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Research Report

Clinically relevant concentrations of ketamine mainlyaffect long-term potentiation rather than basalexcitatory synaptic transmission and do not changepaired-pulse facilitation in mouse hippocampal slices

Patrıcia O. Ribeiroa,b,c,n, Angelo R. Tomec,d, Henrique B. Silvac,Rodrigo A. Cunhac,e, Luıs M. Antunesa,b

aInstituto de Biologia Molecular e Celular, Laboratory Animal Science, Rua do Campo Alegre 823,4150-180 Porto, PortugalbDepartamento de Ciências Veterinárias, Universidade de Trás-os-Montes e Alto Douro, Apartado 1013,5001-801 Vila Real, PortugalcCNC—Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, PortugaldDepartamento das Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade de Coimbra,3001-401 Coimbra, PortugaleFaculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal

a r t i c l e i n f o

Article history:

Accepted 6 March 2014

Ketamine, an analgesic/anesthetic drug, is increasingly popular in clinical practice due to

its analgesic properties and importance for emergency procedures. The impact of ketamine

Available online 15 March 2014

Keywords:

Hippocampus

Ketamine

Synaptic plasticity

Synaptic transmission

1016/j.brainres.2014.03.00sevier B.V. All rights rese

uthor at: Instituto de Bio

opatricia.ribeiro.fafe@gm

a b s t r a c t

on basal excitatory synaptic transmission and synaptic plasticity are not yet fully under-

stood. Therefore we investigated the effects of different concentrations of ketamine on

basal excitatory synaptic transmission and on two forms of synaptic plasticity: paired-

pulse facilitation (PPF) and long-term potentiation (LTP). Evoked field excitatory postsy-

naptic potentials (fEPSP) were recorded in Schaffer fiber – CA1 pyramid synapses of mouse

hippocampal slices and the initial slope of the fEPSP was measured to estimate the

percentage of inhibition of the basal synaptic transmission. Presynaptic volley amplitude,

PPF and LTP induction and maintenance were also calculated. For basal synaptic

transmission and PPF increasing concentrations of ketamine (1, 3, 10, 30, 100, 200, 300

and 600 μM) were applied to each slice and for LTP individual slices were used for each

concentration (3, 10, 30 or 100 μM). Clinically relevant concentrations of ketamine

decreased LTP in a concentration-dependent manner without changing PPF, whereas

basal excitatory synaptic transmission and presynaptic volley amplitude was affected only

with high concentrations of ketamine (300 and 600 μM). These results allow dissociating

4rved.

logia Molecular e Celular, Laboratory Animal Science, Rua do Campo Alegre 823, 4150-180

ail.com (P.O. Ribeiro).

b r a i n r e s e a r c h 1 5 6 0 ( 2 0 1 4 ) 1 0 – 1 7 11

the blockade of LTP from a reduced synaptic input in the action of clinically relevant

concentrations of ketamine in the CA1 region of the mouse hippocampus. Moreover, this

work shows that the effects of ketamine on LTP and on basal synaptic transmission are

dependent of the concentration used.

& 2014 Elsevier B.V. All rights reserved.

Fig. 1 – Schematic representation of a mouse hippocampalslice showing the placement of the stimulating andrecording electrodes. The recording pipette was placedin the stratum radiatum of the CA1, and the stimulatingelectrode was placed in the stratum radiatum near theCA3/CA1 border to stimulate the afferent Schaffercollateral pathway.

1. Introduction

Ketamine, a purported non-competitive glutamate N-methyl-D-aspartate (NMDA) receptor antagonist (Yamamura et al.,1990), is used in veterinary and human clinical anesthesia formore than 45 years (Domino, 2010). It is popular mainly dueto its analgesics properties (Adriaenssens et al., 1999;Michelet et al., 2007) and its importance for emergencyprocedures (Kuznetsova et al., 1984; Rice et al., 2010). How-ever, the use of ketamine has been associated with thedisruption of learning and with psychotic effects such asthe post-anesthetic delirium (Sussman, 1974; Irifune et al.,1991).

Alterations in synaptic efficacy in glutamatergic pathwaysare documented to play a key role in psychopathology(Garcia, 2002). Moreover, activity-dependent synaptic plasti-city is considered a cellular mechanism for learning andmemory (Bliss and Collingridge, 1993). Synaptic plasticityencompasses both short term plasticity, such as paired-pulse facilitation (PPF) and long-term forms of plasticity(Maruki et al., 2001) such as long-term potentiation (LTP),which is proposed to represent a neurophysiological trait oflearning and memory (Lynch, 2004). Hippocampal LTP ismostly dependent on the NMDA receptors (Lynch, 2004),which are considered the main molecular target of ketamine(Davies et al., 1988; Orser et al., 1997). Therefore, it is possiblethat ketamine may impair synaptic plasticity in the hippo-campus. Indeed, an earlier study suggested that dissociativeanesthetics including ketamine (30 mg/kg) abolished LTP inthe rat hippocampus in vivo (Stringer and Guyenet, 1983).However, it is unclear if ketamine selectively affects synapticplasticity rather than synaptic transmission, which wouldrequire comparing the effects of different concentrations ofketamine on synaptic transmission and on synaptic plasti-city. Furthermore, LTP has multiple (pre- and post-synaptic)expression mechanisms (Lynch, 2004), which is particularlyrelevant since it was reported that higher concentrations ofketamine (1000 mM) can decrease the amplitude of NMDApopulation spikes in CA1 hippocampal neurons induced bypaired-pulse stimuli (Wakasugi et al., 1999), a form of shortterm plasticity dependent on presynaptic mechanisms(Kamiya and Zucker, 1994; Zucker and Regehr, 2002). How-ever, it still remains to be established if clinically relevantconcentrations of ketamine indeed affect paired-pulse facil-itation (PPF) in the CA1 region of the hippocampus, whichwould be in agreement with the proposed localization andfunction of presynaptic NMDA receptors in the glutamatergicterminals in the hippocampus (Musante et al., 2011).

The purpose of this study was to evaluate the effect ofdifferent concentrations of ketamine on basal excitatory

synaptic transmission and on two forms of synaptic plasticity(LTP and PPF) in the CA1 area of mice hippocampus.

2. Results

2.1. Effects of ketamine on basal synaptic transmissionand on presynaptic volley amplitude

After 20 min of stable baseline recordings with aCSF, con-secutive application of increasing concentrations of ketamine(1, 3, 10, 30, 100 and 200 μM) did not modify (p40.05) synaptictransmission, as gauged by the lack of alteration of the fEPSPslopes (Fig. 2A). Only at the higher concentrations tested (300and 600 μM) did ketamine decrease synaptic transmission;thus the concentrations of 300 and 600 μM of ketamineinhibited the fEPSP slope by 22.275.3% and 48.177.9%,respectively (n¼4; po0.05) (Fig. 2A, B). It is worth noting thatthe inhibition of synaptic transmission by the higher con-centrations of ketamine was not completely eliminated afterwashout. In fact, following the washout of ketamine, thefEPSPs slopes were 10.672.8% lower than before ketamineadministration (n¼4; po0.05) (Fig. 2A).

Regarding, the effect of ketamine on the presynapticvolley amplitude, we observed that only higher concentra-tions of ketamine significantly decreased fiber volley ampli-tude: thus, 300 and 600 μM of ketamine inhibited theamplitude in 48.978.5% and 76.373.3%, respectively (n¼4;po0.05). This observation argues for a possible effect ofketamine on action potential propagation, in line with the

Fig. 2 – Effects of different concentration of ketamine on basal synaptic transmission. (A) Representative experiment showingthe time-course of the cumulative effects of increasing concentrations of ketamine on field excitatory postsynaptic potential(fEPSP) slope. (B) Concentration–response curve for cumulative inhibitory effects of ketamine fEPSP slopes using the averageresults from four experiments; in the ordinates 0% corresponds to the fEPSP slope before ketamine applications and 100%would represent the complete inhibition of fEPSPs. Significant inhibition of the fEPSP slope was observed with 300 and 600 μMof ketamine (*po0.05). (C) Superimposed fEPSPs showing the cumulative inhibitory effect of the higher concentrations ofketamine on fEPSP slope.

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reported ability of high concentrations of ketamine to affectvoltage-dependent sodium channels (Haeseler et al., 2003;Schnoebel et al., 2005). Accordingly, we observed that theinhibitory effect on fEPSP of the higher concentrations ofketamine (10.171.4% inhibition by 300 μM and 31.172.9%inhibition by 600 μM) was essentially unaltered (p40.05;n¼3) by 50 μM of the selective NMDA receptor antagonist(2R)-amino-5-phosphonovaleric acid, APV (12.171.3% inhibi-tion by 300 μM and 35.673.1% inhibition by 600 μM), whereasAPV (50 μM) did not affect basal synaptic transmission, aspreviously reported (Cunha et al., 2002) but abolished theinduction of long-term potentiation, as also previouslyshown (Harris et al., 1984).

2.2. Effects of ketamine on paired-pulse facilitation

As shown in Fig. 3, none of tested concentrations of ketaminesignificantly (p40.05) affected the paired-pulse facilitation ratio.

2.3. Effects of ketamine on LTP

Under control conditions, HFS effectively triggered LTP sinceit potentiated fEPSP slope to 225.3714.8% (n¼4) 6 min afterHFS (LTP induction) and caused a long-lasting increase ofsynaptic transmission with fEPSP being 143.578.3% larger60 min after HFS (LTP maintenance) compared to fEPSP slopebefore HFS (Fig. 4A). When present at a concentration of 3 μM,ketamine did not significantly (p40.05) modify either LTPinduction or LTP maintenance. However, at a concentration

of 10 μM, ketamine decreased significantly LTP inductionto 158.179.6% (n¼4; po0.01) and LTP maintenance to121.574.1% (po0.01). Moreover, LTP induction was comple-tely blocked by higher concentrations of ketamine, namely30 μM (100.672.4%, n¼4; po0.01) and 100 μM (100.570.8%,n¼4; po0.01) (Fig. 4B and C) and LTP maintenance was alsoabrogated by 30 μM of ketamine (101.370.8%, n¼4; po0.01)(Fig. 4B and D). The log IC50 of ketamine to inhibit LTPinduction (6 min after HFS) and LTP maintenance (60 minafter HFS) were �5.02770.015 and �5.00170.012, respec-tively, with Hill coefficient of �2.8570.42 and �4.1071.90,respectively, as calculated from the concentration–responsecurve shown in Fig. 4C and D.

3. Discussion

This work shows that clinically relevant concentrations ofketamine affect long-term potentiation (LTP) in aconcentration-dependent manner, without affecting paired-pulse facilitation (PPF). Moreover, basal excitatory synaptictransmission and presynaptic volley amplitude were affectedonly by the higher concentrations of ketamine (300 and600 mM).

The concentrations of ketamine tested in the currentstudy included subanesthetics and anesthetics concentra-tions in mice, encompassing plasma concentrations used inclinical human settings. Thus, published human plasmaconcentrations of ketamine for analgesia are around 0.55 mM

Fig. 3 – Effects of different concentration of ketamine on paired-pulse facilitation. (A) Time-course of the effects of increasingcumulative concentrations of ketamine on paired-pulse facilitation measured as a ratio of second pulse slope divided by firstpulse slope (inter-pulse interval of 50 ms). No differences were detected between groups (p40.05). (B) Superimposed fEPSPshowing the cumulative inhibitory effect of the higher concentrations of ketamine on first and second fEPSPs.

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and for anesthesia about 10 mM (White et al., 1980; Grantet al., 1983). In mice, anesthetic doses are around 100–200 mgkg�1 and the corresponding serum concentrations arebetween 30–60 mg ml�1 (Maxwell et al., 2006) or 100–200 mMof ketamine (molecular mass of ketamine hydrochloride:274.19 g mol�1). In humans, approximately 47% of ketamineis bound to plasma proteins and so does not diffuse to thesites of action (Dayton et al., 1983). However, ketamine ishighly lipid soluble and preferentially accumulates in thebrain (Cohen et al., 1973; Maxwell et al., 2006). Thus it ispossible that, during anesthesia, the concentration of keta-mine present in the brain is considerably higher than itsplasma concentration. With that in mind, we studied theeffects of a wide range of relevant concentrations of ketamine(1–600 mM) in hippocampal slices. However, the extrapolationof our experimental results to clinical human practicerequires extreme caution, as differences in drug concentra-tions and exposure times as well as interspecies variationsare all important variables which need to be considered.

Regarding the effects of ketamine on basal excitatorysynaptic transmission, our results showed no effects inducedby 1–200 mM of ketamine at 0.067 Hz. Only the higher con-centrations of ketamine (300 and 600 mM) decreased basalexcitatory synaptic transmission. These results are in agree-ment with the expected impact of ketamine on NMDAreceptors (Davies et al., 1988; Orser et al., 1997), which usuallydo not contribute to basal synaptic transmission and are onlyactivated under certain conditions (Herron et al., 1985; Coanet al., 1987). Indeed, we now directly confirmed that thepharmacological blockade of NMDA receptors did not modifythe inhibition of basal synaptic transmission by the higherketamine concentrations. Instead, the decrease observed inbasal synaptic transmission at the two highest concentra-tions of ketamine could be explained by an inhibition of the

propagation of action potentials since the presynaptic volleyamplitude was also significantly decreased by those twohighest concentrations of ketamine (300 and 600 mM), inaccordance with the reported impact of high ketamine con-centrations on voltage sensitive sodium channels (Haeseleret al., 2003; Schnoebel et al., 2005). Our findings contrast witha report that 10 and 100 μM of ketamine increased the fEPSPamplitude in hippocampal slices at 0.05 and 0.2 Hz in malerats with 7–8 weeks (Narimatsu et al., 2002), a discrepancythat may result from differences between species, age, sexand stimulation frequency.

We also observed that ketamine (1–600 μM) did not affectCA1 hippocampal paired-pulse facilitation. This form ofplasticity occurs through presynaptic mechanisms and itcan be used as a presynaptic index for probability of neuro-transmitter release (Kamiya and Zucker, 1994; Zucker andRegehr, 2002). In control conditions (i.e. in slices superfusedwith aCSF) the response to the second of a pair of stimulationpulses (with 10–50 millisecond inter-pulse intervals) is higherthan that of the first pulse. Residual presynaptic calcium, leftafter the first stimulation, is thought to enhance neurotrans-mitter release in response to the second stimulation (Kamiyaand Zucker, 1994). Thus, PPF increases after manipulationsthat reduce calcium-mediated glutamate release from theSchaffer-collateral-commissural pathway (e.g. Manabe et al.,1993), whereas manipulations that depress CA1 neuronfEPSPs via postsynaptic actions do not alter PPF (Manabeet al., 1993; Zucker and Regehr, 2002). Therefore, our findingssuggest that ketamine does not affect dynamic changes intransmitter release that are required for paired-pulse facil-itation at Schaffer collateral terminals. A previous studyreported that an higher concentration of ketamine (1000 μM)decreased the NMDA population spikes induced by paired-pulse stimuli in the rat hippocampus area CA1 (Wakasugi

Fig. 4 – Effects of ketamine on long-term potentiation (LTP) induced by high frequency stimulation (HFS, 100 pulses at 100 Hz).The control fEPSP slope (corresponding to 100%) was calculated by averaging the fEPSP during the 10 min baseline periodimmediately before HFS. (A) Time-course of the effects of different concentrations of ketamine (ket, 3–100 lM) on LTPamplitude, measured as changes in the slope of the field excitatory postsynaptic potential (fEPSP) response. Eachconcentration of ketamine was applied during the 90 min, 30 min before HFS and 60 min after HFS. Data are the mean valuesof 4 experiments per experimental group. (B) Superimposed fEPSP representative of the inhibitory effect of differentconcentrations of ketamine on LTP: (a) fEPSP just before HFS; (b) fEPSP immediately after HFS; (c) fEPSP 60 min after HFS. (C)Concentration–response curve for LTP induction where the average of fEPSP slope of the first 6 min after HFS was taken as ameasure of LTP induction. Data are mean7SD; *po0.01. (D) Concentration–response curve for LTP maintenance where theaverage of fEPSP slope from 54 to 60 min after HFS was taken as a measure of maintenance of LTP. As 100 μM of ketamineblocked completely the LTP induction, LTP maintenance was not analyzed for this concentration. Data are mean 7SD;*po0.01.

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et al., 1999). However, no studies were found regarding theeffect of clinically relevant concentrations of ketamine in PPFin the hippocampus CA1 region.

Regarding the effect of ketamine on LTP, a long-lastingchange in synaptic efficacy at Schaffer collateral-CA1synapses, we observed that ketamine decreased/inhibitedthe induction and maintenance of LTP in the CA1 region ofthe hippocampus. This effect was concentration dependentand concentrations above 30 μM of ketamine completelyblocked the induction of LTP. This probably results from theability of ketamine to inhibit NMDA receptors (Davies et al.,1988; Orser et al., 1997), which are critically required to for theinduction of LTP in Schaffer fibers-CA1 pyramid synapses(Harris et al., 1984). Accordingly, we noted a pronouncedinhibitory effect of ketamine in the early phase of LTP

induction (up to approximately 6 min after HFS), which isconsistent with the hypothesis that the presence of ketaminedecreased/blocked in a concentration-dependent manner theNMDA receptor-dependent induction of LTP and, conse-quently, the maintenance of LTP (60 min after HFS) was alsoaffected. Our current ex vivo study in slices is in agreementwith a previous in vivo study showing that dissociativeanesthetics, including ketamine, abolish hippocampal LTPin rats (Stringer and Guyenet, 1983).

Overall, the present results are in agreement with theprevalent view that ketamine is mainly an NMDA receptorantagonist (Davies et al., 1988; Yamamura et al., 1990; Orseret al., 1997). In fact, at clinically relevant concentrations,ketamine was mostly effective to inhibit NMDA receptor-dependent processes such as LTP, whereas it was ineffective

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to control synaptic processes that are NMDA receptor-independent processes such a basal synaptic transmissionand PPF in hippocampal slices. This is also in agreement withthe ability of ketamine to depress NMDA receptor-dependenthippocampal LTD in vivo (Desmond et al., 1991) and theNMDA component of the EPSP upon kindling in the dentategyrus (Racine et al., 1991). However, ketamine is a drugknown to potentially affect several different molecular tar-gets (Sinner and Graf, 2008). For instance, it was recentlyreported that ketamine can inhibit HCN1 channels (Chenet al., 2009) and the behavioral anesthetic-like effects ofketamine are reduced in mice with an inducible eliminationof HCN-1 in forebrain neurons (Zhou et al., 2013), as they arein NMDA receptor subunit 1 (NR1) knockout mice (Petrenkoet al., 2004). However, it is unlikely that HCN-1 channels areinvolved in the reported effects of ketamine since HCN-1blockade enhances LTP (rather than inhibiting LTP asobserved for ketamine) (Nolan et al., 2004) and ketaminedepresses hippocampal-dependent memory (Pitsikas et al.2008) as well as epilepsy (Gaspard et al., 2013), whereas HCN-1 constrain learning and memory (Nolan et al., 2004) andhippocampal HCN-1 are down-regulated in epilepsy(McClelland et al., 2011). It is however possible that HCN-1channels contribute for the effect of higher concentrations ofketamine on basal synaptic transmission since this effect isNMDA receptor-independent. An alternative would be aninhibitory effect of ketamine on voltage-sensitive sodiumchannels (Schnoebel et al., 2005), which would be compatiblewith the parallel reduction of fEPSPs and presynaptic volleysand is expected to only occur at high concentrations sinceketamine does not elicit a phasic blockade of sodium chan-nels at lower concentrations (Haeseler et al., 2003).

In conclusion, we have shown that ketamine decreasedlong-term potentiation in CA1 region of the mouse hippo-campus without affecting paired-pulse facilitation. Moreover,only higher concentrations of ketamine affected basal exci-tatory synaptic transmission, which could be a consequenceof a decrease of neuronal activity. These findings allowdissociating the blockade of LTP from a reduced synapticinput in ex vivo conditions. However, we need to keep in mindthat ketamine may display effects on synaptic transmissionand LTP in vivo that may not be recapitulated in an ex vivosetting.

4. Experimental procedure

All procedures were ethically approved by Direcção Geral deAlimentação e Veterinária (Lisbon, Portugal), the Portuguesecompetent authority for animal protection.

4.1. Hippocampal slice preparation

All experiments were performed on hippocampal slices from5–6 months old females BALB/c mice, except the pharmaco-logical testing of the impact of NMDA receptor blockade thatwas tested in 3 CD1 mice. Females were chosen since theyhave been shown more sensitive than males to the psycho-tomimetic and neurotoxic effects of ketamine (Jevtovic-Todorovic et al., 2001). The animals were euthanized by

cervical dislocation followed by decapitation, the brain wasrapidly removed and the hippocampi dissected free in ice-cold artificial cerebrospinal fluid (aCSF) of the followingcomposition (mM): NaCl 124, KCl 3, NaH2PO4 1.25, glucose10, NaHCO3 26, MgSO4 1, CaCl2 2, gassed with 95% O2 and 5%CO2 (pH¼7.4). Slices (400 μm thick) were cut perpendicular tothe long axis of the hippocampus with a McIlwain tissuechopper (Mickle Laboratory Engineering Co Ltd, Guildford, UK)and maintained for at least 60 min in gassed aCSF solution atroom temperature (23–251).

4.2. Electrophysiological recordings

A single slice was placed in a submerged recording chamber(1 mL capacity) and superfused at a rate of 3 mL/min with aCSFcontinuously bubbled with 95% O2 and 5% CO2, which wasmaintained at a constant temperature of 32.070.1 1C. Electricalstimulation was applied through a bipolar tungsten electrodeplaced over the Schaffer collateral/commissural fibers. Stimula-tion was applied through a constant current output unit (GrassPhotoelectric Stimulus Isolating Unit 6), connected to a GrassS44 stimulator. The duration of the stimulus was 0.1 ms and itsintensity was adjusted to evoke a field excitatory postsynapticpotential (fEPSP) with 40–50% of their maximal amplitude. Thestimulus range was 4–6mA. Under basal conditions, the stimuliwere delivered at a frequency of 0.067 Hz. Evoked fEPSPs wererecorded from the stratum radiatum layer of the hippocampusCA1 area (Fig. 1) using a glass micropipette filled with 4 M NaCl(2–5 MΩ resistance). Signals were amplified 1000-fold, andfiltered below at 5 Hz and above at 3 kHz, using an ISO-80amplifier (World Precision Instruments), and digitally recordedat 10 kHz using a ADC-42 analog-to-digital converter (PicoTechnologies) connected to a Pentium-based PC system run-ning the 1.3 version of the WinLTP program (Anderson andCollingridge, 2001). All data were stored as averages of eightconsecutive responses. Offline analysis was performed with theWinLTP program without any anti-aliasing/offline filtering, andresponses were quantified as the initial slope of the averagefEPSPs. Additionally, the amplitude of fiber volley was alsomeasured when studying basal synaptic transmission.

To access paired pulse facilitation (PPF), two consecutivepulses were applied with a 50 ms inter-pulse interval. PPFquantification was carried out as the ratio of the fEPSP slopesof the second response over that of the first response in eachpair of stimuli.

Long term potentiation (LTP) was induced with a highfrequency stimulation train (HFS; 100 pulses at 100 Hz) 30 minafter establishing a stable baseline at 0.067 Hz. LTP inductionwas quantified as the ratio of averaged fEPSP slopes of thefirst 6 min after HFS over the averaged fEPSP slopes duringthe 10 min before HFS and LTP maintenance was quantifiedas the ratio of the averaged fEPSP slope from 54 to 60 minafter HFS over the averaged fEPSP slope 10 min before HFS.

4.3. Drugs used and their administration

Ketamine (2-[2-chlorophenyl]-[1-methylamine]-cyclohexa-none) hydrochloride solution (Ketalars) was obtained fromPfizer Company (Porto Salvo, Oeiras, Portugal) and was dilutedin aCSF to obtain the desired concentration. For paired-pulse

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facilitation (PPF) and basal synaptic transmission experi-ments, each slice was cumulatively exposed to increasingconcentrations of ketamine (1, 3, 10, 30, 100, 200, 300 and600 μM). When we tested the ability of the NMDA receptorantagonist (2R)-amino-5-phosphonovaleric acid (APV) toinfluence the effects of ketamine, we first carried out thesequential application of the two highest concentrations ofketamine (300 and 600 μM) and, after washout, we superfusedAPV (50 μM) and re-tested the impact of ketamine (300 and600 μM) in the presence of 50 μM APV. For LTP experiments,each slice was exposed to only one concentration of ketamine(3, 10, 30 or 100 μM) during 90 min (30 min before HFS and60 min after HFS). Slices of the control group were superfusedonly with aCSF.

4.4. Statistical analysis

All data (effects of different concentrations of ketamine on basalsynaptic transmission, presynaptic volley amplitude, PPF, LTPinduction and LTP maintenance) were analyzed using the non-parametric Kruskal–Wallis test followed by Dunn's multiplecomparison test, using SPSS 19 for Windows (IBM Corporation,Armonk, NY), except the impact of APV on the on the inhibitoryeffects of ketamine on basal synaptic transmission, which wereanalyzed with a paired Student's t test. The logarithm of theconcentration of ketamine that produces 50% of maximalinhibition (log IC50) was calculated by fitting our data to a fourparameter Hill equation (Y¼Bottomþ(Top-Bottom))/(1þ(10\widehat Log EC50/10 \widehat X) \widehat Hill Slope), withnon-weighted samples, and with the following constraint:bottom¼100% (to LTP induction and LTP maintenance). Non-linear regression analyses were done using the GraphPad-Prismsoftware version 5.0 (GraphPad Software, Inc., San Diego, CA). Allresults are expressed as mean 7SD. Pr0.05 was consideredstatistically significant.

Conflict of interest

None declared.

Author's contribution

P.R.: Collection and interpretation of data and writing of thefirst draft of the paper.

A.T.: Study design and revision of the manuscript.H.S.: Data collection and data analysis.R.C.: Critical revision of the manuscript with important

intellectual content.L.A.: Study design, revision of the manuscript and final

approval of the submitted version.

Acknowledgments

This work was supported by Fundação para a Ciência e aTecnologia (FCT) and co-funded by COMPETE (01-0124-FEDER-009497), through the Project Grants PTDC/CVT/099022/2008 and

PTDC/SAU-NSC/122254/2010 and through a personal Ph.D.Grant (SFRH /BD/48883/2008) to Patrícia O. Ribeiro.

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