genetic changes; for instance, Bdnf +/2 and Bdnf KIV mice exhibit normal and increased emotionality, resectively (31, 32). Disrution of forebrain-secific BDNF leads to higher emotionality when combined with exosure to chronic stress in female mice (33, 34). Conversely, low ventral striatum BDNF can have antideressant-like effects (35). Our observation in humans of reduced exression of BDNF in the amygdala and of reduced TRKB in the subgenual anterior cingulate cortex suggests that altered BDNF signaling may reresent a comlex integration of currently unidentified ustream events (e.g., stress factors, develomental trajectories, and genetic variation), which result in similar core downstream changes (i.e., reduced markers of dendritic inhibition) and that are further moderated by numerous factors (e.g., sex, brain region, and brain activity). Finally, it is also becoming evident that the observed findings are not secific to major deression, since similar reductions in BDNF and SST exression have been reorted in studies of other neurosychiatric (e.g., schizohrenia [36, 37] and biolar disorder [38]) and neurological (e.g., Alzheimer s disease and Huntington s disease) disorders. Thus, our findings may more accurately reflect a molecular and cellular endohenotye that imlicates BDNF signaling and GABA microcircuitry and that has its own etiological factors. However, the restricted scoe on markers affecting dendritic inhibition that we observed differs from observations in studies of other diseases in which changes occur in the context of other core athologies, such as robust PV-related GABA dysfunction in schizohrenia (39) or neurodegenerative rocesses in Alzheimer s disease (40). Investigating the etiological factors and henotyic oututs of these resective molecular and cellular endohenotyes outside the restriction of the categorical definitions of sychiatric and neurological illnesses may rovide dimensional insight into relevant roximal athohysiological mechanisms to be targeted for theraeutic uroses, while their atterns of co-occurrences may be informative of mechanisms underlying clusters of symtoms that are enriched in clinically defined disorders. Received Feb. 21, 2012; revision received May 28, 2012; acceted June 21, 2012 (doi: /ai.aj ). From the Deartment of Psychiatry and the Center for Neuroscience, University of Pittsburgh, Pittsburgh; the Université Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry, France; the Genes, Cognition, and Psychosis Program, NIMH, Bethesda, Md.; and the Lieber Institute for Brain Develoment, Johns Hokins University School of Medicine, Baltimore. Address corresondence to Dr. Sibille (sibilleel@umc. edu). Dr. Lewis currently receives investigator-initiated research suort from Bristol-Myers Squibb, the Bristol-Myers Squibb Foundation, Curridium, Ltd., and Pfizer; he has also reviously served as a consultant to Bristol-Myers Squibb in the areas of target identification and validation and new comound develoment. The other authors reort no financial relationshis with commercial interest. Dr. Tri and Hyunjung Oh contributed equally to this article. 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