Progress in Neuro-Psychopharmacology & Biological Psychiatry 51 (2014) 9–15

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Ketamine reverses stress-induced depression-like behavior andincreased GABA levels in the anterior cingulate:An 11.7 T 1H-MRS study in rats

Shane A. Perrine a,⁎, Farhad Ghoddoussi b, Mark S. Michaels a, Imran S. Sheikh c,George McKelvey b, Matthew P. Galloway a,b

a Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, MI, USAb Department of Anesthesiology, Wayne State University School of Medicine, Detroit, MI, USAc Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA, USA

Abbreviations: 1H-MRS, proton-magnetic resogamma-aminobutyric acid; CUS, chronic unpredictab⁎ Corresponding author at: Department of Psychiatry

Wayne State University School of Medicine, 2353 ScotMI, 48201, USA. Tel.: +1 313 577 9989; fax: +1 313 5

E-mail address: sperrine@med.wayne.edu (S.A. Perrin

0278-5846/$ – see front matter © 2013 Published by Elsehttp://dx.doi.org/10.1016/j.pnpbp.2013.11.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 July 2013Received in revised form 6 November 2013Accepted 6 November 2013Available online 15 November 2013

Keywords:Chronic unpredictable stressGamma-aminobutyric acidProton-magnetic resonance spectroscopy

Gamma-aminobutyric acid (GABA) is the major inhibitory amino acid neurotransmitter in the brain and isprimarily responsible for modulating excitatory tone. Clinical neuroimaging studies show decreased GABA levelsin the anterior cingulate of patients with mood disorders, including major depressive disorder. Chronicunpredictable stress (CUS) is an animalmodel thought to mimic the stressful events that may precipitate clinicaldepression in humans. In this study male Sprague–Dawley rats were subjected to a modified CUS paradigm thatused a random pattern of unpredictable stressors twice daily for 10 days to explore the early developmentalstages of depression-like endophenotypes. Control rats were handled daily for 10 days. Some rats from eachtreatment group received an injection of ketamine (40 mg/kg) after the final stressor. One day following thefinal stressor rats were tested for behavioral effects in the forced swim test and then euthanized to collecttrunk blood and anterior cingulate brain samples. GABA levels were measured in anterior cingulate samplesex vivo using proton magnetic resonance spectroscopy (1H-MRS) at 11.7 T. Animals subjected to CUS hadlower body weights, higher levels of blood corticosterone, and increased immobility in the forced swim test;all of which suggest that the stress paradigm induced a depression-like phenotype. GABA levels in the anteriorcingulate were significantly increased in the stressed animals compared to controls. Administration of ketamineon the last day of treatment blunted the depression-like behavior and increased GABA levels in the anteriorcingulate following CUS. These data indicate that stress disrupts GABAergic signaling, which may over timelead to symptoms of depression and ultimately lower basal levels of cortical 1H-MRS GABA that are seen inhumans with depression. Furthermore, the data suggests that ketamine modulates cortical GABA levels as amechanism of its antidepressant activity.

© 2013 Published by Elsevier Inc.

1. Introduction

Depression is one of the high prevalence psychiatric disordersand often precipitated by stressful life events (Kessler et al., 2007).The symptoms of depression include anhedonia, appetite disturbance,sleep disturbance, hopelessness, avolition and anergia. These disruptdaily life and normal function, and unfortunately, many peoplewith depression do not respond to traditional pharmacotherapiesor psychotherapy. Two lines of clinical research on the neurobiology

nance spectroscopy; GABA,le stress.and Behavioral Neurosciences,t Hall, 540 E Canfield, Detroit,77 9958.e).

vier Inc.

and treatment of depression precipitated the present pre-clinicalstudy.

The first line of research involves several clinical proton-magneticresonance spectroscopy (1H-MRS) studies, which have shown thatanterior cingulate and/or occipital cortex levels of the inhibitoryneurotransmitter gamma-aminobutyric acid (GABA) are decreased inmedication-free, recovered depressed individuals (Bhagwagar et al.,2007, 2008), in people with major depressive disorder (Hasler et al.,2007; Sanacora et al., 1999, 2004), and in individuals with treatmentresistant depression (Price et al., 2009). Following serotonin-selectivereuptake inhibitor treatment and electroconvulsive therapy, corticalGABA levels increase (Sanacora et al., 2002, 2003); however, aftercognitive behavioral therapy, cortical GABA levels remain decreased inpatients with depression (Sanacora et al., 2006). These studies areconsistent, and a meta-analysis (Yildiz-Yesiloglu and Ankerst, 2006)agreeswith this notion that cortical GABA levels are decreased in people

Table 1Chronic unpredictable stress (CUS) paradigm.

1st stressor 2nd stressor

Day Time Type Time Type

−3 to 0 Weighed and handled once daily1 10 AM Swim stress (4 min) 1 PM Cage rotation (50 min)2 3 PM Lights off (2 h) 7 PM Isolation housing overnight3 7 PM Cage rotation (50 min) 7 PM Lights on overnight4 11 AM Cold isolation (1 h) 7 PM No food/water overnight5 12 PM Lights off (3 h) 4 PM Restraint stress (1 h)6 1 PM Swim stress (3 min) 7 PM Lights on overnight7 7 PM Light on overnight 7 PM No food/water overnight8 10 AM Cage rotation (20 min) 3 PM Cold isolation (15 min)9 7 PM No food/water overnight 7 PM Lights on overnight10 10 AM Restraint stress (1 h) 3 PM Pre-swim (15 min)a,b

11 3 PM Forced swim testa 3:05 PM Euthanize and collectsamples

Control animals were weighed and handled once daily during the stress treatment.a All animals received a 15 minute pre-swim followed 24 h later by the forced

swim test.b Animals received 1 ml/kg saline or 40 mg/kg ketamine (IP) 3 h after the pre-swim.

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with depression; additionally, pharmacotherapies which provideantidepressant effects return GABA to similar levels as in healthycontrols.

The second line of research involves numerous case studies andclinical findings that suggest glutamate receptor ligands representa novel pharmacotherapy for the treatment of depression (reviewedby Hashimoto, 2010; Krystal, 2007; Mathew et al., 2008; Skolnicket al., 2009). Pharmacotherapeutics, behavioral therapies and elec-troconvulsive therapy are used to manage the symptoms of depres-sion, yet these treatments are often ineffective, have delayed onsetto efficacy, require repeated administration, and produce unwantedside effects. In efforts to overcome these clinical difficulties and findnovel pharmacotherapies for depression, researchers have shownthat a single dose of ketamine, a glutamate N-methyl-D-aspartate(NMDA) receptor antagonist causes antidepressant effects that lastup to two weeks in treatment-resistant depression (Berman et al.,2000; Maeng and Zarate, 2007; Mathew et al., 2005; Zarate et al.,2006). Ketamine also produces antidepressant-like actions in animalmodels of antidepressant-like behavior (Garcia et al., 2008a,b;Ghasemi et al., 2010; Maeng et al., 2008; Popik et al., 2008; Sofia andHarakal, 1975; Yilmaz et al., 2002) and in animal models of depression(Garcia et al., 2009; Li et al., 2011; Ma et al., 2013; Rezin et al., 2009).Both human and animal studies indicate that modulation of ionotropicglutamate receptors, which likely reside on cortical GABAergic inter-neurons (Homayoun and Moghaddam, 2007), is a novel target to treatdepression.

While these findings suggest the involvement of GABA in the patho-physiology of depression and theutility of glutamate ligands as pharma-cotherapies for depression, themechanism(s) of action that relate thesephenomena are just being discovered (Autry et al., 2011; Li et al., 2010;Maeng et al., 2008). In this study a chronic unpredictable stress(CUS) paradigm (Cox et al., 2011; Ortiz et al., 1996) was used to explorethe alteration in GABA levels in the anterior cingulate cortex ofmale ratsusing ex vivo 1H-MRS, as well as, determine the utility of ketamine toreverse the behavioral and neurochemical plasticity that develop afterthe CUS paradigm. The hypotheses of this study is that CUS will yield adepressed phenotype and a decrease in levels of GABA in the anteriorcingulate cortex and that a sub-anesthetic dose of ketamine will havean antidepressant-like effect and normalize the change in GABA levelsin CUS-treated animals.

Fig. 1. Chronic unpredictable stress (CUS) (A) decreases weight gain and (B) increasesplasma corticosterone compared to control rats. Data are presented as the mean ± SEM.Group sample sizes include control = 6 and CUS = 8 for the weight gain data andcontrol = 6 and CUS = 7 for the ELISA analysis. *P b 0.05.

2. Methods

2.1. Animal care and the chronic unpredictable stress (CUS) paradigm

All procedures involving animals were approved by the WayneState University (WSU) Institutional Animal Care and Use Committeeand followed guidelines laid out in the Guide for the Care and Use ofLaboratory Animals (Institute of Laboratory Animal Resources (U.S.),1996). The WSU Division of Laboratory Animal Research is accreditedby the Association for Assessment and Accreditation of LaboratoryAnimal Care (AALAC) International. Male Sprague Dawley rats werepurchased from Charles River Laboratory (Portage, MI) and grouphoused in microisolator cages (standard for rats) in a temperature(21–23 °C), humidity (30–50%) and light (on 7 AM–off 7 PM) controlledenvironment with free access to water and standard rat chow. Followingsix days of acclimation, rats were exposed to a random (unpredictable)pattern of mild stressors twice daily for 10 days (i.e. chronic unpredict-able stress) or handled daily for 10 days as control (Doyle andYamamoto, 2010; Johnson and Yamamoto, 2009; Ortiz et al., 1996).Table 1 outlines the CUS paradigm. Rat weights were recorded dailyand weight gain (mean ± standard error of the mean) is presentedin Fig. 1A. Weight gain was calculated by averaging each animal'sweight on a given day minus the average of the weights recordedfor the 3 days before CUS.

2.2. Ketamine injection

Ketamine (40 mg/kg) or saline (0.9% NaCl; 1 ml/kg) was injectedintraperitoneally 3 h after the pre-swim (on day 10). Depression-likebehavior was tested 21 h later using the forced swim test.

2.3. Forced swim test

All animals were tested for depression-like behavior as previouslydescribed (Perrine et al., 2008). Each rat was subjected to a 15 minutepre-swim on day 10 of the CUS paradigm or on day 10 of handling(control). Twenty-four hours later (or 21 h after ketamine/saline

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injection) rats were subjected to a 5 minute swim test. An acrylic glasscylinder (45 cm height × 21 cm inner diameter) was filled with roomtemperature (~22 °C) water to 35 cm in depth and used for swimmingtest. Rats were dried with a towel following each swim and returned totheir home cage. The 5 minute swim test was recorded by digital videoand each swim test was scored by three raters, two of whomwere blindto the treatment conditions. The average of the three raters' scores wasused for the final analysis. For scoring, a 5 min sampling technique wasused and immobility, swimming and climbing scores were determined.Immobility scores were used to assess depression-like behavior, wherean increase in immobility behavior reflects a depressed phenotype.

2.4. Tissue and blood collection

Animals were euthanized immediately after the forced swim test.Potential stress-induced effects of the forced swim test were expectedto be transient and not affect baseline levels of GABA or other neuro-chemical. Trunk blood was collected in heparin-coated BD Vacutainertubes (BD, Franklin Lakes, NJ), and tubes gently mixed and stored onice for up to 1 h. Blood samples were centrifuged for 20 min at~2000 rpm at room temperature; plasma (supernatant) was collectedand stored at −80 °C for measurement of corticosterone. UsingThe Rat Brain in Stereotaxic Coordinates 6th edition (Paxinos andWatson, 2007) as a guide, a tissue sample of the anterior cingulatecortex of each rat was collected. The anterior cingulate was selectedbecause 1) 1H-MRS-measured GABA levels in this region are altered inhumans with depression, and 2) the (sub-genual) anterior cingulatecortex is involved in neuroendocrine and autonomic dysregulation aswell as abnormal motivational and emotional behaviors observed inmajor depressive disorder (reviewed by Drevets et al., 2008). Afterdecapitation brains were rapidly removed, placed into an ice-coldbrain matrix, and a 2 mm thick coronal slice ranging approximatelyfrom bregma 2.28 mm to bregma 0.48 mm was dissected. The coronalslice was placed flat with the anterior side up on a solid block of carbondioxide (dry ice). Using a 1.5 mm diameter tissue punch, a singlecylindrical tissue punch was taken from the medial anterior cingulatecortex (including Cg1 and Cg2). Tissue punches were stored untreatedat−80 °C until 1H-MRS measurement.

2.5. Enzyme linked immuno-sorbent assay (ELISA)

For quantitative measurement of plasma corticosterone, a commer-cially available ELISA kit (Neogen Corp., Lexington, KY) was used in ac-cordance with the manufacturer methods and as described previously(Cox et al., 2011). Corticosterone was extracted from plasma samplesby adding ethyl ether to the sample, agitating by vortex, allowing thephases to separate, evaporating the organic phase with a stream of N2

and reconstituting the residue in extraction buffer provided with thekit. Next, the sample and the corticosterone horseradish peroxidaseconjugate were added to the antibody-coated plate and incubated for1 h at room temperature on an orbital shaker at low speed. The platewas rinsed with wash buffer to remove unbound material, and thebound conjugate was detected by absorbance at 650 nm after additionof tetramethylbenzidine plus H2O2. Corticosterone standards were pre-pared and run in parallel to quantify the concentration of corticosteronein each sample. Corticosterone data are shown as ng/ml, and weredetermined in control and CUS groups only and did not include samplesfrom ketamine treated groups.

2.6. High resolution-magic angle spinning proton-magnetic resonancespectroscopy (1H-MRS)

High resolution-magic angle spinning 1H-MRS is a specializedversion of spectroscopy ideally suited for ex vivo analysis of frozenintact tissue samples, and it provides a highly resolved proton reso-nance spectrum that includes several key neurochemicals including

GABA. The details of the methodology have been published by ourlaboratories (Ghoddoussi et al., 2010; Knox et al., 2010; O'Leary-Moore et al., 2007; Perrine et al., 2009, 2010, 2011) and will be brief-ly descried here. After weighing samples (~3 mg), the frozen intactpiece of anterior cingulate was placed into a 10 μl Bruker zirconiumrotor with 5 μl ice-cold buffer (pH 7.4, 100 mM K2HPO4/KH2PO4,200 mM HCOONa, 1 g/l NaN3 diluted 50% with D2O containing3 mM trimethylsilylpropinoate — an internal reference). A vertical8.9 cm bore Bruker 11.7 T magnet with an Avance DRX-500 console(Bruker Biospin Corp., Billerica, MA) was used to analyze the sampleby placing the rotor including tissue sample and buffer into a multi-nuclear Bruker magic angle spinning probe. Samples temperaturewas maintained at 4 °C and rotors were spun at 4.2 ± 0.002 kHz at54.7° relative to the static magnetic field B0. A 1-D Carr–Purcell–Meiboom–Gill pulse sequence with a pre-saturation pulse for watersuppression and semi-automated shimming procedure to compen-sate for field inhomogeneities was used to acquire data usingBruker-XWINNMR (version 3.6). Raw spectral data were analyzedwith a custom LCModel version 6.1-4 utilizing a basis set consistingof a linear combination of 27 individual neurochemical spectra andnon-specific lipid signals designed to fit the brain tissue spectrumand quantify concentrations for neurochemicals with resonancepeaks between 1.0 and 4.2 ppm (Provencher, 1993). Neurochemicalsignals were corrected for tissue weight resulting in nmol neuro-chemical/mg tissue wet weight. Cramér–Rao bounds provided anestimate the precision of the LCModel fit to the spectral data andwere typically 10% or less for most neurochemicals and acceptableunder 25%.

2.7. Data analysis

Data are presented as themean ± standard error of themean (SEM)and group sizes are described in the figure legends. Graphs wereconstructedwith GraphPad Prism (version 5) and statistical parametersdetermined with SPSS (version 17). Weight data were analyzed usingtwo-way analysis of variance (ANOVA) with treatment and day asfactors, and Bonferroni posttestwere used for followupanalysis. Plasmacorticosterone datawere analyzed by one-tailed t-test. Forced swim testdata and 1H-MRS data were analyzed separately by one-way ANOVAfollowed by Newman–Keuls multiple comparison posttest. In all cases,the confidence interval was set at 95%with P b 0.05 to obtain statisticalsignificance.

3. Results

3.1. Ten days of chronic unpredictable stress increases immobility behaviorin the forced swim test in rats

As shown in Fig. 1A, CUS treatment disrupts weight regulation,which is a symptom characteristic of clinical depression and an effectobserved in a previous study from our laboratory using this model(Cox et al., 2011). At the beginning of the experiment (i.e. 3 days beforeCUS), body weights for control (275 ± 7) and CUS (281 ± 6) groupswere not significantly different (P N 0.05). Two-way ANOVA revealeda significant effect of CUS treatment (F1,156 = 53.95, P b 0.0001),day (F12,156 = 19.94, P b 0.0001), and treatment × day interaction(F12,156 = 2.20, P = 0.014) between groups. Bonferroni posttest analy-sis showed that all animals gained weight daily; however, CUS treatedrats had significantly (P b 0.05) less weight gain on treatment days 6(t = 2.94), 7 (t = 3.90), 8 (t = 3.16), 9 (t = 4.26), and 10 (t = 3.23)compared to controls.

Fig. 1B shows that CUS increases plasma corticosterone levels, whichmodels the increase in cortisol observed in clinical depression, aswell asreplicates findings from a previous study from our laboratory using thismodel (Cox et al., 2011). One-tailed t-test revealed a significant differ-ence (t11 = 2.06, P = 0.032) between group means with higher levels

Table 2Effects of chronic unpredictable stress with or without 40 mg/kg ketamine on swimmingand climbing behavior in the forced swim test.

Swimming Climbing

Control (handling) 34 ± 2.9 12 ± 1.1Chronic unpredictable stress 26 ± 2.4 8 ± 1.8Control + ketamine 29 ± 3.1 16 ± 1.7CUS + ketamine 31 ± 2.1 13 ± 1.2

Data are presented as the mean ± SEM and units are based on scoring criteria.CUS = chronic unpredictable stress. Group sample sizes for the forced swim testinclude control = 5, CUS = 6, control + ketamine = 6, and CUS + ketamine = 7.

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of plasma corticosterone in CUS treated rats compared to controls.Control levels of corticosterone reported here are higher than thosepreviously reported by our laboratory (Cox et al., 2011) likely due tothe mild stress induced by the forced swim test immediately beforeeuthanasia in the present study.

3.2. Ketamine attenuates forced swim test behavior and blocks the increasein anterior cingulate GABA caused by ten days of chronic unpredictablestress in rats

A one-way ANOVA of the results shown in Fig. 2 revealed a signifi-cant difference (F3,20 = 5.46, P = 0.007) among group means on im-mobility behavior in the forced swim test. Newman–Keuls multiplecomparison test showed that the CUS treated rats were significantly dif-ferent (P b 0.05) from control rats (q = 4.86), as well as CUS treatedrats that received an injection of 40 mg/kg ketamine after CUS and be-fore behavioral testing (q = 4.41). In other words, ketamine reversedthe CUS-induced depression-like behavior in the forced swim test.Interestingly, ketamine treatment alone did not significantly affectimmobility, indicating that ketamine only affected behavior in animalssubjected to the CUS animal model of depression. Swimming andclimbing behaviors did not significantly differ among the groups(Table 2).

Fig. 3 illustrates the changes inGABA levels in the anterior cingulate ofrats undergoing CUS treatment with or without 40 mg/kg ketamine in-jection. One-way ANOVA revealed a significant difference (F3,23 = 8.67,P = 0.0005) among group means on levels of GABA as measuredby 1H-MRS ex vivo. Newman–Keuls multiple comparison testshowed that GABA levels were increased significantly (P b 0.05) inCUS treated rats compared to controls (q = 3.57). Additionally,rats receiving ketamine injection after CUS had significantly less GABAlevels in the anterior cingulate compared to CUS treated animals(q = 3.56). Finally, ketamine alone significantly decreased GABA levelscompared to controls (q = 3.57).

The effects of CUS with or without ketamine treatment on otherneurochemicals of the 1H-MRS spectrum were measured in anteriorcingulate tissues in addition to the hypothesis-driven investigationof GABA (Table 3). Besides GABA neurochemicals of the 1H-MRSspectra included betaine, creatine, glutamine, glutamate, glycerylphosphoryl choline, glutathione, myo-inositol, lactate, N-acetylaspartate,N-acetylaspartyl-glutamate, phosphorylethanolamine, succinate, andtaurine. Using one-way ANOVA for a given neurochemical, statistical

Fig. 2. The increase in immobility behavior in the forced swim test caused by chronicunpredictable stress (CUS) is attenuated by treatment with 40 mg/kg ketamine (IP).Additionally, 40 mg/kg ketamine decreases GABA levels but not immobility behavior innaïve (handling-control) rats. Data are presented as the mean ± SEM. Group samplesizes include control = 5, CUS = 6, control + ketamine = 6, and CUS + ketamine = 7.*P b 0.05, **P b 0.01.

analysis revealed no significant main effects and no significant effectbetween control and CUS or control (handling) and ketamine(control + ketamine).

4. Discussion

Two lines of clinical research on the neurobiology and treatment ofdepression precipitated this study. A meta-analysis of neuroimagingdata from humans with depression shows that cortical GABA levels(anterior cingulate and/or occipital cortex) are consistently decreased(Yildiz-Yesiloglu and Ankerst, 2006), and pharmacotherapeutic treat-ment outcomes from humans show that ketamine can rapidly relievedepressive symptoms in people with treatment-resistant depression(Berman et al., 2000; Zarate et al., 2006 and reviewed by Hashimoto,2010; Krystal, 2007; Mathew et al., 2008; Skolnick et al., 2009). Theseindependent lines of research suggest the involvement of GABA inthe pathophysiology and glutamate in the treatment of depression,however, the mechanism linking these findings has not been addressedheretofore. In this study, the CUSparadigmwas used tomodel the initialphase of stress-induced depression (i.e., produce a depressive-likephenotype) in rats to determine if themechanism of action of ketamineinvolves normalization of altered cortical GABA levels.

The CUS model and the similar paradigm chronic mild stress (CMS)have been extensively used to study depression-like behaviors andthe neurobiological and neurochemical underpinnings that cause thedisorder (reviewed by Willner, 1997, 2005; Willner et al., 1992).The CMS model incorporates a longer duration of stress exposure(e.g. 4–6 weeks) than CUS (e.g. 10–14 days) and may model morechronic phenotypes than CUS, but both paradigms use the same typeand frequency of stressors to model stress-induced depressive

Fig. 3. The increase in 1H-MRS GABA levels after chronic unpredictable stress (CUS) isattenuated by a single administration of 40 mg/kg ketamine (IP). Additionally, 40 mg/kgketamine decreases GABA levels in naïve (handling-control) rats. Data are pre-sented as the mean ± SEM. Group sample sizes include control = 6, CUS = 8,control + ketamine = 5, CUS + ketamine = 8. *P b 0.05.

Table 3Effects of chronic unpredictable stress with or without 40 mg/kg ketamine on other neurochemicals of the 1H-MRS spectra.

Control (handling) Chronic unpredictable stress Control + ketamine CUS + ketamine

Betaine 1.20 ± 0.18 0.76 ± 0.07 0.90 ± 0.21 0.97 ± 0.13Creatine 4.21 ± 0.12 4.28 ± 0.11 4.01 ± 0.30 4.24 ± 0.15Glutamine 2.61 ± 0.31 2.78 ± 0.14 2.57 ± 0.09 2.61 ± 0.23Glutamate 7.99 ± 0.29 8.05 ± 0.29 7.58 ± 0.34 8.01 ± 0.28GPC 0.88 ± 0.12 0.89 ± 0.13 0.90 ± 0.17 0.86 ± 0.09Glutathione 0.78 ± 0.07 0.98 ± 0.06 0.83 ± 0.09 0.93 ± 0.08myo-Inositol 4.13 ± 0.29 4.42 ± 0.22 3.96 ± 0.31 4.09 ± 0.20Lactate 6.90 ± 0.11 7.47 ± 0.23 7.19 ± 0.69 7.34 ± 0.24N-acetylaspartate 5.32 ± 0.22 5.51 ± 0.09 5.23 ± 0.26 5.23 ± 0.26NAAG 1.01 ± 0.03 0.99 ± 0.06 0.93 ± 0.06 1.06 ± 0.07PEA 2.60 ± 0.21 2.95 ± 0.11 2.26 ± 0.25 2.63 ± 0.14Succinate 0.33 ± 0.01 0.36 ± 0.03 0.31 ± 0.01 0.35 ± 0.03Taurine 6.43 ± 0.51 6.66 ± 0.21 5.97 ± 0.38 6.45 ± 0.34Glutamine/glutamate 0.33 ± 0.04 0.34 ± 0.02 0.35 ± 0.01 0.32 ± 0.03Glutamate/GABA 3.65 ± 0.22 4.16 ± 0.31 3.30 ± 0.29 3.69 ± 0.28

Data are presented as the mean ± SEM and units are nmol neurochemical/mg tissue (nmol/mg). CUS = chronic unpredictable stress, GABA = gamma aminobutyric acid,GPC = glyceryl phosphoryl choline, NAAG = N-acetylaspartyl-glutamate, and PEA = phosphorylethanolamine. Group sample sizes include control = 6, CUS = 8,control + ketamine = 5, CUS + ketamine = 8 for the 1H-MRS analysis.

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phenotypes. Our laboratory has previously shown that in rats CUSdecreases weight gain as well as increases plasma corticosteronewhich correlates with serotonin turnover (i.e. increased serotonergicactivity) in the hypothalamus (Cox et al., 2011). The data in Fig. 1 arein line with these results, and although CUS may not model allaspects of clinical depression, the results provide construct validityof the CUS model in that two of the physiological symptoms, includ-ing disrupted weight regulation and increased plasma corticoste-rone, that are common in depression were observed in CUS-treatedrats. Additionally, CUS induces a depressive-like phenotype in theforced swim test evident by an increase in immobility behavior(Fig. 2) and a decrease in both swimming and climbing behaviors(Table 2). Levels of GABA from the anterior cingulate of theserats were determined using ex vivo high resolution-magic anglespinning proton-magnetic resonance spectroscopy (1H-MRS) at11.7 T in order to determine the neurochemical underpinnings ofthe behavior.

The stress paradigm used herein was modeled after studies byDuman and colleagues, who described the effects of CUS exposure as afunction of exposure period (Banasr and Duman, 2007, 2008; Banasret al., 2010). We selected 10 days exposure to specifically probe for anemerging phenotype that would presage the effects of the 5–6 weeksdescribed by Hemanth Kumar and colleagues (Hemanth Kumar et al.,2012). The fact that our experimental paradigm altered physiological(body weight, corticosterone) and behavioral (forced swim test)parameters is consistent with a stress response and validates our novelobservations regarding cortical GABA and its reversal by an acute, single,sub-anesthetic dose of ketamine. Since a true animalmodel of clinical de-pression is non-existent no matter how long a rodent animal is stressed,our investigations are limited to those phenotypeswe can actuallymodeland in this case we are studying one of the antecedent vulnerabilities ofclinical depression (10 days stress).

Chronic unpredictable stress increases GABA levels in the anteriorcingulate cortex of rats (Fig. 3). The direction of change is inconsistentwith our a priori hypothesis that was based on clinical observationsshowing that drug-naïve patients with certain depression sub-typeshave decreased rather than increased cortical GABA. Potential reasonsfor this discrepancy include differences in species, brain region, meth-odology, time-course of stress-mediated effects, and dissimilarity be-tween clinical depression and CUS. Although rats commonly modelhuman neurobiology very well, differences in GABA pharmacology be-tween the species may exist and account for the difference seen herein.Methodological details may account for the observed difference. For ex-ample, the brain region of interest being measured may differ betweenstudies. A study investigating the amino acid neurotransmitter levels by1H-MRS in the occipital cortex of rats exposed to chronic stress found no

difference in levels of glutamate or GABA (Valentine et al., 2011). Also,rats subjected to the forced swim test have increased choline/creatineratio in dorsolateral pre-frontal cortex but decreased choline/creatineratio in the left hippocampus (Hong et al., 2007, 2009). It should benoted that, in the present study, cortical levels of GABA could havebeen influenced by the forced swim test, in addition to the CUS orketamine effect; further studies are needed to address this concern.Although CUS has good construct and predictive validity as ananimal model of depression, differences between clinical depression,which is a chronic condition with an unknown etiology, and thismodel, which is a stress-induced state that mimics certain aspects(i.e. endophenotypes) of the human condition, may contribute tothe observed differences. For example, the animals used in thisstudy are fully developed adult rats with no genetic or pre-disposingcondition, known to affect MRS-related neurochemistry. Young rats(6–8 weeks) subjected to chronic forced swim stress show reducedhippocampal glutamate/GABA ratio as well as reduced pre-frontal–cortical glutamate and hippocampal glutamate and N-acetylaspartate(NAA) (Li et al., 2008). Rats bred for learned helplessness show in-creased pre-frontal cortical and hippocampal glutamate/GABA ratio(Sartorius et al., 2007). Further research is necessary to understandthe effects of CUS on cortical GABA levels and to determine the differ-ences between clinical and pre-clinical findings. One study using CMSin vesicular glutamate transporter 1 knockout mice found disruptionsin cortical and hippocampal glutamate, GABA, vesicular GABA trans-porter, glutamic acid decarboxylase and excitatory amino acid trans-porter 1 implicating a crucial role of VGLUT1 in the depressivephenotype induced by chronic stress (Garcia-Garcia et al., 2009).A recent similar 1H-MRS study to ours found that 6 weeks of CMS de-creases glutamate, glutamine, and GABA levels in both hippocampusand pre-frontal cortex (Hemanth Kumar et al., 2012), which suggeststhat extended exposure to stress changes GABA and, in light of ourresults, suggests a temporally dynamic effect of stress on GABAhomeostasis. However, it should be noted that our shorter CUS para-digmmay be modeling a different plasticity than the relatively long-term CMS paradigm.

The results presented here indicate that abnormal GABA levels areassociated with depressive phenotypes; however, the aberrant controlof cortical GABA does not fully explain the pathophysiology of depres-sion.While our data do not clearly define the role of GABA in this animalmodel of depression, the deficit may occur either in the cycling(i.e. anabolism, catabolism, and transporting) or in neurotransmittersignaling function of GABA. For example, reduced counts of glial cellsand smaller neuronal size in the pre-frontal and anterior cingulatecortex have been reported in postmortem brains from persons with de-pression (Cotter et al., 2001; Ongur et al., 1998; Rajkowska et al., 1999).

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Given the role of glial cells in the metabolic cycling of GABA andglutamate, reduced glial density may contribute to alterationsin 1H-MRS measured GABA as well as glutamate and glutamine(Hasler et al., 2007; Price et al., 2009). Loss of cortical glial cells orchronic stress induces depressive-like behaviors (Banasr andDuman, 2008), and chronic stress decreases glial metabolism andglutamate/GABA-glutamine cycling evident in part by reductions of13C-GABA in the pre-frontal cortex (Banasr et al., 2010). Similarly,in normal rats phenelzine increases cortical GABA and disruptsglutamine–glutamate cycling suggesting a modulatory effect of theantidepressant on the neurobiology of amino acid neurotransmitters(Yang and Shen, 2005). The consequence of disrupted GABA levelsmay relate to abnormal neurotransmitter signaling in key circuits.For example, an increase in the ratio of glutamate (excitatory) toGABA (inhibitory) has been observed using 1H-MRS in humanswith depression and suggested to be a reflection of altered brainfunction (Sanacora et al., 2004). Finally, it is notable that abnormalGABA may be a reflection (i.e. biomarker) of trait vulnerability tomood disorders rather than the speculated role of cortical GABA as aneurochemical correlate (or regulator) of an altered mood state(Bhagwagar et al., 2007). Although a clear understanding of the role ofGABA in depression remains to be established, published clinical andpre-clinical data agree with the results shown here that cortical GABAis mechanistically germane to human depression and animal modelsof depression.

Ketamine attenuates the CUS-induced depressive (behavioral)phenotype seen in the forced swim test (Fig. 2) and reverses theCUS-induced increase in cortical GABA (Fig. 3) one day after a singledrug administration. These observations are consistent with previ-ous reports that ketamine has antidepressant effects in humansand animal models (Berman et al., 2000; Garcia et al., 2009; Sofiaand Harakal, 1975; Yilmaz et al., 2002) and that antidepressantscan reverse the disruption of cortical GABA (an endophenotype)observed in depression (Sanacora et al., 2002). Other molecularstudies have shown that the mechanism of ketamine action involvesnot only direct inhibition of NMDA receptors, but also increasedAMPA receptor phosphorylation and subsequent plasma membranetranslocation (Maeng et al., 2008), and blockade of AMPA receptorsabolishes the rapid and long-lasting antidepressant-like behavior(Koike et al., 2011). The acute ketamine effects on depressive-likebehavior also involve activation of mammalian target of rapamycin(mTOR) and subsequent synaptic formation and development inthe pre-frontal cortex (Li et al., 2010, 2011) and. Researchers havealso shown that ketamine reverses the inhibition of complexes I, III,and IV of the mitochondrial respiratory chain caused by CMS(Rezin et al., 2009). The acute but not long-lasting antidepressanteffects of ketamine are dependent on BDNF activation (Garcia et al.,2008a,b; Machado-Vieira et al., 2009), which is regulated by eukary-otic elongation factor 2 (eEF2; a.k.a. CaMKIII) following NMDAreceptor inhibition (Autry et al., 2011; Monteggia et al., 2012). Stillother studies have shown that antidepressant doses of ketamineincrease creatine kinase activity in striatum and cerebral cortex(Assis et al., 2009). Although these studies and more (as reviewedby Duman et al., 2012; Pilc et al., 2013) are unraveling the antide-pressant action of ketamine, the mechanism by which inhibition ofglutamate NMDA receptor function leads to alterations in anteriorcingulate GABA levels and how this affects behavior remain to bedetermined. It is tempting to speculate that ketamine normalizesthe CUS-induced increase in GABA levels in anterior cingulate(Fig. 3) by restoring impaired GABA metabolism, particularly inglia. Additionally, CUS has been shown to decrease glial metabolismand amino acid (i.e. GABA, glutamate, and glutamine) cycling in thepre-frontal cortex of rats, an effect that is reversible by the glutamatemodulator riluzole (Banasr et al., 2010). Given the metabolic rela-tionship among GABA, glutamate and glutamine synthesis, a disrup-tion in the glutamate/GABA-glutamine cycle would likely result in an

imbalance in GABAergic and glutamatergic signaling. Therefore,although the molecular target of ketamine is the glutamate NMDAreceptor, the antidepressant-like effects may result from normalizedamino acid cycling between cortical neurons and astrocytes.The clinical diagnostic and therapeutic implications of this studyindicate that GABA levels may indicate depressive state and thatnovel pharmacotherapies could be designed to target glutamate/GABA-glutamine cycling (Salvadore et al., 2012).

Acknowledgments

This work was partially supported by the National Institutes ofHealth (NIH)/National Institute on Drug Abuse (NIDA) grant K01-DA024760 to SAP. Additional support was provided by NIH/NIDA R01-DA016736, the Joe Young Sr. Fund for Psychiatry Research, and theFMRE Fund for Anesthesiology Research to MPG. The authors wouldlike to thank previous reviewers for their helpful comments andsuggestions.

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