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Neuropharmacology 63 (2012) 1161e1171

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Neurochemical and molecular characterization of ketamine-inducedexperimental psychosis model in miceq

Manavi Chatterjee a, Rajkumar Verma a, Surajit Ganguly b,**, Gautam Palit a,*aDivision of Pharmacology, Central Drug Research Institute, Lucknow e 226001, Uttar Pradesh, IndiabChronic Disease Biology Group, Institute of Molecular Medicine, 254 Okhla Industrial Estate, Phase 3, New Delhi e 110020, India

a r t i c l e i n f o

Article history:Received 13 December 2011Received in revised form24 May 2012Accepted 25 May 2012

Keywords:KetamineSchizophreniaNeurotransmittersAmino acidsReceptorsMice model

Abbreviations: 5HIAA, 5-hydroxyindoleacetic aciserotonin 1A receptor; 5HT2AR, serotonin 2A recereceptor; Ach, acetylcholine; AChE, acetylcholinestdopamine transporter; DHBA, 3,4-dihydroxybenzyyphenylacetic acid; GABA, g-amino butyric acid;transporter; GLT-1, glutamate transporter-1; GlyT-1, glhigh performance liquid chromatography-electrochevanillic acid; MAO, monoamine oxidase; NA, noradreaspartate; nNOS, neuronal nitric oxide synthase; NR1ANR2A, NMDA receptor subunit 2A; NR2B, NMDA recehydroxylase; a-7nAChR, alpha-7-nictonic acetylcholinq CDRI communication no: 8261.* Corresponding author. Tel.: þ91 522 2612411

2623405, þ91 522 2623938.** Corresponding author. Tel.: þ91 11 41708321, þ91

E-mail addresses: surajitg@immindia.org (S. Gan(G. Palit).

0028-3908/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2012.05.041

a b s t r a c t

Ketamine, an NMDA receptor antagonist has been shown to induce aberrant behaviour phenotypes inrodents, some of which are known to simulate the behaviour abnormalities observed in patientssuffering from schizophrenia. Thus, developing ketamine-induced animal models became an importanttool of choice to study the mechanistic details of some critical symptoms associated with schizophrenia.In this study, our goal was to characterize and correlate the ketamine-induced changes in the behaviouralphenotypes to the changes in neurochemical and molecular profile(s) in the brain tissues implicated inthe pathophysiology of schizophrenia. We studied the effects of ketamine in mice using ‘acute’ and‘chronic’ treatment regimens along with the ‘drug withdrawal’ effects on their biochemical andmolecular parameters in the pre-frontal cortex, hippocampus, and striatum. Our results demonstratedthat the acute and chronic ketamine administration, differentially and site specifically, modulated thelevels of acetylcholine, dopamine, serotonin and noradrenaline. In addition, the chronic ketamine dosesdramatically suppressed the levels of glycine among some of the amino acids examined and inducedalternations in gene expression of the key neurotransmitter receptor systems, including some membersof the dopamine and the serotonin receptor families. The acute and chronic ketamine treatment induced“signature” neurochemical and gene-expression patterns that are implicated in the pathophysiology ofschizophrenia. Our analyses tend to support the “chronic ketamine” mice model for experimentalpsychosis as a tool for deeper investigation of the mechanistic paradigm associated with the schizo-phrenia spectrum disorder and for screening next-generation antipsychotic drugs.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Schizophrenia is a chronic, debilitating psychiatric disorderaffecting a large number of geriatric populations worldwide (Reus,

d; 5HT, serotonin; 5HT1AR,ptor; 5HT2CR, serotonin 2Cerase; DA, dopmaine; DAT,lamine; DOPAC, dihydrox-GLAST, glutamate aspartateycine transporter; HPLC-ECD,mical detector; HVA, homo-naline; NMDA, N-methyl D-, NMDA receptor subunit 1A;ptor subunit 2B; TH, tyrosinee receptor.

418x4303; fax: þ91 522

9999797944.guly), gpalitcdri@gmail.com

All rights reserved.

2008). Despite this huge social and economic burden, very littleprogress has been made in developing new antipsychotics witha combined effect encompassing a wide array of symptomsincluding the so-called negative symptoms. This, in part, is attrib-uted to the lack of appropriate animal models that can simulate thegross behaviour symptoms associated with the schizophreniaspectrum disorder (Ellenbroek et al., 1989; Corbett et al., 1993).

Though used extensively in modelling psychosis, amphetaminedoes not induce the “negative” symptoms of schizophrenia, whichinclude emotional withdrawal and depressive effects (Sams-Dodd,1998). In contrast, sub-anesthetic doses of NMDA receptor antag-onists, such as phencylidine (PCP), MK-801 and ketamine, werereported to induce a wider spectrum of behavioural responses thatencompass positive, negative, and cognitive schizophrenia-likesymptoms in healthy human volunteers (Javitt and Zukin, 1991)and rodents (Chatterjee et al., 2011).

We previously reported that single-dose administration (acutetreatment) of ketamine at 100mg/kg (i.p.) induced hyperlocomotorresponses in mice along with reduced ‘transfer-latency time’ in the

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e11711162

passive avoidance test (Chatterjee et al., 2011). However, the dosagedid not have any significant effect in the forced swim test (negativesymptoms). In contrast, chronic administration of ketamine notonly produced significant ‘hyperactivity’ response and decreasedthe latency period in the passive avoidance test, but also enhancedimmobility period in those animals during the forced swim test.This fundamental difference in the ‘negative’ or ‘depression-like’behavioural phenotype between the ‘acute’ and the ‘chronic’ ket-amine treated animal models led us to investigate the modulationsin the molecular parameters as a function of the ketamine treat-ment regimens. Therefore, in the present study, we have attemptedto evaluate the construct validity of the ketamine model bycomparative estimations of various biochemical and molecularmarkers and their tissue-specific regulations and tried to correlatethe possible mechanism underlying the ketamine-induced behav-ioural phenotypes.

2. Materials and methods

2.1. Animals

Adult male Swiss albino mice weighing 30e35 g were used in this study. Theywere housed three to four per cage at room temperature (22 � 2 �C) and 12/12-hlight/dark (8:00 a.m. to 8:00 p.m.) cycle. They were allowed to acclimatize for atleast one week prior to all experimental procedures. The animals had free access toregular food pellets and water ad libitum. All the experimental procedures usedwere approved by Institutional Animal Ethical Committee which follows theguidelines of CPCSEA (Committee for the Purpose of Control and Supervision ofExperiments on Animals), India.

2.2. Drugs and treatment schedule

Ketamine was purchased from Ranbaxy, India. All other compounds wereprocured from SigmaeAldrich, UK, unless specified.

Mice were randomly selected and distributed into different groups of 12 animalseach. The experiments, where mice were treated with either vehicle or ketamine(100 mg/kg, i.p.) 30 min before sacrificing, are denoted as ‘acute’ studies. ‘Chronic’studies are the ones in which mice were challenged continuously with ketamine(100mg/kg/day i.p.) for 10 days andwere sacrificed on the 11th day. For ‘withdrawal’studies, mice were continuously treated with either the vehicle or ketamine(100 mg/kg/day i.p.) for 10 days; the drug/vehicle was then withdrawn on the 11thday and the animals were subsequently sacrificed on the 21st day.

For the control groups, we initially performed the experiments with single-time‘vehicle’ injection for the ‘acute’ studies and 10 times injections of the vehicle for the‘chronic’ studies. Since, after comparative analysis of some critical parametersexamined in this study, no significant effects were observed for single versusmultiple times (10�) vehicle administration in the animals (SupplementaryFigure 1), we opted to use the 10 times vehicle injected animal sets as the‘control’ groups where both the acute and the chronic groups were included foranalysis. This allowed us to reduce the number of animal usage as preferred by theInstitutional Animal Ethical Committee. Hence, all our data included here, wereanalysed using 10 times vehicle-injected animals as the ‘control’ group.

2.3. Biochemical estimations

2.3.1. Estimation of monoamine oxidaseMice brains were dissected and homogenized at 4 �C in a buffer containing 0.3%

dithiothreitol, 1 mM ATP, 0.1 mM GTP, 0.1 mM PIPES, 0.5 mM MgCl2, 2 mM EGTA,0.1 mM EDTA, pH 7.2. Then, 0.5% Triton X-100 were added to each homogenate andwere incubated on ice for 2 h and finally centrifuged at 15,000 rpm at 4 �C for 10min.The supernatants were used as the enzyme sources for monoamine oxidase (MAO).

A modified version of the Lowry method was used for protein estimation(Markwell et al., 1978). MAO assays were performed using a commercially availablekit as described previously (Chatterjee et al., 2012b). Tyraminewas used as substratefor MAO-A and benzylamine for MAO-B. The assays were conducted at roomtemperature (25 �C). Briefly, aliquots of the brain homogenates were incubated ina reaction mixture of 200 mM Amplex Red, 1 mM substrate, and 1 U/mL HRP for60min. The product was measured by a fluorescence microplate reader set at560 � 10 (excitation) and 590 � 10 nm (emission).

2.3.2. Estimation of acetylcholineConcentrations of acetylcholine (ACh) were measured using a commercially

available kit (Molecular Probes, Inc.) with minor modifications (Chatterjee et al.,2012b). Tissue extracts, as described above, were incubated in Na/K buffer con-taining 100 mM Amplex Red, 200 mU horseradish peroxidase, 20 mU choline

oxidase, and 5 U acetylcholinesterase (AChE). Formation of the assay end-product,resorufin, was determined following a three-step enzymatic reaction catalyzed byAChE, choline oxidase, and hydrogen peroxidase. The fluorescence of resorufin (Ex:540 nm, Em: 590 nm) was monitored in a microplate fluorimeter following a 30 minincubation period. The concentration of ACh was determined from a standard curve(0e2 mM ACh chloride).

2.3.3. Estimation of acetylcholinesterase activityThe esterase activity was measured as described previously (Ellman et al., 1961).

Briefly, the assay was performed by adding 2.9 mL of 0.1 mM sodium phosphatebuffer (pH 8.0) to 50 ml of the tissue homogenate and incubating themixture at 37 �Cfor 5 min. After incubation, 40 ml of acetylthiocholine iodide (154.38 mM) and 10 mlof DTNB (10 mM) were added to the reaction mixture and the formation of thio-nitrobenzoic acid was recorded at 412 nm for 150 s at 30 s intervals using UVspectrophotometer. The AChE activity was quantified by measuring the concentra-tion of thionitrobenzoic acid (extinction coefficient 1.36 � 104/molar/cm).

2.3.4. Nitrite estimationNitrite was determined by spectrophotometric method using Griess reagent as

described previously (Chatterjee et al., 2012b). Equal volume of brain homogenateand Griess reagent were mixed. The mixture was incubated at 37 �C for 10 min andthe absorbance was measured at 542 nm.

2.3.5. Estimation of monoamines using HPLC-ECDAnimals were sacrificed by cervical dislocation and the brains were removed.

Frontal cortex, hippocampus and striatum regions of the brain were dissected andthe wet weights of the tissues were recorded. The tissues were homogenizedseparately in 0.17 M HClO4 and the supernatant collected after centrifugation(35,000 g for 20 min). The clear supernatants were used for the analysis of dopa-mine, serotonin and noradrenaline by high performance liquid chromatography(HPLC) coupled with an electrochemical detection (ECD) system, as describedpreviously (Kim et al., 1987). 3,4-dihydroxybenzylamine (DHBA) was used asinternal standard (25 ng/mL). 20 mL of the acid extract was injected into the HPLCsystem. Monoamines were detected by oxidation at þ0.8 V with glassy carbonworking electrode and Ag/AgCl as reference electrode. Concentrations of neuro-transmitters were calculated from the standard curve generated by using cocktail ofstandards along with the internal standard at a concentration of 20e100 ng/mL.

2.3.6. Estimation of amino acids using HPLC-ECDConcentrations of glutamate, glycine and GABA were determined using HPLC-

ECD. The mobile phase (0.1 M NaH2PO4, 0.1 mM EDTA and 15% methanol,pH ¼ 5.6 with H3PO4) was filtered through a 0.2 mm filter and degassed usingUltrasonicator, before pumping at a flow rate of 1.2 mL/min. The amino acids in thetissue samples were converted into their derivatives following a published proce-dure with minor modifications (Donzanti and Yamamoto, 1988). The stock reagentsfor the derivatization reaction consisted of 27 mg of o-phthalaldehyde (OPA, Sigma)dissolved in 1mL of methanol with 5 mL of b-mercaptoethanol (bME) and 9mL 0.1 Msodium tetraborate (pH 9.3). The working OPA/bME reagent was freshly prepared bydiluting 2.5 mL of the working OPA/bME in 7.5 mL tetraborate buffer. Pre-columnamino acid derivatization reaction was performed by mixing equal volumes of thestandard amino acids or tissue homogenate and the working OPAebME solution for2 min before injecting onto the analytical column. Stock solutions of amino acidstandards were prepared at 1 mg/mL in 50% methanol. Working solutions wereprepared in 0.05 M perchloric acid. Amino acids were quantified from the standardcurve generated using cocktails of standard amino acid derivatives.

2.3.7. RNA isolation and PCR studiesTotal RNAwas isolated from brain tissues (cortex, striatum and hippocampus) by

Trizol Reagent (Life technologies) using RNA isolation method described previouslywith minor modifications (Chomczynski and Sacchi, 1987; Verma et al., 2010).

Single stranded cDNAs was generated from 5 mg of total cellular RNA usingRETROscript kit (Ambion Inc., USA) following manufacturer’s instruction. cDNAswere annealed at 94 �C (5 min) and amplified for 30e35 cycles using the followingprogram: 94 �C for 1 min; respective annealing temperature for specific genes(Table 1) for 1 min; 72 �C for 1 min followed by a final extension at 72 �C for 10 min.The PCR products were resolved in 1.2% agarose gel containing EtBr 5 mg/mL.Selected gene amplicons of all the samples from each experiment were resolvedsimultaneously in the same gel having a 40-lanes (slots) capacity. Hence, the sameimage of b-actin PCR amplicon, which was used for normalization, has beenincluded in some figures. The intensities of the PCR products were measured usingBiovis gel documentation software. The results were expressed as ratio of theinvestigated PCR amplicon to the b-actin amplicon.

3. Statistical analysis

The comparisons among different groups were made using theStudent t-test or one-way analysis of variance (ANOVA) followed byNewmaneKeuls multiple comparison test, wherever applicable.

Table 1List of primer sequences.

Gene Sequence Annealingtemp (�C)

D1 FP:50-CAGTCCATGCCAAGAATTGCC-30

RP:50-AATCGATGCAGAATGGCTGGG-3060

D2 FP:50-GCAGTCGAGCTTTCAGAGCC-30

RP:50-TCTGCGGCTCATCGTCTTAAG-3063

DAT FP:50-TCCCTGACAAGCTTCTCC-30

RP:50-GCCAGGACAATGCCAAGA-3064

TH FP:50-GCACTATGCCCACCCCCAG-30

RP:50-TCGTCAGACACCCGACGCA-3060

NMDA 1A FP:50-TGACAACAAGCGCGGACCCA-30

RP:50-GGACTGCCTGTGCCACCACG-3060

NMDA 2A FP:50-GACTACAGCCTGGAGGCAAG-30

RP:50-AGGTGAACTTCACAGTTCTG-3052.1

NMDA 2B FP:50-GGATCTACCAGTCTAACA-30

RP:50-GATAGTTAGTGATCCCACTG-3051.8

GLT-1 FP:50-ATTGGTGCAGCCAGTATTCC-30

RP:50-AATCGCCCACTACATTGACC-3056

GLAST FP:50-TGTCTTCTCCATGTGCTTCG-30

RP:50-CAAGAAGAGGATGCCCAGAG-3057

GlyT-1 FP:50-CCTGGGGGTCTGGCGGATCA-30

RP:50-CCACCCCATGCACAGCCCAG-3060

nAChR FP:50-GTGGAACATGTCTGAGTACCCCGGAGTGAA-30

RP:50-GAGTCTGCAGGCAGCAAGAATACCAGCA-3060

AChE FP:50-GATCCCTCGCTGAACTACACC-30

RP:50- GGTTCTTCCAGTGCACCATGTAGGAG-3060

5HT1A FP:50-CCCCCCAAGAAGAGCCTGAA-30

RP:50-GGCAGCCAGCAGAGGATGAA-3060

5HT2A FP:50-CATGCCTCTCCATTCTTCATCTCCAGGAA-30

RP:50-CAAGGTGGCTTCTTTCTGAAGTGACTTGA-3060

5HT2C FP:50-CGTCGGCGTCGTGGAGATCG-30

RP:50-CAAGGAGTGAGCGCACCGCA-3060

nNOS FP:50-CTTCCGAAGCTTCTGGCAACAGCGACAATT-30

RP:50-GGACTCAGATCTAAGGCGGTTGGTCACTTC-3060

b-actin FP:50-GCTGTGTTGTCCCTGTAT-30

RP:50-CCGCTCATTGCCATAGTG-3055

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e1171 1163

The F/P significance values were calculated using GraphPad Prismsoftware version 5. The values are represented as mean � SEM. Pvalues of <0.05, <0.01 and <0.001 were considered statisticallysignificant.

4. Results

4.1. Effects of ketamine on dopamine content and its metabolites

In order to determine the effects of ketamine treatment ondopamine (DA) turnover, we compared the levels of total DAcontent in various brain regions in “acute”, “chronic” ketamine“withdrawal” groups as described in the Methods. Our resultsindicate that in the cortex region, there was a 60% increase in theDA levels as a function of the acute ketamine challenge. Chronicketamine treatment for 10 days resulted in further increase of theDA content (88%), and this high DA levels persisted for 10 days post-drug withdrawal (Fig. 1a). In the striatal regions of the brain,a significant increase [F(3, 44) ¼ 9.030; P < 0.01] of 130% in DAlevels was observed with both acute and chronic treatment ofketamine. However, unlike in the cortex, DA levels reduced to 91%in the striatal region during the drug withdrawal phase. In contrast,the DA concentration in the hippocampus did not change signifi-cantly after acute and chronic ketamine treatments, but an increasein the DA levels was observed after the drug withdrawal. Thus, theresults indicate that the DA turnover was drastically modulated asa function of ketamine treatment in the cortex and striatum regionirrespective of the treatment regimen.

In addition, we also examined the concentrations of DA metab-olites in the brain tissue samples to determine theDA turnover ratio.In the cortex region, concentration of 3,4-dihydroxyphenylacetic

acid (DOPAC), one of the major DA metabolites, was increased by3-fold in both the acute and the chronic groups, but surprisingly itincreased dramatically by about 20-fold in the withdrawal group(Fig. 1b). In the striatum, a significant increase was observed only inthe acute groups [w4-fold; F(3, 44)¼17.37; P<0.001]. However, theDOPAC levels declined to about 2-fold after chronic treatment andincreased by about 8-fold in the ‘drug withdrawal’ group. In thehippocampal regions, the DOPAC concentrations increased uponacute ketamine treatment and start to decline to the basal levelsupon chronic treatment and in the post-drug withdrawal period(Table 2).

Homovanillic acid (HVA), the downstream metabolite of DOPACand 3-methoxytyramine and hence DA, has been used asa peripheral marker for dopaminergic activity in the centralnervous system. So, we estimated the levels of HVA in the cortex,striatum and hippocampus. A significant increase in the corticalHVA levels, after both acute (P< 0.01) and chronic (P< 0.001), [F(3,44) ¼ 44.23] ketamine treatments, was observed (Fig. 1c). Thesedata were consistent with the trends observed in DA (Fig. 1a) andDOPAC (Fig. 1b) production. However, HVA increase did not persistafter the drug withdrawal and flattened out close to basal levels(Fig. 1c). Similarly, in the striatal tissues [F(3,44)¼ 10.61; P< 0.001]and the hippocampus [F(3,44) ¼ 31.56; P < 0.001], HVA levels wereincreased in acute groups, but no significant effects on HVAconcentrations were observed in the chronic and the withdrawalgroups (Table 3).

4.2. Effects of ketamine on serotonin and its metabolism

Serotonin and serotonergic receptor systems are widely regar-ded as the key molecular components associated with the patho-physiology of schizophrenia. Decades of fundamental research ondrug discovery efforts with antipsychotic candidate drugs havingaffinity towards serotonergic receptor(s), and hallucinogens likeLSD, led to the strengthening of serotonergic hypothesis of theschizophrenia pathogenesis. Hence, it became apparent that thechanges in serotonin levels and the expression of the relatedreceptors be examined in the ketamine-induced animal model aswell. We estimated the level of serotonin along with its metabolite,5HIAA, in the specific regions of mice brain. As clearly evident fromFig. 2a, serotonin levels in the chronically ketamine treated groupshot up drastically by about 4-fold, as compared to the controlgroup, in the cortex region of the brain. This activation of serotoninwas further exacerbated (w8-fold) upon ketaminewithdrawal [F(3,44) ¼ 1.94; P < 0.05; Fig. 2a]. However, no such changes wereevident in the ‘acute’ ketamine treated group. These findings arequite significant, as they tend to indicate that ketamine-effect onthe dopamine levels are more rapid and are associated with theearly cellular events, eventually leading to the modulation of theserotonin levels at a later phase. Similarly, in striatal region, a 4-foldincrease was observed in chronic ketamine treated groups [F(3,44) ¼ 5.95; P < 0.001], but the effects did not persist after the drugwithdrawal. In contrast, no significant changes in the serotoninlevels were observed in the hippocampal tissues.

Further, we estimated the levels of 5HT metabolite, 5-hydroxyindoleacetic acid (5HIAA) to determine the effects of ket-amine on serotonin turnover. As shown in Fig. 2b, our resultssuggest an increased 5HIAA formation in the cortex [F(3, 44)¼ 7.39;P < 0.001] and striatal regions [F(3, 44) ¼ 10.67; P < 0.05 in theacute and P< 0.001 in the chronic group] as a function of acute andchronic ketamine administration and this activation closely followsthe trend observed in the case of 5HT. Moreover, it is to be notedthat the enhanced oxidation of 5HT to 5HIAA in the cortical areas inresponse to the acute and the chronic ketamine treatments wasdiminished in 10 days post-drug withdrawal period (Table 4). This

Fig. 1. Ketamine modulates dopamine content and its metabolites. Bars indicate the concentration of (a) DA, (b) DOPAC and (c) HVA in the brain regions as mentioned. Experimentalgroups are the acute ketamine treated, chronic ketamine treated and ketamine withdrawn after 10 days as described in the Methods and materials section. Values are calculated asmean � S.E.M. with n ¼ 12 in each group. ***P < 0.001, **P < 0.01, *P < 0.05 versus control group.

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e11711164

decrease in concentration of 5HIAA might indicate a possibleblockade of the 5HT catabolizing machinery leading to an increasein the 5HT levels during the ketamine withdrawal phase. Theincrease in 5HTconcentration in the cortex of the ‘drug withdrawal’group (Fig. 2a) perhaps supports this possibility.

4.3. Effects of ketamine on gene-expression of dopamine andserotonin receptors and transporter systems

Drastic increase in the concentration of dopamine (DA) andserotonin (5HT) as a function of ketamine administration led us toinvestigate the gene-expression profile of some crucial componentsof the dopaminergic and serotonergic-signalling machinery insearch for markers that might define the mechanistic basis ofketamine-induced experimental psychosis model. As both DA and5HT were consistently modulated in the cortex in chronicallyadministered ketamine groups, the gene-expression analysis waslimited to using tissues from the cortex region in mice that werechronically challenged with ketamine. For dopaminergic system,DA receptors D1 and D2, DA transporter (DAT), and DA synthesizingenzyme, tyrosine hydroxylase (TH), were selected for expressionanalysis using PCR (Fig. 3a). The results show an enhancedexpression of both D1 [F(1, 6) ¼ 15.71; P < 0.01] and D2 [F(1,

Table 2Effects of ketamine on DOPAC/DA turnover.

DOPAC/DA Control Acuteketamine

Chronicketamine

Withdrawalketamine

Cortex 0.096768 0.228253 0.176149 0.3207Striatum 0.060642 0.105549 0.096287 0.273358Hippocampus 0.216301 0.551181 0.355854 0.233493

6) ¼ 4.603; P < 0.05] receptors together with the DAT [F(1,6)¼ 2.23; P< 0.05] and TH [F(1, 6)¼ 56.16; P< 0.001] in the cortex.For evaluating the expression of serotonergic receptor subtypes,5HT1A, 5HT2A and 5HT2C were specifically selected for analysis.The selection was based on the role that these receptors play inregulating the serotonin firing and their involvement in the path-ophysiology of schizophrenia (Simpson et al., 1996; Yasuno et al.,2003; Yuen et al., 2008; Jensen et al., 2010). Our results indicatethat the expression of 5HT1A receptor gene was activated by about2-fold in response to the chronic ketamine treatment in the cortextissues [F(1, 6) ¼ 2.78; P < 0.001; Fig. 3b]. However, a modestincrease was observed in case of 2A-receptors. The trend wasreversed in case of the 5HT2C receptor as its expression was sup-pressed by chronic ketamine treatment [F(1, 6) ¼ 236.8; P < 0.001;Fig. 3b]. These results indicate that the functions of the 5HTreceptor subtypes are specific and exert opposing effects inresponse to ketamine.

4.4. Effects of ketamine on noradrenalin (NA) content

As noradrenaline (NA) is the downstream product of DAmetabolism and DA concentration was found to be dramaticallyactivated by ketamine treatment (Fig. 1), it became important to

Table 3Effects of ketamine on HVA/DA turnover.

HVA/DA Control Acuteketamine

Chronicketamine

Withdrawalketamine

Cortex 3.14073 3.919145 5.518225 0.176215Striatum 0.353454 0.812765 0.180295 0.082026Hippocampus 1.667302 2.347814 0.9572 0.047368

Fig. 2. Ketamine regulates concentration of serotonin and its downstream metabolites. Levels of (a) 5HT and (b) 5HIAA content in various brain regions for each experimentalgroups as indicated. Results are represented as mean � S.E.M. with n ¼ 12 in each group. ***P < 0.001, **P < 0.01, *P < 0.05 versus control group.

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e1171 1165

check the status of NA production in the cortex, striatum andhippocampus. As expected, patterns of NA levels followed closelythe distribution of DA concentration across the tissues of the ‘acute’,the ‘chronic’ and the ‘withdrawal’ groups analysed (Fig. 4). Theresults show a two-fold increase in the NA levels after acute andchronic ketamine treatments. However, drastic increase (w10 fold;Fig. 4) was observed in the ketamine ‘withdrawal’ group, primarilyin the cortex [F(3, 28) ¼ 135.80; (P < 0.001)] and hippocampal [F(3,28) ¼ 3.97; (P < 0.001)] tissues. Thus, the changes in the levels ofNA had a close resemblance to the changes in the DA pattern,implicating an expected linear metabolic relationship between DAand NA.

4.5. Effects of ketamine on monoamine oxidase (MAO) activities

In order to rule out any interference of the housekeepingcellular metabolic activities as the reason behind the increased DA,5HT and NA content, we evaluated the effects of ketamine onmonoamine oxidases, MAO-A and MAO-B, activities in specifiedbrain tissues of mice. Our observations indicate a significant [F(2,21)¼ 27.48; P< 0.001] increase in the enzyme activity of MAO-A inthe striatal region of acute ketamine group. In contrast, uponchronic treatment, the enzyme activity diminished below thecontrol levels (P < 0.001; Fig. 5a). However, no change of MAO-Aactivity was observed in the cortex and hippocampal areas. Asimilar pattern of response was observed in the MAO-B enzymeactivity (Fig. 5b). So, except in the striatal tissues [F(2, 21) ¼ 419.2;P < 0.001], all other regions examined showed no modulation ofthe total MAO activities and did not seem to contribute to theketamine-induced changes in the neurotransmitter levels.

4.6. Effects of ketamine on acetylcholine and acetylcholinesteraseenzyme activity

Cortical acetylcholine (ACh) is known to mediate the detection,selection and processing of stimuli. Changes in the ACh concen-tration have been associated with visual hallucination or reality

Table 4Effect of ketamine on 5HIAA/5HT turnover.

5HIAA/5HT Control Acuteketamine

Chronicketamine

Withdrawalketamine group

Cortex 0.469799 1.02895 4.9025 0.302273Striatum 1.684574 1.795422 1.812693 0.33887Hippocampus 6.501493 10.02232 14.69256 8.292898

distortion. Evidence also suggests the involvement of ACh metab-olism in the cognitive functions (Hasselmo, 2006). Hence, weestimated the levels of ACh along with the acetylcholinesterase(AChE) activity in the ketamine treated animals. As expected,a significant increase [F(2, 21) ¼ 24.89; P < 0.001] was observed inthe cortex of the acute and the chronic ketamine treated groups(Fig. 6a). However, no change was observed in the striatal and thehippocampal regions.

Ketamine-induced AChE activity was found to be much higher(about 4-fold higher) in the cortex of the acute group [F(2,12) ¼ 47.14; P < 0.001] than the control, though the activation wasmuch lower (about 2-fold than the control group) in the chronicgroup (Fig. 6b). These results are consistent with our estimation ofthe ACh content, as acute ketamine treatment activates AChEdramatically, probably leading to the low ACh content. However, inthe chronically ketamine treated groups, this activation was lower,leading to an increase in ACh levels (Fig. 6b). This persistence ofhigh concentration of ACh is probably due to the rapid recycling byelevated levels of AChE activity with respect to the control. More-over, significantly higher levels of AChE (P< 0.001) was observed inthe hippocampal [F(2, 12) ¼ 31.08; P < 0.001] and striatal regions[F(2, 12) ¼ 25.03; P < 0.001] than in the cortex (Fig. 6b) in bothacute and chronic conditions, probably justifying the suppressionof the ACh levels in these tissues.

4.7. Effects of ketamine on the glutamate, glycine and GABAcontents

Role of hypofunctional glutamatergic transmission via interplaybetween NMDA receptor and GABA receptor systems in the path-ophysiology of schizophrenia has been widely reported (Olney andFarber, 1995; Coyle, 2006; Stahl, 2007). Hence, estimation of thechanges in the glutamate, glycine and gamma-aminobutyric acid(GABA), if any, becomes relevant in the context of acute and chronicdoses of ketamine administration. The glutamate content in micebrain showed a significant increase of about 40%, [F(2, 21) ¼ 6.42;P < 0.001] in cortex region as a function of acute and chronic ket-amine treatments (Fig. 7a). In fact, the basal concentration ofglutamate in the cortex of the control animals was higher (about 3-fold) per mg of total protein than in the striatum and hippocampusextracts. However, the chronic and acute ketamine administrationresulted in reduction of glutamine levels in the striatum [F(2,21) ¼ 15.0; P < 0.01] and hippocampus [F(2, 21) ¼ 53.97; P < 0.01]areas (Fig. 7a).

Glycine is known to act as an obligatory co-agonist by binding tothe NMDA receptor site away from the ion-channel. The binding of

Fig. 3. Gene-expression profiles of some selective dopamine and serotonin receptors and transporter systems as a function of ketamine administration. Gene expressions of (a)dopamine D1 and D2 receptor, dopamine transporter (DAT) and tyrosine hydroxylase (b) serotonin receptors 5HT1A, 5HT2A and 5HT2C receptors in cortex region of mice inresponse to the chronic ketamine treatment. As described in the Methods and materials section, gene-expression values of the indicated genes are measured as a ratio of theintensity of the chosen amplicon to the intensity of the b-actin amplicon. Results are represented as mean � S.E.M. with n ¼ 4 in each group. ***P < 0.001, **P < 0.01, *P < 0.05 versuscontrol group. The results are derived from three independent experiments.

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e11711166

both glycine and glutamate is necessary for proper functioning ofthe NMDA receptor. Increasing NMDA function with glycineagonists has been thought to be a potential new strategy for themanagement of schizophrenia, particularly for the treatment of thenegative symptomatology. Hence, we examined the status ofglycine in the chronic ketamine model. Our observations indicatea significant decrease of about 60e70% in glycine levels of cortex

Fig. 4. Ketamine-induced changes in noradrenalin (NA) content. Bar diagram repre-sents effects of ketamine administration on NA content in various brain regions.Experimental groups are indicated by respective shades as described. Results arerepresented as mean � S.E.M. with n ¼ 12 in each group. ***P < 0.001, **P < 0.01,*P < 0.05 versus control group.

[F(1, 8) ¼ 49.88; P < 0.001], striatum [F(1, 8) ¼ 5.71; P < 0.01] andhippocampus [F(1, 8) ¼ 3.03; P < 0.001] regions (Fig. 7b). We alsoanalysed the levels of GABA concentration. Amodest increase in thecortex [F(1, 8) ¼ 3.66; P < 0.01] and without much significantdifference in the striatum and hippocampus was observed (Fig. 7c).However, unlike the total glutamate concentration, basal GABAlevels were found to be higher in the striatum and hippocampusthan in the cortical tissues of the ‘control’ animal group.

4.8. Effects of ketamine on mRNA expression of nicotinic receptor,cholinesterase, NMDA subunits and glutamate transporters inresponse to ketamine

An increased expression of alpha-7-nictonic acetylcholinereceptors was observed after chronic ketamine administration incortex [F(1, 6) ¼ 2.26; P < 0.01] (Fig. 8a). However, no significantchanges were observed on the acetylcholinesterase expression inthe cortex. The gene-expression of NMDA subunits, NR1A [F(1,6) ¼ 6.43; P < 0.001] and NR2B [F(1, 6) ¼ 20.04; P < 0.05] werefound to be increased in the cortex after chronic ketamineadministration, but no significant effects were observed with NR2Asubunit (Fig. 8b). In addition, we also analysed the expression levelsof Glutamate transporters (Verma et al., 2010), specifically GLT-1and GLAST but no significant changes were observed (Fig. 8c).

4.9. Effect of ketamine on nitrite content

In order to address the effect of ketamine on nitric oxide (NO)metabolism, we estimated the nitrite levels as a measure of NOconcentration. A drastic increase in nitrite levels was observed inthe hippocampal regions after acute ketamine treatment, but not inchronic treated groups [F(2, 15) ¼ 13.66; P < 0.001] (Fig. 9). Amodest reduction of nitrite was observed after both acute andchronic ketamine treatment in cortical areas.

Fig. 5. Effect of ketamine on monoamine oxidase (MAO) activities. Bar diagram represents the effects of acute and chronic ketamine administration on (a) MAO-A and (b) MAO-Bactivity in brain regions indicated. Results are represented as mean � S.E.M. with n ¼ 8 in each group. ***P < 0.001, **P < 0.01 vs control group, ###P < 0.001 vs acute ketaminegroup.

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e1171 1167

4.10. Effects of ketamine on mRNA expression of glycine transporterand neuronal nitric oxide synthase

We also investigated the effect of ketamine on the expression ofglycine transporter, GlyT-1 and nitric oxide synthase (nNOS) genes.As shown in Fig. 10, chronic ketamine administration did not haveany significant effect on the GlyT-1 expression. However, corticalnNOS expression was suppressed by about 4-fold in ketaminetreated animals [F(1, 6) ¼ 88.04; P < 0.05] (Fig. 10). This mightreflect the changes in the NO production in the cortex (Fig. 9).

5. Discussion

In the present study, we have investigated the changes in anarray of molecular parameters in response to acute and chronicketamine administration in mice. The goal was to identify thesignature molecular modulations that might correlate with theketamine-induced behavioural phenotypes identified in ourprevious studies (Chatterjee et al., 2011). The molecular parametersevaluated were selected from published literature describing theirassociationwith psychosis-related disorders in animal models and/or in clinical setup, with an aim to evaluate the construct validity ofthe ketamine-induced models. We also included the withdrawaleffects of ketamine in our studies to dissociate the molecular

Fig. 6. Ketamine mediated changes in acetylcholine concentration and acetylcholinesterasadministration on (a) ACh content and (b) AChE activity in various brain regions. Results are r###P < 0.001 versus acute ketamine group.

changes that persisted even after the release of chronic ketamineabuse, implicating their probable involvement in the irreversible,“long-term” effects.

The comparative analyses were primarily conducted between(a) acute and chronic ketamine doses versus control (vehicleinjected for 10 days); and (b) acute versus chronic treatments. Inthe absence of any significant difference between the two controls(single saline injection versus 10 times saline injections;Supplementary Figure 1), as described in the Methods and mate-rials section, we carried out all our analyses using the 10 days ofsaline injected “control” groups. Our results demonstrated thatketamine-induced perturbations of the investigated neurotrans-mitter levels and related receptor systems that are implicated inpathophysiology of schizophrenia. These pathways include dopa-minergic, serotonergic and cholinergic systems.

It is to be noted that one of the striking differences between theeffects of the acute and the chronic doses of ketamine on animalbehaviour phenotypes, based on the performance in the forcedswim test, was the development of the negative behaviour symp-toms in the chronic model (Chatterjee et al., 2011, 2012a). Ourcurrent biochemical data provide us an opportunity to deducemolecular feature(s) that correlates to such a behavioural pheno-type. Most prominent among the investigated molecular parame-ters was an approximately 3-fold increase in the total 5HT content

e enzyme activity. Bar diagrams represent the effects of acute and chronic ketamineepresented as mean � S.E.M. with n ¼ 5e8 in each group. ***P < 0.001 vs control group,

Fig. 7. Chronic dose of ketamine modulates the glutamate, glycine and GABA contents. Bar diagrams represent changes in (a) glutamate; (b) glycine; and (c) GABA concentration atvarious brain regions, as indicated, in animals chronically injected with ketamine as described in Materials and Methods section. Results are represented as mean � S.E.M. withn ¼ 8 in each group. ***P < 0.001, **P < 0.01 versus control group.

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in the cortex of the ‘chronic’ group as compared to the ‘acute’ group(Fig. 2a). 5HT levels were also found to be significantly higher in thestriatum of the ‘chronic’ group. This activation of 5HT metabolismin the cortex and the striatum in response to the chronic ketaminetreatment was reflected by a massive surge in the 5HIAA produc-tion (Fig. 2b). Thus, the development of the negative symptoms inthe ‘chronic’ model could probably be attributed to the persistentactivation of the dopaminergic turnover in the striatum and cortex

Fig. 8. Gene-expression analyses of nicotinic receptor, cholinesterase, NMDA subunits andanalysis of (a) nicotinic acetylcholine receptor and acetylcholinesterase; (b) NMDA receptorfrontal cortex of chronically ketamine treated mice. The results are expressed as ratio of relan ¼ 4 in each group. ***P < 0.001 versus control group. The results are representative of th

(Fig. 1, Tables 2 and 3), possibly contributing to an enhanced 5HTproduction (Fig. 2) and hence, activation of the overall serotonergicsystem in the cortex (Jensen et al., 2010).

Consistent with these observations, chronic ketamine treatmentlowered the total glycine concentration in the cortex, striatum andhippocampus (Fig. 7b). This data is significant in the context of the‘chronic’ model validation, as glycine has been linked to the nega-tive symptoms of schizophrenia and glycine transporter has been

glutamate transporters in response to ketamine. Reverse transcriptase coupled PCRsubunits: NR1A, 2A, 2B; and (c) glutamate transporters, GLT-1 and GLAST; in the pre-tive intensity of gene of interest to b-actin. Values are expressed as mean � S.E.M. withree independent experiments.

Fig. 9. Effect of ketamine on nitrite content. Bar diagram representing the effects ofacute and chronic ketamine administration on nitrite content in various brain regions.Values are calculated as mean � S.E.M. with n ¼ 6 in each group. ***P < 0.001 versuscontrol group.

Fig. 11. Scheme describing the probable molecular relationships to the “positive”behavioural phenotype in mice. Antagonistic effect of acute ketamine administrationblocks NMDA receptor located on GABAergic neurons, eventually leading to constitu-tively active D2 and 5HT2 receptors in striatal areas, translating into positive symp-toms of psychosis i.e., hyperactivity in mice.

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e1171 1169

considered to be one of the critical drug targets (Javitt et al., 1994;Hons et al., 2010).

Our earlier work showed increased locomotor activity andstereotypy counts in both the acute and the chronic ketaminetreated mice (Chatterjee et al., 2011). It has also been reported thatthe dopaminergic pathways are critical for the control of suchlocomotor activities (Salamone et al., 2005; Benturquia et al., 2008).Our present data on the DA content and turnover (Fig. 1, Tables 2and 3) in the brain tissues examined, particularly in the striatum,tend to support a ketamine-induced hyperdopaminergic state. Thisdata was further supported by enhanced activity of dopaminemetabolizing enzymes like, monoamine oxidases, in the striatal

Fig. 10. Gene-expression profiles of glycine transporter and neuronal nitric oxide synthase insynthase (nNOS), in the cortex tissue of mice is analysed by PCR as described in the Methodgroup. The results are expressed as the ratio of the intensity of the indicated amplicon to the*P < 0.05 versus control group. The results are representative of three independent experim

tissue. In addition, an elevated 5HT2 receptor density and increased5HT turnover were also observed in the striatum (Figs. 2 and 3 andTable 4). Taken together, these results seem to be in agreementwiththe previous reports that the regulation of striatal DA release mightbe mediated via enhanced 5HT2 receptor activities (Gudelsky et al.,1994).

Moreover, ketamine-induced behavioural effects have beenpartially attributed to the blockade of NMDA receptors located oninhibitory GABAergic neurons in the limbic and subcortical brainregions (Moghaddam et al., 1997; Duncan et al., 1998; Nakao et al.,2003). Thus, the release of this inhibitory actions of the GABAergicneurons has been reported to increase the neuronal activity via

response to ketamine. Expression of (a) glycine transporter-1; (b) neuronal nitric oxides and materials section. ‘Chronic’ ketamine treated group is compared with the controlb-actin PCR product. Values are expressed as mean � S.E.M. with n ¼ 4 in each group.ents.

Fig. 12. Possible mechanism of action of ketamine on hippocampal neurons and itsrole in learning and memory functions. Ketamine acts as an antagonist to a-7nAChRand NMDA receptors. Blocking a-7nAChR by ketamine leads to a decrease in glutamaterelease. Inhibition of NMDA receptor mediated activity by ketamine mediate reductionin the NO content as well.

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e11711170

increased glutamate and dopamine release in the limbic striatalregions (Gass et al., 1993; Moghaddam et al., 1997; Duncan et al.,1998; Gao et al., 1998; Lorrain et al., 2003; Balla et al., 2009; Honset al., 2010). Our results tend to corroborate these findings (Fig. 11).

Acetylcholine, which plays a critical synaptic role in the initialstages ofmemory formation (Hasselmo, 2006), was also found to besuppressed in the hippocampus as a function of the acute ketaminetreatment (Fig. 6a). This data could be correlated to our previousobservation that the acute ketamine treatment leads to poor

Fig. 13. Schematic representation of the cellular and molecular e

performance in the passive avoidance test reflecting disruption inmemory acquisition and retention (Chatterjee et al., 2011). Activa-tion of acetylcholinesterase enzyme (AChE) in the cortical andhippocampal areas (Fig. 6b) suggesting an increased ACh turnover,further lends credence to the ketamine-induced cognitiveaberration.

Ketamine has also been reported to act as an antagonist tonicotinic acetylcholine receptor a-7nAChR (Coates and Flood,2001). Acetylcholine via a-7nAChR regulates glutamate release,which in turn, is believed to activate NMDA receptors. ActivatedNMDA receptors are thought to mediate influx of calcium ions byactivating calcium-dependent neuronal nitric oxide synthase(nNOS) leading to elevated levels of NO and ultimately causingenhanced glutamate release from the presynaptic cells throughmultiple mechanisms (O’Dell et al., 1991). Our results on theantagonistic effects of chronic ketamine administration on NMDAreceptors and a-7nAChR seem to fit the above model, as outlined inFig. 12, by drastically suppressing the nNOS expression (Fig. 10) andhence, NO synthesis (Fig. 9).

Overall, our findings suggest that ‘chronic’ ketamine adminis-tration may produce psychosis like symptoms by acting on a widearray of molecular parameters and the effect on some of which,including DA and 5HT levels, persisted even after ketamine with-drawal. In contrast, the ‘acute’ ketamine mediated effects weretransient and failed to induce negative symptoms, as reportedpreviously (Chatterjee et al., 2011). The ‘chronic’ ketamine micemodel possesses excellent construct validity as a model for exper-imental psychosis. As summarized in Fig. 13, the following eventsare deduced from our data: 1) Ketamine inhibits NMDA receptor inthe GABAergic neurons leading to the enhanced DA and 5HTrelease. Excess 5HT in turn leads to hyperactivation of 5HT1Areceptors, as was demonstrated in schizophrenic patients (Simpsonet al., 1996), reducing NMDA subunit transport to membraneaffecting synaptic plasticity (Yasuno et al., 2003; Yuen et al., 2008;Zhuo, 2009). 2) Ketamine blocks nicotinic cholinergic receptors(Scheller et al., 1996) and thereby suppresses glutamate release. 3)Blockade of these receptors, induces increased ACh release

vents in response to the chronic administration of ketamine.

M. Chatterjee et al. / Neuropharmacology 63 (2012) 1161e1171 1171

activating cholinesterase enzyme which, in turn, interferes withhippocampal memory formation. 4) Ketamine down-regulatesneuronal nitric oxide synthase expression, a key factor in synapticplasticity (Keilhoff et al., 2004).

6. Conclusions

We have characterized and validated the ketamine-inducedanimal model for experimental psychosis mimicking some of thesymptoms of schizophrenia. Our analyses of multiple molecularand biochemical parameters led to the identification of chronicketamine administration as the dose of choice for the developmentof the working model. Though the interpretation of such a widerange of data are complex and any attempt to deduce the mecha-nisms leave multiple gaps in the backdrop of extensive cross-talksamong the pathways, the results provide us with an opportunity tonarrow down the factors that might correlate with a cross-sectionof the psychosis symptoms. This report will help to incorporatethose molecular signatures as end-point assay tools or markers forscreening new generation antipsychotics in future.

Acknowledgements

Authors are grateful to the DBT and CSIR, New Delhi, India forproviding financial support. We sincerely acknowledge Mrs. Shi-bani Sengupta for her technical assistance. We also thank ourcolleague, Dr. S. Kar of Institute of Molecular Medicine, New Delhi,India, for critical reading of the manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.neuropharm.2012.05.041.

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