Using the BOLD MR signal to differentiate the stereoisomers of ketamine in the rat

www.elsevier.com/locate/ynimg

NeuroImage 32 (2006) 1733 – 1746

Using the BOLD MR signal to differentiate the stereoisomers of

ketamine in the rat

Clare L. Littlewood,a,* Diana Cash,a Aisling L. Dixon,a Sophie L. Dix,b Craig T. White,b

Michael J. O’Neill,b Mark Tricklebank,b and Steven C.R. Williamsa

aKing’s College London, Neuroimaging Research Group, Institute of Psychiatry, PO42 De Crespigny Park, London SE5 8AF, UKbEli Lilly and Co. Ltd., Erl Wood Manor, Windlesham, Surrey, GU20 6PH, UK

Received 6 January 2006; revised 10 April 2006; accepted 3 May 2006

Available online 11 July 2006

Rationale: Ketamine is a chiral molecule that is reported to model

aspects of schizophrenia.

Objectives: To investigate the stereospecificity of the isomers of

ketamine using pharmacological magnetic resonance imaging (phMRI)

in order to further understand ketamine’s pharmacodynamic actions.

Method: Responses to 25 mg kg�1 S(+) isomer, R(�) isomer and

racemic ketamine in independent groups of Sprague–Dawley rats were

investigated using a prepulse inhibition paradigm, locomotor observa-

tions, MRI and 2-deoxyglucose techniques.

Results: Racemic ketamine and the S(+) isomer were both capable of

disrupting sensorimotor gating as measured using prepulse inhibition

and produced a longer period of hyperlocomotion comparative to the

R(�) isomer. In contrast, large alterations in the BOLD MR signal

were observed with R(�) isomer, whereas S(+) isomer and racemate

precipitated more localized BOLD signal changes predominantly in

cortical, hippocampal and hindbrain regions. Glucose utilization rates

in conscious animals are in agreement with previously published data

and verify the BOLD responses in the racemic group. However, no

significant changes in glucose utilization were observed in the

anesthetized cohort.

Conclusions: Ketamine and its isomers have stereospecific effects on

sensorimotor gating and locomotion that correlate with the enantiom-

er’s affinity for the NMDA receptor. It would appear that anesthesia, as

required for preclinical MRI procedures, may interact with and

potentially attenuate the drug’s response. Although analysis of the

main effect of isomers in comparison to each other or the racemate

offers an alternative analysis method that should be less susceptible to

anesthetic interactions, only the R(�) isomer comparative to the

racemate offers significant differences of interest.

D 2006 Elsevier Inc. All rights reserved.

1053-8119/$ - see front matter D 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.neuroimage.2006.05.022

* Corresponding author. Fax: +44 20 7919 2116.

E-mail address: [email protected] (C.L. Littlewood).

Available online on ScienceDirect (www.sciencedirect.com).

Introduction

Ketamine hydrochloride is a well known sedative and

psychomimetic agent which is believed to exert its effects via

non-competitive NMDA receptor antagonism (Anis et al., 1983;

Duncan et al., 1999; Lodge and Johnson, 1990). At subanesthetic

doses, ketamine is purported to model a variety of the symptoms

associated with schizophrenia (Breier et al., 1997; Duncan et al.,

2001; Krystal et al., 1999). In rodents, racemic ketamine can

disrupt sensorimotor gating (Mansbach et al., 2001), a process

known to be disrupted in schizophrenics (Braff et al., 2001), as

measured with a prepulse inhibition (PPI) paradigm. Ketamine also

precipitates hyperlocomotion in rodents, thought to be a model of

psychosis (Carlsson and Carlsson, 1990; Corbett et al., 1995).

Ketamine has a chiral center and as such is made up of the two

‘‘mirror image’’ optical isomers, S(+) and R(�), named after their

rotation of polarized light. Although both compounds possess the

same constituent groups, their positions in space allow different 3-

dimensional interactions with their environment which can alter

their underlying pharmacology. In vivo and in vitro work has

elucidated that the S(+) isomer has a much higher affinity for the

phencyclidine (PCP) site on the NMDA receptor (Klepstad et al.,

1990; Oye et al., 1992) as well as A and n opioid receptors (Hirota

et al., 1999; Hustveit et al., 1995). Ex vivo experimentation has

also elucidated stereospecific effects upon dopamine efflux in the

nucleus accumbens (Hancock and Stamford, 1999) and caudate

putamen (Nishimura and Sato, 1999) of the rat. These experiments

suggest that S(+) isomer has a greater potency to mobilize DA

storage pools presynaptically in the nucleus accumbens whereas

the S(+) isomer is believed to act stereoselectively upon blockade

of DA uptake in the caudate putamen. In vitro studies have also

demonstrated that S(+) isomer is up to eight times more potent at

inhibiting DA transporters than the R(�) form (Nishimura and

Sato, 1999). In contrast, the R(�) isomer shows a higher degree of

affinity for the j site (Klepstad et al., 1990). In addition, R(�)isomer displayed stereoselective enhancement of serotonin (5HT)

efflux in the dorsal raphe nucleus (Tso et al., 2004). No

http://dx.doi.org/10.1016/j.neuroimage.2006.05.022

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C.L. Littlewood et al. / NeuroImage 32 (2006) 1733–17461734

stereoselectivity is apparent at acetyl choline receptors (Durieux

and Nietgen, 1997; Sasaki et al., 2000) or noradrenaline and

serotonin transporter proteins (Nishimura and Sato, 1999).

At equimolar, subanesthetic doses, the S(+) isomer is a better

analgesic and causes more perceptual disturbances than the R(�)form (Klepstad et al., 1990; Mathisen et al., 1995; Oye et al., 1992;

Vollenweider et al., 1997). A deeper level of anesthesia is achieved

with the S(+) thanwithR(�) isomer as indicated by its greater ability

to slow down EEGwaves in man (Schuttler et al., 1987;White et al.,

1985). The psychic emergence reactions at these doses are reported

to be equivalent for both isomers (White et al., 1985). The potential

of S(+) isomer to produce perceptual disturbances and analgesia

correlates with its stereoselectivity at the PCP binding site (Klepstad

et al., 1990) and with ketamine’s ability to suppress spontaneous

calcium oscillations, believed to be initiated by calcium’s entry

through NMDA receptors (Sinner et al., 2005). It is therefore widely

believed that the NMDA receptor mediates these properties of

ketamine at both subanesthetic and anesthetic doses.

Early studies investigating ketamine precipitated hyperlocomo-

tion (Ryder et al., 1978) suggest a rank order of potency equivalent

to that of psychotic symptoms in man. As publications discussing

the effects of the ketamine isomers upon the behavioral responses

of the rodent are sparse, one of the purposes of the current

publication was to further investigate the effects of the isomers of

ketamine upon PPI and locomotion, which are both common

testing paradigms used in models of schizophrenia. Further to this,

the neuronal origins of these behaviors would be most interesting.

Blood Oxygen Level Dependent (BOLD) signal originates from

the fact that there is an overcompensation in blood flow to an

activated area, thus decreasing the paramagnetic deoxyhemoglobin

concentration and altering the local magnetic field (Bandettini et

al., 1992; Ogawa et al., 1992). Examining the neural actions of

drugs by measuring BOLD signal changes is a popular technique

(Leslie and James, 2000; Salmeron and Stein, 2002) and has been

used to investigate a variety of compounds including sulpiride

(Preece et al., 2001), ketamine (Burdett et al., 1995), quinelorane

(Ireland et al., 2005), MK-801 (Houston et al., 2001), amphetamine

(Dixon et al., 2005) and heroin (Xu et al., 2000). The major

advantage of such a technique is that it allows in vivo imaging of

the whole brain non-invasively.

Using PPI and hyperlocomotion to verify the face and

predictive validity of ketamine isomers to model some of the

symptomatology of schizophrenia, it was hypothesized that BOLD

contrast could be utilized to further investigate the stereospecific

effects of ketamine. We were expecting BOLD activation to

correlate with the psychotic profile of the isomers as measured with

locomotor testing. Although ketamine has been previously

investigated with in vivo neuroimaging techniques in rodents

(Burdett et al., 1995), the enantiomers of this drug have never been

investigated in such a manner. In fact, we purport that this is the

first communication of ‘‘chiral’’ phMRI. Further to this, the

comparison of the isomers with the racemic mixture offers a new

technique to analyze the stereospecificity of a drug, regardless of

any systemic or anesthetic interactions.

As the employment of phMRI to investigate chiral interactions

of drugs is a novel technique, the established neuroimaging

technique for the quantification of glucose utilization rates, namely14C-2-deoxyglucose (2-DG) autoradiography, was also undertaken

in a separate cohort of animals. This technique allows the glucose

utilization rate of brain regions to be quantified by the use of a

radioactive analogue of glucose.

Materials and methods

All experiments were conducted to comply with the Animals

(Experimental Procedures) Act, 1986, and the local ethical

requirements. Male adult Sprague–Dawley rats were used in four

separate experiments: PPI investigations (40 animals, 280 T 11 g

[mean T SD], Harlan, UK), locomotor testing (40 animals, 319 T25 g [mean T SD], Charles River, UK), phMRI (40 animals, 306 T20 g [mean T SD], Charles River, UK) and 2-DG experimentation

(38 animals, 319 T 20 g [mean T SD], Charles River, UK). Animals

were housed in groups of 3–5 prior to the start of experiments in a

temperature-controlled environment (20–22-C) with food and

water available ad libitum.

Pure enantiomers of ketamine hydrochloride (Sigma, UK) were

isolated using semi-preparative HPLC. The column was a 15 cm �21.2 mm Chiralcel OJ-H (Chiral Technologies), the mobile phase

was 85% heptane, 15% ethanol each with 0.2% isopropylamine

and the flow rate 25 ml min�1. Column loading for each injection

was approximately 70 mg. Fractions were collected by UV

detection at 260 nm.

For all experiments, solutions were freshly prepared at the start

of each test day. For the PPI experiments, racemic ketamine

solutions were prepared in 5% glucose and the enantiomers were

dissolved in sterile water with the pH adjusted as described below.

In this case, 5% glucose was used for the vehicle group. For all

other experiments, ketamine and its isomers were dissolved in a

solution of 0.9% saline and 1 M HCl, with the pH adjusted to 5–7

using 1 M NaOH before injection; 0.9% saline was utilized for the

vehicle group. Animals received doses subcutaneously (sc) 1 ml

kg�1 doses of either 25 mg kg�1 ketamine racemate, 25 mg kg�1

S(+) isomer, 25 mg kg�1 R(�) isomer or vehicle. An additional

group was included in the PPI investigations which received 3 mg

kg�1 of the competitive NMDA antagonist, SDZ 220,581 (Tocris,

UK) dissolved in 5% glucose and equimolar NaOH.

Prepulse inhibition

PPI testing was conducted within sound-attenuated startle

chambers controlled by SDI software (SR-LAB, San Diego

Instruments, CA). Acoustic impulses could be delivered via a

speaker, and movement was recorded using a piezoelectric

accelerometer. The amplitude of the whole body startle to an

acoustic pulse was defined as the maximum of 100 one-

millisecond accelerometer readings collected from pulse onset.

Each animal underwent a habituation session 24 h before the

experiment commenced. This consisted of a 15 min period of white

noise at 75 dB followed by eight startle trials (40 ms of 120 dB).

Results from these startle trials were used to create matched

treatment groups. On the day of testing, animals were administered

vehicle (n = 8), 25 mg kg�1 racemate (n = 8), 25 mg kg�1 S(+)

isomer (n = 8), 25 mg kg�1 R(�) isomer (n = 8) or a positive

control of 3 mg kg�1 SDZ 220,581 (n = 8) 30 min prior to

experimentation. They were then subjected to a 5 min acclimation

period of white noise followed by 6 startle trials. This was followed

by pseudorandomized sequence of 8 prepulse–pulse +4 dB trials

(ppP4: 40 ms of 79 dB prepulse, 120 ms interstimulus interval

(ISI), 40 ms of 120 dB pulse), 8 prepulse–pulse +8 dB trials

(ppP8: 40 ms of 83 dB prepulse, 120 ms ISI, 40 ms of 120 dB

pulse), 8 prepulse–pulse +16 dB trials (ppP16: 40 ms of 91 dB

prepulse, 120 ms ISI, 40 ms of 120 dB pulse), 4 prepulse +8 dB

trials (40 ms of 83 dB prepulse), 4 prepulse +16 dB trials (40 ms of

C.L. Littlewood et al. / NeuroImage 32 (2006) 1733–1746 1735

91 dB prepulse), 8 no stimulus trials and 8 pulse trials (P: 40 ms of

120 dB). Six startle trials finished the testing period. For all trials,

the intertrial interval averaged at 15 s (range 10–25 s). Each test

session lasted 25 min, and the pre-treatment time included the

acclimation period.

Prepulse inhibition at each prepulse intensity was calculated

using the mean of each trial type and the following formula: 100 *

[(P � ppP) / P]. Startle amplitude was calculated from the mean for

the 8 pulse (P) trials. Data were analyzed using a mixed analysis of

variance (ANOVA). In all statistical analyses, a P value of 0.05 or

less was considered significant. Significant main effects were

followed by planned comparisons comparison against the vehicle

group; significant interactions were further analyzed with an

analysis of simple effects and, if significant, planned comparisons

against the vehicle group. Planned comparisons were also made

between the 2 enantiomers.

Locomotor activity

Using video tracking software (Ethovision, Noldus Information

Technology), locomotion was assessed as the movement of the

animal within its testing environment. Animals were habituated to

the testing chambers for 3 days prior to ketamine administration.

For each animal, testing was performed at roughly the same time of

day. On the day of dosing, animals randomly received either

vehicle (n = 9), 25 mg kg�1 racemate (n = 9), or 25 mg kg�1 S(+)

isomer (n = 11) or 25 mg kg�1 R(�) isomer (n = 11) 30 min into

the testing period.

Data recorded by the video tracking software were collected in

1 min time slots as total distance moved (cm) and then averaged

over ten-minute time bins. Data from one subject were excluded

from the analysis as a proportion of values from this rat were

classified as outliers using Grubbs test at P = 0.01 significance

level. A General Linear Model analysis of variance (SPSS 13.0

statistical package) examined within and between group effects. In

order to assess whether the Greenhouse–Geisser adjustment was

required, Mauchly’s test of sphericity was performed to determine

whether the sphericity of the data had been violated. Further to this,

post hoc comparisons of the relative changes between groups were

determined using a Tukeys comparison test. From these tests, any

groups that were identified to be significantly different from

control were further investigated using an independent t test at each

time point. Alternatively, the non-parametric Mann–Whitney U

test was used if Levene’s test for the equality of variances

identified time points where significant differences in variance

existed. In all statistical analyses, a P value of 0.05 or less was

considered significant.

Pharmacological MRI

All MRI protocols were performed using a 4.7 T (T) Oxford

200/300 MkII (Oxford Instruments) superconducting magnet; hpag

18 (Oxford Instruments) combined gradients and shims (max 100

mT/m, 12 cm bore size); q63 (Varian) quadrature birdcage coil; and

VNMR 6.1B (Varian) software. To induce anesthesia, animals were

subjected to 4% isoflurane in 0.9 l/min medical air and 0.1 l/min

medical oxygen within an induction chamber. Once unresponsive,

the animal was secured into a stereotaxic head frame and placed

into the center of the magnet. Anesthesia was maintained at 1.6%

isoflurane ventilated with the same gases supplied through a face

mask for the remainder of the experiment. To allow subcutaneous

administration of drugs during the image acquisition, a cannula

was inserted into each subject and attached to tubing which ran

outside the scanner. Each animal’s physiology was constantly

assessed using a rectal probe, respiration monitor and tail pulse

cuff sensor (Harvard Apparatus, UK) that interfaced with a PC

running Biopac MP100 software (Linton Instruments, UK). An

MRI compatible homeothermic blanket (Harvard, UK) responded

to any alterations in the body temperature of the animal that were

identified by the rectal probe and maintained temperature around

37-C.Subjects were scanned using a continuous, three-echo, gradient-

echo (GE) MR sequence (TE = 5, 10, 15 ms; TR = 940 ms;

acquisition matrix = 64 � 64 � 40; FOV = 4 cm2, yielding an

isotropic voxel resolution of 0.5 � 0.5 � 0.5 mm) allowing brain

volumes of 40 slices to be acquired every minute. After 30 such

scans, subjects randomly received either vehicle (n = 10), 25 mg

kg�1 ketamine racemate (n = 10), 25 mg kg�1 S(+) isomer (n = 10)

or 25 mg kg�1 R(�) isomer (n = 10). A further 120 scans were

then acquired.

phMRI: pre-processing

Extensive pre-processing prior to statistical analysis was

conducted on all GE images. Mean-echo images of the GE data,

with an effective TE of 10 ms, were produced by the summation

of the 5, 10 and 15 ms images (Ireland et al., 2005). Vascular

masks (derived by thresholding the raw images time series above

15% coefficient of variance) were applied to the data in an

attempt to remove signal changes associated with macroscopic

vessels, thus minimizing the contamination of surrounding tissue

as a consequence of spatial smoothing (Hlustik et al., 1998). An

motion correction algorithm, correcting for translational and

rotational movements during data acquisition [SPM99 (Institute

of Neurology, UK)], was applied to the image time series from

each animal. Following this, intra-cerebral structures were

outlined using an automated Brain Extraction Tool [(Smith,

2002); part of the FMRIB Software Library, Oxford, UK] and

utilized to create an extra-cerebral mask which was applied to

each subject’s time course. Each subject’s images were then

spatially normalized (SPM99) to a rat brain template created by

averaging the realigned time series of gradient-echo images from

a randomly chosen single subject (Friston et al., 1995). Finally,

Gaussian smoothing using a Full Width Half Maximum kernel of

1 mm (2� in-plane resolution) which imposes a normal

distribution on the data (Worsley and Friston, 1995) was applied

to the normalized time series.

phMRI: statistical analysis

Fixed effects multi-subject analyses [using a simple off

(baseline)/on (drug) paradigm] (SPM99) were primarily used to

identify any aberrant changes in a subject’s activation profile. From

this procedure, 16 subjects were identified and included in an

independent components analysis using MELODIC [Version 2;

FMRIB Software Library (Beckmann, 2002)]. Any components

with aberrant time courses that deviated above a designated

threshold (>0.5 abrupt change in signal that was deemed to be

unrelated to the drug profile or a gross unilateral signal) were

designated noise components and thus removed so that the

resultant data could be used for all further analyses (Thomas et

al., 2002). No more than 5 components were removed from any


one subject. Fixed effects multi-group analyses were then

conducted in SPM99 with no global normalization applied to the

data. This process undertakes a voxel by voxel analysis of brain

volumes from each subject per time point and identifies voxels

displaying temporal changes in signal intensity that correlate with a

specified input function. The input function used here was derived

from the pharmacodynamic results of the locomotor activity

experiment and consisted of a range of values from 0 (no effect)

to 0.5 (maximum effect) as shown in Fig. 2. In this case, the input

paradigm was derived from the ketamine racemate locomotor

curve to allow consistent comparison across groups. To account for

possible motion artefacts, the realignment (movement-correction)

parameters in the x, y and z planes were entered as covariates of no

interest.

The outputs of such analyses are Statistical Parametric Maps

(SPMs) which identify statistically significant changes in BOLD

contrast that correlate with the input function. To calculate group

differences in BOLD signal, the contrasts between these SPMs

were examined. To determine any drug-induced differences in

BOLD signal, contrasts defining the vehicle group as �1 and the

drug group as +1 were performed. In a similar manner, differences

between drug groups have been examined. Statistical significance

for the SPMs was set at P < 0.05, corrected for multiple

comparisons. All resulting SPMs were superimposed onto a co-

registered, high resolution anatomical image (spin echo, TE 40 ms,

TR 2 s, 0.25 � 0.25 � 0.5 mm voxel) of the animal that was used

as a template in spatial normalization.

Region of interest (ROI) analyses were then conducted using

the MarsBar toolbox for SPM99 (Brett et al., 2002). These ROIs

were selected on the basis of prior SPM analyses (Littlewood et al.,

2006; Paxinos and Watson, 1998) as well as the results observed

currently and constructed manually on coronal sections from the

template image. ROIs were also placed in areas of known variance

(e.g. ventricles) and in an arbitrary control region where no BOLD

contrast changes were observed. Signal intensities of these regions

were extracted for each subject over the time period of interest,

scaled to the mean signal intensity of the baseline data (0–30 min)

Fig. 1. Disruption of sensorimotor gating as measured using prepulse

inhibition of acoustic startle. Animals were administered 25 mg kg�1

ketamine racemate, S(+) isomer, R(�) isomer, 3 mg kg�1 SDZ 220,581 or

vehicle. Significant main effects for comparisons against vehicle are

indicated by * along the x axis; significant effects from the interaction term

for comparisons against vehicle are marked by * on the graph. Planned

comparisons between the enantiomers reaching significance are delineated

with #. *P < 0.05, **P < 0.0001.

and then scaled to an arbitrary value (100). Average time courses

from each region per group were plotted over 10 min intervals.

Physiology was also statistically investigated using the same

protocol as defined for locomotor activity.

Glucose utilization measurements

Anesthesia was induced in animals using 4% isoflurane in

medical oxygen, and once unresponsive to paw pinch, the

animals were transferred to a face mask where isoflurane

delivery was reduced to 2–3% for surgery. Cannulae were

inserted into the femoral artery and vein to allow blood sampling

and 2-DG administration, respectively. Local anesthetic was

applied, and the surgical area was sutured closed. A subcutane-

ous cannula was secured into the scruff of the animal to allow

drug administration.

In the first instance, experiments were conducted in

conscious animals to replicate the findings of previous

laboratories and verify the technique’s ability to identify

significant alterations in glucose utilization (GU). Animals were

restrained within a loose-fitting plaster cast (3M), and once this

had hardened, anesthesia was terminated. These animals were

then monitored for 2 h before autoradiography commenced.

Following this experimentation, GU in anesthetized animals was

assessed to mimic the experimental conditions required for

phMRI. These animals were reduced to 1.6% isoflurane and

allowed 1 h to stabilize before the 14C-2-DG technique was

initiated. Their temperature was maintained using a rectal probe

and homeothermic blanket (Harvard Apparatus, UK).

One minute after subcutaneous injection of either 25 mg kg�1

ketamine (n = 6) or vehicle (n = 6) into conscious rats, 14C-2-DG

infusion was initiated following the protocol of Sokoloff et al.

(Sokoloff et al., 1977). The same procedure was also utilized to

study the effects of vehicle (n = 5), 25 mg kg�1 racemate (n = 6),

25 mg kg�1 S(+) isomer (n = 6) or 25 mg kg�1 R(�) isomer

(n = 6) in isoflurane-anesthetized rats. 10 ACi/100 g of 14C-2-

DG was administered at a constant infusion rate over 30 s.

Fourteen timed arterial blood samples were collected into

lithium heparinized polyethylene microfuge tubes (Beckman

Coulter), immediately centrifuged for 1 min at 10,000 rpm and

the plasma removed. Quantification of the plasma glucose level

and 14C-2-DG concentration was carried out using the YSI 2300

Blood glucose analyzer (YSI) or PerkinElmer Wallac 1414

liquid scintillation counter (WinSpectral), respectively. After the

14th blood sample at 45 min post-14C-2-DG administration, the

animals were decapitated and the brains rapidly removed, frozen

in cold isopentane (�40-C) and stored at �70-C until post-

processing.

Brains were cryosectioned into 20 Am slices and thaw-mounted

onto pre-heated glass coverslips that were rapidly transferred to a

hot plate, maintained at 60-C. These coverslips were then secured

onto a cardboard base and exposed to X-ray film (Kodak Biomax

MR) along with 14C-2-DG microscale calibrated standards (31–

883 nCi/g, Amersham). After 6 days of exposure, autoradiographs

were developed. Digital capturing of the images was performed

using a lightbox (Northern Lights Illuminator set at 500), RT Spot

Slider camera (aperture f8; Height 36.1 cm) with 55 mm lens

(Micro Nikkor) and Spot Basic program. One subject was excluded

due to gross morphometric abnormalities, and no data were

available for two subjects due to thawing issues prior to

cryosectioning.


GU maps (Amol/100 g/min) were constructed within the MCID

software analysis package (Interfocus, UK), and region of interest

(ROI) analysis was performed upon these maps. ROIs were chosen

based on the results of the phMRI study as well as previously

published 2-DG investigations into the metabolic effects of

ketamine in rodents (Crosby et al., 1982; Duncan et al., 1999;

Miyamoto et al., 2000). For each area identified for analysis, GU

values from manually drawn ROIs were collected from both

hemispheres, wherever possible, and from a minimum of 2 slices.

At least 4 samples were collected per area per rat and averaged

across each group. Significant differences per brain region were

determined using analysis of variance statistical comparisons,

where P < 0.05 was considered significant.

Results

Prepulse inhibition

The enantiomers of ketamine have stereoselective effects on

sensorimotor gating as measured by PPI. The ANOVA revealed a

significant main effect between groups (F[4,35] = 9.8, P <

0.0001) and an interaction of group and prepulse level (F[8,70] =

3.6, P < 0.01). Planned comparisons following the main effect of

treatment identified the S(+) isomer, racemic ketamine and SDZ

220,581 as significantly different from vehicle (F[1,35] = 22.4,

P < 0.0001; F[1,35] = 26.6, P < 0.0001; F[1,35] = 16.6, P < 0.001;

respectively), while there was no effect of the R(�) isomer

(F[1,35] = 2.7, P > 0.1). Analysis of simple effects following the

significant interaction of group by treatment demonstrated a

significant effect of the treatment on the +4 dB prepulse

(F[4,35] = 5.7, P < 0.01), the +8 dB prepulse (F[4,35] = 12.2,

P < 0.0001) and the +16 dB prepulse (F[1,35] = 9.7, P < 0.0001).

This was followed by planned comparisons against the vehicle

group at each prepulse intensity. Further planned comparisons

were performed between the ketamine enantiomers. Significant

differences between groups are shown in Fig. 1. The S(+) isomer,

ketamine racemate and SDZ 220,581 were all found to produce

significant disruption of PPI across prepulse intensities. In

contrast, with the R(�) isomer, a small but significant disruption

Fig. 2. Effect of sc injection of 25 mg kg�1 ketamine racemate or its stereoisomers

modeled on this behavioral data used as an input function for phMRI analyses (

(TSEM). *P < 0.05 25 mg kg�1 ketamine racemate vs. saline; ¨P < 0.05 25 mg k

measured with post hoc independent t test (or Mann–Whitney U if equality of v

of PPI was only present when the prepulse tone was 8 dB above

background noise.

Locomotor activity

Ketamine and its isomers produced highly significant

increases in locomotor activity (Fig. 2), with observed significant

main effects of groups (F[3,3] = 8.521, P < 0.001), time � group

interaction (F[3,12.669] = 5.092, P < 0.001) and time (F[3,4.223] =

30.214, P < 0.001). The Greenhouse–Geisser adjustment was

included in the analysis to compensate for any violations of the

sphericity of the data. Tukeys comparison test identified 25 mg

kg�1 ketamine racemate (P < 0.01), 25 mg kg�1 S(+) isomer

(P < 0.01) and 25 mg kg�1 R(�) isomer (P < 0.05) as

significantly different from the vehicle group. No other dosage

groups were found to be significantly different from any other. To

inspect which time points were different from vehicle in the drug-

administered groups, post hoc independent t tests (P < 0.05; or

Mann–Whitney U, dependent on equality of variances) identified

that both the 25 mg kg�1 ketamine racemate and S(+) isomer

were significantly different from vehicle until 60 min post-

injection. However, the group administered 25 mg kg�1 R(�)isomer differed from the control group until only 40 min post-

injection.

Pharmacological MRI

The stereospecificity and differences between the effects of the

two isomers are obvious from the BOLD contrast maps (Fig. 3).

Increases in BOLD signal were observed in a number of brain

regions following 25 mg kg�1 ketamine racemate administration

(Fig. 3A). Correlation of cortical areas including the cingulate,

somatosensory and retrosplenial cortex with the locomotor input

function is apparent with 25 mg kg�1 ketamine. There is also some

evidence of hippocampal, thalamic, hindbrain and cerebellar

BOLD signal changes following ketamine racemate injection.

SPM maps derived from the 25 mg kg�1 S(+) isomer dataset

suggest a similar activation profile, particularly in limbic regions

but with reduced BOLD contrast in hindbrain and frontal areas

(Fig. 3B).

on locomotor activity in Sprague–Dawley rats (left panel) and the paradigm

right panel). Total distance moved in 10 min is displayed as group means

g�1 S(+) isomer vs. saline; # P < 0.05 25 mg kg�1 R(�) isomer vs. saline as

ariances is violated).


The 25 mg kg�1 R(�) isomer displayed large changes in

BOLD contrast covering nearly the entire brain area imaged (Fig.

3C). To ensure that these changes were not derived as a result of

using an input function based on the racemic locomotor profile,

an input paradigm modeled on the locomotion curve for the 25

mg kg�1 R(�) isomer group was used in a separate analysis and

Fig. 4. Temperature (top left), pulse (bottom left) and respiration (top right) measurements collected during the functional MR imaging protocol. Values were

collected every minute from a subset of animals undergoing scanning and averaged into 10 min time blocks. The graph displays these means T standard error ofthe mean. No significant differences were observed between groups.


was found to make a negligible difference to the resultant SPM

map.

Further analyses of the main effect of the isomers in respect to

the racemate and each other were also undertaken. Negligible

BOLD contrast changes were observed in the S(+) isomer group in

comparison to racemate (Fig. 3E) or S(+) isomer in comparison

with R(�) isomer (Fig. 3F). However, this further analysis

highlighted the stereospecific effect of 25 mg kg�1 R(�) isomer

within the nucleus accumbens (Fig. 3D). An analysis such as this

helps reduce systemic influences on BOLD contrast as both the

racemate and isomers should affect the physiology and anesthesia

of an animal in a similar pattern that may not be paralleled in a

vehicle group.

There were no significant changes in the physiology of the

animals post-injection. Physiological datawere not available for post

hoc analysis from all animals, but pulse measurements were

recorded for at least 7 animals in each group. However, there

appeared to be a depression of pulse post-ketamine or its enantiomers

that was not apparent in the vehicle group (Fig. 4). No significant

differences in the global brain signal were identified, suggesting no

global differences between drug and vehicle groups (Fig. 5F).

Fig. 3. SPM{t} distribution maps representing BOLD signal change overlaid on

represent significant correlation (thresholded at P < 0.05 corrected for multiple com

in comparison to vehicle animals. Red represents positive correlation, blue represen

racemate (t > 4.38) with locomotor profile (Fig. 2, right panel, 110 min) compared w

with locomotor profile compared with saline. (C) Correlations of 25 mg kg�1 R

Correlations of 25 mg kg�1 R(�) isomer with locomotor time course compared wi

S(+) isomer with locomotor time course compared with 25 mg kg�1 ketamine racem

time course compared with 25 mg kg�1 S(+) isomer (t > 4.3).

ROI analysis further illustrated the similarity in the response to

both S(+) and racemic ketamine in cortical areas (Fig. 5B). Signal

intensity changes were not large, but the temporal course produced

show definite alterations between drug and saline administration in

the nucleus accumbens, hippocampus, cortex and ventricles (Figs.

5A–D). No differences were observed in an area that was designated

as not activated as a consequence of the SPM{t} maps produced

(Fig. 5E).

Glucose utilization measurements

In agreement with previous publications (Crosby et al., 1982;

Duncan et al., 1999; Miyamoto et al., 2000), one-way analysis of

variance identified a large number of brain areas with signifi-

cantly altered cerebral rate of glucose utilization (GU) after 25

mg kg�1 ketamine racemate in conscious rats (Fig. 6, top). Areas

with altered GU after racemic ketamine include: cingulate cortex,

retrosplenial cortex, somatosensory cortex, hippocampal areas,

inferior colliculus, medial geniculate, nucleus accumbens, ventral

pallidum, caudate putamen, nuclei of the amygdala and thalamic

areas.

co-registered anatomical templates (n = 10 rats per group). Colored pixels

parisons) of signal time course with input function (locomotor time course)

ts negative correlation. (A) Significant correlations of 25 mg kg�1 ketamine

ith saline. (B) Significant correlations of 25 mg kg�1 S(+) isomer (t > 4.31)

(�) isomer with locomotor profile in comparison to saline (t > 4.31). D:

th 25 mg kg�1 ketamine racemate (t > 4.35). (E) Correlations of 25 mg kg�1

ate (t > 4.37). (F) Correlations of 25 mg kg�1 R(�) isomer with locomotor


To further investigate the influence of anesthesia upon glucose

utilization and any potential alteration of neurovascular coupling,

GU was also investigated under the same anesthetic regimen as

the phMRI experiment. No significant changes in glucose

utilization were found in any of the brain regions studied (Fig.

6, bottom).

Discussion

The aim of this study was to utilize a recent imaging method

(phMRI) to observe the effects of racemic ketamine, as well as

the stereospecificity of the isomers which correlated with a

behavioral input function. In addition, endeavors were made to

validate these results using behavior and autoradiography.

Although interesting differences in the BOLD contrast profiles

Fig. 5. Time course of signal intensity in six regions of interest (ROIs) as presente

All signal intensities have been scaled to arbitrary units and grand mean scaled to t

are displayed as group means (TSEM) over 10 min intervals.

obtained for the isomers and racemate comparative to saline were

observed, little difference was found for the isomers in

comparison with each other. Furthermore, the neuronal origin

of the BOLD signal could not be conclusively verified using GU

measurements.

It is purported that this is the first communication of isomeric

differences in the disruption of sensorimotor gating by ketamine.

As can be visualized in Fig. 1, S(+) isomer, racemic ketamine and

the competitive NMDA antagonist SDZ 220,581 produce disrup-

tion of PPI. The inclusion of the SDZ 220,581 dosing group

provided a positive assay control that may suggest NMDA

antagonism involvement in the PPI disruption elicited by ketamine

and its isomers. These results also suggest that ketamine, and to

varying degrees its isomers, can model the sensorimotor gating

deficits observed in schizophrenic patients. However, this is a very

complicated arena insomuch that ketamine appears unable to

d overlaid onto the co-registered spin echo anatomical template (left panel).

he mean of the baseline (0–30 min) of each time series. Signal time courses

Fig. 5 (continued).


disrupt PPI in healthy volunteers (Abel et al., 2003; Duncan et al.,

2001).

Investigation of the hyperlocomotion precipitated by ketamine

and its isomers in the current study has displayed a similar profile

between all the ketamine forms, though lasting for a shorter time

period when the R(�) isomer is administered. However, no

significant differences were observed between the different chiral

forms of ketamine whereas all ketamine isoforms were signifi-

cantly different from vehicle. Although the locomotor profile

described in this paper complements previous work carried out

with the enantiomers of ketamine (Ryder et al., 1978), no

significant stereospecific effects of the isomers can be reported in

contrast to work carried out in mice, where the S(+) isomer was

found to be more psychomimetic than the R(�) isomer as

measured using ataxia, head weaving and c-fos expression

(Nishizawa et al., 2000). Although hyperlocomotion is often

used as a model of psychosis, it must be noted that non-psychotic

agents are also capable of eliciting hyperlocomotor activity

(Grottick et al., 2000).

In contrast to its lesser effect on locomotor behavior,

administration of the R(�) isomer affected a much larger cerebral

BOLD contrast response than was observed with either the

racemate or the S(+) isomer in comparison to vehicle. On the

other hand, the similarity of the SPM maps derived from data

collected after the S(+) isomer or racemic drug is striking. If the

data from selected ROIs (Fig. 5) are studied, the similarity of the

temporal signal profiles between the S(+) isomer and racemate,

especially in the hippocampus and cerebral cortex, adds further

weight to this observation. These results are also consistent with

our previous study (Littlewood et al., 2006, in press) in which

cortical and hippocampal areas were found to be activated by 25

mg kg�1 racemic ketamine. In this previous study, however,

limbic areas including the nucleus accumbens and ventral

pallidum were also found to have correlation with the locomotor

Fig. 6. Quantitative 2-deoxyglucose uptake (Amol 100 g�1 min�1) across a variety of regions of interest in Sprague–Dawley rats. Data are expressed as group

means (TSEM). The top figure displays glucose utilization in conscious restrained animals after 25 mg kg�1 ketamine racemate subcutaneously, whereas the

bottom chart displays glucose utilization in isoflurane anesthetized rats after 25 mg kg�1 ketamine racemate and its isomers subcutaneously. *P < 0.05

ketamine racemate comparative to saline; **P < 0.001 ketamine racemate comparative to saline.


input paradigm. It is imperative to remember that BOLD

responses are being compared to the saline-injected ‘‘control’’

group. Even under anesthesia, we cannot conclusively argue that

identical brain activation profiles will be evident in both the

control groups. Therefore, the BOLD contrasts obtained will be

influenced by these potentially differing ‘‘baseline’’ states. These

differences may also originate from the slightly different input

profiles used for the two analyses as well as the different time

courses investigated (110 min vs. 150 min) and potential

anesthetic interactions discussed in detail later.

Furthermore, it would appear from the signal intensity

graphs (Fig. 5) that ketamine administration acted to attenuate

the trend for a decline in signal intensity that can be clearly

seen in all groups in the baseline state rather than cause an

elevation in signal intensity. This may suggest a potential

decline in the physiological status of the animal not detected by

the monitoring system employed or scanner drift that is being

counteracted by administration of ketamine. In addition, it

substantiates the hypothesis of anesthetic interaction in the

phMRI protocol.


GU was also calculated after a racemic dose of ketamine (Fig.

6, top), and an agreement was found with previous studies (Crosby

et al., 1982; Duncan et al., 1999; Miyamoto et al., 2000).

Significant alterations in GU were observed in a variety of brain

regions studied. Many of these areas correlate with the areas

displaying altered BOLD contrast, including hippocampal areas,

retrosplenial cortex, anterior cingulate, thalamic areas and somato-

sensory cortex. In contrast, GU calculations undertaken in

isoflurane anesthetized rodents lacked any significant findings.

This may suggest interaction of the anesthetic paradigm with GU

or potential dissociation of activation–metabolism coupling

mechanisms, especially in light of work carried out in humans

that observed psychomimetic effects and metabolic hyperfrontality

in awake, healthy volunteers administered S(+) isomer whereas a

state of relaxation and decreased GU in the temporomedial cortex

and left insula were reported after the R(�) isomer (Vollenweider

et al., 1997). However, the existence of significant BOLD contrast

changes in the absence of GU alterations after ketamine and its

isomers may suggest that changes in activity are being attenuated

by the anesthetic agent employed so that only small modulations

are occurring. These are too small to be detected by GU techniques

but are visible as small, though significant alterations in BOLD

contrast. It could also be purported that BOLD contrast changes

originate from blood flow effects that are not necessarily coupled

to brain activation.

Unlike the S(+) isomer and racemate, R(�) isomer produced

large BOLD signal changes across most of the brain. Systemic

origins for this large signal cannot be totally excluded, although an

ROI placed within a non-activated area (Fig. 5E) shows no drug

response, indicating that this large BOLD contrast may not be a

systemic effect. It might be hypothesized that the reduced NMDA

antagonism expected with this equimolar dose of R(�) isomer may

be causing less of an interaction with the anesthetic agent

isoflurane (see below). As ketamine-induced cerebral blood flow

changes have been shown to be related to neuronal activation in

rats and man (Cavazzuti et al., 1987; Langsjo et al., 2003, 2004), it

may be assumed that, although the changes observed are

widespread, they are neuronally related.

Another possibility to explain the large BOLD signal changes

observed in the R(�) isomer group is the slower hepatic clearance

of R(�) isomer relative to the S(+) enantiomer (Kharasch and

Labroo, 1992) and the differential metabolite concentrations of the

2 isomers in plasma and brain fractions (Marietta et al., 1977;

Ryder et al., 1978). The differential pharmacokinetics of the

isomers may alter the receptor binding kinetics of the isomers, and

thus the temporal neuronal characteristics may reflect this potential

longer, though less robust, receptor binding within the R(�) isomer

group. To help further elucidate the differential neuronal activation

profiles of the isomers, it would be of interest to replicate the

current study using equianalgesic doses.

For MRI studies to be conducted in rodents, anesthesia or

sedation is usually required in order to reduce motion artefacts.

Thus, in this study, we used low doses of the inhalation anesthetic

isoflurane throughout the imaging session. At higher doses, such as

100 mg kg�1, ketamine (we used 25 mg kg�1 ketamine in the

current study) itself acts as an anesthetic and so there is a

possibility for interaction between ketamine and the other

anesthetic agents. In this case, isoflurane has been utilized in

order to allow non-invasive sedation with the potential for repeated

anesthesia. Isoflurane has been reported to increase the local charge

transfer at GABAergic synapses and decrease charge transfer at

glutamatergic synapses in rat hippocampal cultures (de Sousa et al.,

2000). Added to evidence that ketamine anesthesia can be

potentiated using GABA agonists muscimol and diazepam in mice

(Irifune et al., 2000), it may be postulated that isoflurane

administration may attenuate cerebral responses to subanesthetic

doses of ketamine, potentially via a GABAergic mechanism. Even

though there is no evidence that isoflurane interacts directly with

the NMDA receptor, there is evidence that it can alter glutama-

tergic transmission probably by a presynaptic mechanism (Berg-

Johnsen and Langmoen, 1992; Maclver et al., 1996; Pearce et al.,

1989). Therefore, it is possible that isoflurane may interact with

ketamine via either the GABA or glutamate neurotransmitter

systems. Halothane, an inhalation anesthetic pharmacologically

similar to isoflurane, was found to suppress the ketamine-induced

expression of c-fos in the posterior cingulate/retrosplenial cortex;

preliminary results with isoflurane suggested an equivalent

attenuation (Nakao et al., 1996). In agreement with these findings,

we found no changes in GU in isoflurane-anesthetized rats, after

ketamine or its isomers.

A possible interaction of isoflurane and ketamine via the

dopaminergic system is also another consideration for this study.

Ketamine has actually been reported to have a greater affinity for the

high-affinity D2 receptor than the NMDA receptor (Seeman et al.,

2005). and isoflurane is also known to inhibit the high affinity state

of the D2 receptor (Seeman and Kapur, 2003). Work is ongoing

within the laboratory to replicate these studies using alpha-

chloralose sedation, although interactions may still not be excluded.

There were no significant differences between the groups in the

physiological parameters (Fig. 4). Therefore, it is unlikely that any

physiological effects of these compounds significantly interfered

with the observed cerebral BOLD contrast changes. A phMRI

investigation of cocaine and its derivative cocaine methiodide

(which cannot cross the blood brain barrier but produces equivalent

cardiovascular effects), in which both compounds significantly

altered blood pressure but only cocaine produced meaningful

BOLD contrast changes (Luo et al., 2003), indicates that blood

pressure has little influence on BOLD signal. The BOLD signal

changes calculated here are small, but the areas which display

significant correlation with the locomotor profile of racemic

ketamine are areas which have been previously identified to be

of interest in the acute profile of ketamine (Burdett et al., 1995;

Duncan et al., 1999; Olney et al., 1991). Inhalation anesthetics

have previously been used successfully both within our group

(Cash et al., 2003; Jones et al., 2005; Roberts, 2004) and by other

laboratories (Dixon et al., 2005; Hewitt et al., 2005; Steward et al.,

2004) to investigate BOLD responses to CNS-active compounds.

Therefore, it is proposed that, although the S(+) isomer and

ketamine racemate display BOLD signal changes in brain areas of

interest, the interaction with anesthesia limits any conclusions

derived from this work.

A visual examination of the 2-DG results (Fig. 6, bottom)

suggests a trend, albeit non-significant, for the S(+) isomer and

racemate to have reduced glucose utilization, whereas the R(�)isomer has very similar effects to the control group. It has been

previously reported that intrathecal application of S(+) isomer or

racemic ketamine could potentiate morphine analgesia at doses

that did not cause motor impairment, whereas the R(�) isomer

could not (Joo et al., 2000). It could be that, in our study, due to

its lesser interaction with anesthesia, R(�) isomer has a greater

psychotic-like activity resulting in the widespread BOLD

response. A large BOLD response to a psychostimulant has


previously been reported by an alternative laboratory using

amphetamine challenge (Dixon et al., 2005), further validating

this hypothesis.

At equimolar doses, the S(+) isomer produces a prolonged

hyperlocomotor response. However, at equianalgesic doses, R(�)isomer is actually more potent (Marietta et al., 1977; Ryder et al.,

1978). It may be that the relative NMDA blockade between the

different molecules in sedated animals has a more equianalgesic

profile rather than the equimolar response observed in conscious

animals. The stereospecific interactions of R(�) isomer with other

receptor subtypes other than the NMDA receptor must also be

considered. For example, R(�) isomer has been demonstrated to

stereoselectively interact with j recognition sites (Klepstad et al.,

1990). It may be that the large correlation of BOLD contrast with

the locomotor input paradigm may be related to the isomer’s jreceptor affinity. j ligands have been previously been shown to be

able to attenuate hyperlocomotion elicited by cocaine and

amphetamine, suggesting that j receptors have a role in locomotor

activity (Hascoet et al., 1995; McCracken et al., 1999). Using a

behavioral covariate of interest within the BOLD analysis method

offers a useful strategy to identify changes in BOLD contrast

changes that correlate with the behavioral time course of the drug.

However, signal intensity changes elicited by ketamine that deviate

from that of the locomotor input function could be inadvertently

missed. Furthermore, the locomotor input profile was derived from

data collected in the conscious rather than anesthetized state. With

the anesthetic interaction that seems apparent in the current study,

the use of an input function in MRI analysis that has not been

obtained in an anesthetized cohort may be questioned. However,

the premise of the current study was to identify BOLD contrast

changes that correlated with the time course of induced psychosis

as previously discussed (Littlewood et al., 2006).

To eliminate the confounding effect of anesthesia in this study,

a further analysis of phMRI data was undertaken. This involved

contrasting the isomers to the racemate rather than the vehicle

group to identify stereoselective responses. In such an analysis, as

animals are more physiologically comparable, a greater confidence

can be placed on the neuronal origin of signal changes. However, it

is unknown what interactions the S(+) and R(�) isomers may have

with each other in the racemic mixture, thus complicating

interpretation of an analysis of this nature. Negligible BOLD

contrast changes were observed with S(+) isomer comparative to

racemate (Fig. 3E) and the S(+) isomer comparative to the R(�)isomer (Fig. 3F). Therefore, although large differences exist in the

BOLD contrast profiles of the different enantiomers when

compared to vehicle, negligible statistical differences can be

observed when the isomers are directly compared, helping to

further illustrate the complex interactions the isomers of ketamine

have with each other.

However, correlation with the locomotor input function was

identified in the nucleus accumbens of the R(�) isomer group, with

respect to the racemate (Fig. 3D). As the analysis was probing for

brain regions correlating with the behavioral response of the

compound, this area would not be unexpected as the nucleus

accumbens is believed to be involved in ketamine-induced hyper-

locomotion (Hunt et al., 2005; Irifune et al., 1991). Given that

ligands of j receptors can produce responses similar to those of

ketamine (Brent, 1991) and are believed to influence the nucleus

accumbens (Guitart and Farre, 1998), the observed BOLD contrast

may be partially related to R(�) isomer’s affinity for the j receptor

(Klepstad et al., 1990). Moreover, certain j antagonists have

shortened NMDA-antagonist-induced cognitive dysfunction and

hyperlocomotion in rats (Maj et al., 1996; Ogawa et al., 1994).

These antagonists can also block cocaine-induced c-fos expression

in the nucleus accumbens (Maurice and Romieu, 2004) and

ketamine-induced c-fos expression in the posterior cingulate/

retrosplenial cortex (Nakao et al., 2002). No normal physiological

function of the j receptor is known, but it would appear that this

class of receptor is involved in psychotropic drug responses

(Debonnel and de Montigny, 1996). In fact, j receptor binding has

been shown to be reduced in post-mortem schizophrenic brain

samples, adding further evidence for j receptor involvement in

psychosis (Helmeste et al., 1996).

Conclusions

The stereoselectivity of the ketamine enantiomers has been

investigated using PPI, hyperlocomotion and phMRI techniques.

PPI identified the S(+) isomer as a potent sensorimotor disrupting

agent, and locomotor activity demonstrated the potential psychot-

ic activity of all the ketamine isoforms. However, the BOLD

contrast results reported here suggest an interaction with the

required anesthesia profile that has been substantiated using 2-DG

autoradiography. Although this anesthetic confound may limit the

interpretation of the results, phMRI can be potentially utilized to

identify stereospecific effects of isomers, a method first described

here. Furthermore, it is purported that comparison of the isomers

of ketamine with the racemate, rather than the vehicle group, may

offer an alternative method to investigate isomeric differences

while minimizing any anesthetic interaction.

Acknowledgments

This research was generously funded by a BBSRC CASE

studentship in collaboration with Eli Lilly and Co. The University

of London Intercollegiate Research Service scheme permitted

access to the MR imaging spectrometer located at Queen Mary

College London where it is managed by Dr. Alasdair Preston.

Preliminary data relating to this study have been previously

presented at the British Association of Psychopharmacology

Summer Meeting 2005 and the Society for Neuroscience 35th

Annual Meeting.

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