Neurobiology of Learning and Memory 110 (2014) 47–54

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Neurobiology of Learning and Memory

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

Forebrain glycine transporter 1 deletion enhances sensitivity to CS–USdiscontiguity in classical conditioning

http://dx.doi.org/10.1016/j.nlm.2014.01.0141074-7427/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Legacy Research Institute, 1225 NE 2nd Avenue,Portland, OR 97232, USA. Fax: +1 503 413 5465.

E-mail addresses: byee@downeurobiology.org, yee@hotmail.ch (B.K. Yee).1 Contributed equally.

Philipp Singer a,b,1, Sylvain Dubroqua a,b,1, Benjamin K. Yee a,b,⇑a Laboratory of Behavioural Neurobiology, Swiss Federal Institute of Technology (ETH) Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerlandb Legacy Research Institute, 1225 NE 2nd Avenue, Portland, OR 97232, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 January 2013Revised 15 January 2014Accepted 20 January 2014Available online 27 January 2014

Keywords:Conditioned freezingGlyT1MemoryNMDA receptorSLC6A9Trace conditioning

The deletion of glycine transporter 1 (GlyT1) in forebrain neurons can apparently strengthen Pavlovianaversive conditioning, but this phenotype is not expressed if conditioning followed non-reinforced pre-exposures of the to-be-conditioned stimulus (CS). To examine whether GlyT1 disruption may onlyenhance aversive associative learning under conditions that most favour the formation of CS–US excit-atory link, we evaluated the impact of GlyT1 disruption on the trace conditioning procedure wherebya trace interval between a tone CS and a shock US was introduced during conditioning. CS and US occur-rences were thus rendered discontiguous, which was expected to impede conditioning compared withcontiguous CS–US pairing. Conditioned freezing to the CS was measured in a retention test conducted48 h after conditioning. The genetic disruption significantly modified the temporal dynamics of the freez-ing response over the course of the 8-min presentation of the CS, although the immediate conditionedresponse to the CS was unaffected. The separation between ‘‘trace’’ and ‘‘no-trace’’ conditions was aug-mented in the mutant mice, but this only became apparent in mid-session; and the augmentation canbe attributed to the combined effects of (i) weaker conditioned freezing in the mutant relative to controlsubjects in the ‘‘trace’’ condition, and (ii) stronger conditioned freezing in mutants relative controls in the‘‘no-trace’’ condition. The demonstrated increased sensitivity to the effect of CS–US temporal discontigu-ity further highlights the importance of GlyT1-dependent mechanisms in the regulation of associativelearning.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction of NMDAR is implicated. GlyT1 inhibition has been attempted as a

Disruption or blockade of glycine transporter 1 (GlyT1) is aneffective way to elevate extracellular glycine levels in the vicinityof N-methyl-D-aspartate receptors (NMDARs) and thereby topotentiate the activation of NMDARs upon presynaptic release ofglutamate (Bergeron, Meyer, Coyle, & Greene, 1998). It is becausethe binding of glycine (or D-serine) to the glycine-B site on theNR1 subunit is required for NMDAR channel activation by gluta-mate stimulation (Kleckner & Dingledine, 1988). Importantly,glycine-B site occupancy is normally regulated by GlyT1s (co-expressed with NMDARs, Smith, Borden, Hartig, Branchek, & Wein-shank, 1992), which maintain synaptic glycine concentration atsub-saturation levels (Berger, Dieudonne, & Ascher, 1998; Berger-on et al., 1998; Supplisson & Bergman, 1997). GlyT1 is thereforea possible drug target for diseases in which a functional deficiency

therapy against the negative and cognitive symptoms of schizo-phrenia, which do not respond to current medication targetingdopamine D2 receptors (Javitt, 2009, 2012). Yet, the efficacy ofGlyT1-inhibiting drugs against the cognitive symptoms of schizo-phrenia remains controversial (Harvey & Yee, 2013) despite thepromise of negative symptoms alleviation in early clinical trials(Pinard et al., 2010).

Preclinical models are instrumental in defining the scope ofpossible treatment efficacy (Möhler et al., 2011). The most relevantmouse model to date involves the conditional deletion of GlyT1after birth by introduction of CaMKII-driven expression of Crerecombinase to delete the GlyT1 gene preferentially in forebrainneurons (Yee et al., 2006). A number of cognitive phenotypes havebeen reported in the GlyT1fl/fl:CaMKII-Cre+/� mutant mice acrossdifferent tests of learning and memory (Möhler et al., 2011). Evi-dence for enhanced learning has been reported across differentPavlovian paradigms (Yee et al., 2006) in which the mutants miceconsistently showed a stronger conditioned response (CR) to a con-ditioned stimulus (CS) that had previously been paired with anaversive unconditioned stimulus (US). This led to the authors’

48 P. Singer et al. / Neurobiology of Learning and Memory 110 (2014) 47–54

speculation that the genetic disruption enhanced the acquisitionand/or the memory strength of learned CS–US association. How-ever, this interpretation is unsatisfactory because it is not able toaccount for the lack of enhanced CR when conditioning was pre-ceded by repeated non-reinforced exposures to the to-be-condi-tioned CS (Yee et al., 2006). Hence, whether Pavlovianconditioning was enhanced or not is critically dependent on theconditions in which learning occur.

Indeed, the mutant mice were more sensitive to CS pre-expo-sure – exhibiting weaker CR compared to mutant mice withoutCS pre-exposure (i.e., giving rise to latent inhibition, Lubow &Moore, 1959), even though the number of CS pre-exposures wasinsufficient to weaken CR expression in the controls (i.e., they didnot show latent inhibition). Instead of referring to the conditionedfreezing phenotype as a broad strengthening of CS–US associativelinks, a more fitting description may involve modifications to thecognitive processes that govern the selective acquisition and/orexpression of Pavlovian associations. Selectivity of learning is cen-tral to all theories of associative learning (e.g. Mackintosh, 1975;Pearce & Hall, 1980; Wagner, 1981), without which the adaptivevalue of associative learning would be seriously undermined.

Here, we tested whether the conditional GlyT1 deletion wouldenhance the animals’ sensitivity to another critical parameter ofCS–US associability, namely, the temporal proximity between theCS and the subsequent US. Pavlov (1927) was the first to describethat CR is weaker when CS and US are interspersed by a temporalgap, referred to as the ‘trace’ interval, during conditioning. By con-trast, CS–US learning is favoured when the US follows the CS clo-sely in time (i.e., without any CS–US trace interval). A linkagebetween the expression of latent inhibition and the trace condi-tioning effect has been justified on theoretical grounds (Ayres, Al-bert, & Bombace, 1987; DeVietti, Bauste, Nutt, & Barrett, 1987) andsupported by experimental evidence. Specifically, latent inhibitionand the trace conditioning effect are similarly disrupted by sys-temic amphetamine (Norman & Cassaday, 2003; Weiner, Lubow,& Feldon, 1988) and selective disruption of hippocampal a5GABA-A receptors (Gerdjikov et al., 2008; Yee et al., 2004).

The present study tested if GlyT1fl/fl:CaMKII-Cre+/� mutant micewith a phenotype of enhanced sensitivity to the non-reinforcedhistory of a potential CS (Yee et al., 2006) might also express a phe-notype of increased sensitivity to CS–US temporal discontiguity. Ifso, the differential expression of CR acquired under the trace andno-trace conditions would be larger in the mutant than controlmice.

2. Materials and methods

2.1. Subjects

A homozygous Glyt1tm1.2 fl/fl colony was established andmaintained on a pure C57BL/6 background as described before(Yee et al., 2006). Forebrain neuron specific deletion of GlyT1was achieved by CamKIIaCre-mediated recombination of the flo-xed GlyT1 allele. Appropriate heterozygous Cre mice were matedwith Glyt1tm1.2 fl/fl mice to generate the desired mutant and con-trol littermates. Animals of both sexes were employed in the pres-ent study. The mice were weaned at 21 days old, and littermates ofthe same sex were kept in groups of four to six in Makrolon Type-III cages (Techniplast, Milan, Italy). The subjects were housed in atemperature- and humidity-controlled (at 22 �C and 55% R.H.) ani-mal vivarium under a reversed light-dark cycle with lights off from07.00 to 19.00 h. Testing was always conducted in the dark phaseof the cycle. The animals were maintained under ad libitum waterand food (Kliba 3430, Klibamuhlen, Kaiseraugst, Switzerland)throughout the study. All procedures described here had been

previously approved by the Cantonal Veterinary Office of Zurich,which conformed to the ethical standards stipulated in the SwissFederal Act on Animal Protection (1978) and Swiss Animal Protec-tion Ordinance (1981) in accordance with the European CouncilDirective 86/609/EEC (1986). All efforts had been made to alleviateanimal suffering and minimize the number of animals used.

2.2. Trace conditioning in the conditioned freezing paradigm

Here, expression of the trace conditioning effect was measuredusing the conditioned freezing paradigm based on establishedparameters known to induce a robust trace conditioning effect inmice (Yee et al., 2004). The apparatus consisted of two sets of fourconditioning chambers. The two sets were distinct from each other,and were installed in separate testing rooms, providing two dis-tinct contexts as fully described before (Yee et al., 2006). The firstset of chambers (context ‘A’) comprised four Coulbourn Instru-ments (Allentown, PA, USA) operant chambers (Model E10-10),each equipped with a grid floor made of stainless steel rods(4 mm in diameter) spaced at an interval of 10 mm centre to cen-tre, and through which scrambled electric shocks (the US, set at0.3 mA) could be delivered (model E13-14; Coulbourn Instru-ments). A transparent Plexiglas enclosure confined the animals toa rectangular region (17.5 � 13 cm). The inside of the chamberswas illuminated by a house light (2.8 W) positioned on the panelwall, 21 cm above the grid floor. The second set of chambers (con-text ‘B’) comprised four cylindrical (19 cm in diameter) enclosuresmade of clear Plexiglas resting on a metal mesh floor. Illuminationinside the chamber was provided by an infrared light source in-stead of visible light. The CS was an 86-dBA tone provided by a son-alert (model SC628; Mallory, Indianapolis, IN, USA). Each of theeight chambers contained a miniature digital camera mounted30 cm directly above the centre of the area of interest. The algo-rithm of the freezing response detection procedure has been vali-dated and fully described before (Richmond et al., 1998). Adetailed description of the trace conditioning procedure has beenpublished elsewhere (Yee et al., 2004). The subjects of each groupwere randomly allocated into one of two conditions of CS–US pair-ing differing in the (trace) interval between CS offset and US onset:either 0 or 20 s (‘no-trace’ and ‘trace’ conditions, respectively). Therespective group sizes were as follows: no-trace condition:mutants ¼ 12ð6$ þ 6#Þ, controls ¼ 6ð8$ þ 8#Þ; trace condition:mutants ¼ 13ð7$ þ 6#Þ, controls ¼ 17ð10$ þ 7#Þ. On day 1, theanimals were given three trials of CS–US pairings (CS: 30 s, US:1 s) in context A. Each trial was preceded and followed by a120 s inter-trial interval. 24 h later, the CR to the conditioning con-text was evaluated by returning the animals to context A for a per-iod of 8 min. Another 24 h later, conditioned freezing to the tone CSwas assessed in the neutral context B. The test session began with a2 min acclimatization period, followed by the presentation of theCS for 8 min; and then the CS was turned off and the animals leftin the chamber for an additional 2 min.

2.3. Statistical analysis

All data were analysed by parametric analysis of variance (AN-OVA) with the between-subject factors genotype, sex and trace,which referred to the CS–US trace interval (‘‘trace’’ vs. ‘‘no-trace’’)employed during conditioning. Repeated measures factor withthe inclusion of the within-subject factor time bins was performedin the analysis of data obtained on the CS-test day to provide atemporal profile of the phenotype. Fisher’s Least Significant Differ-ence (LSD) post hoc pair-wise comparisons and planned contrastanalysis were performed to assist interpretation of statistically sig-nificant effects emerged from an overall ANOVA. In addition, sup-plementary restricted ANOVAs and one-sample t-tests were

P. Singer et al. / Neurobiology of Learning and Memory 110 (2014) 47–54 49

applied to subsets of data. All statistical analyses were carried outusing SPSS for Windows (version 19, SPSS Inc., Chicago IL, USA)implemented on a PC running the Windows 7 operating system.

3. Results

3.1. Conditioning (day 1)

Freezing was operationally defined as the number of secondsthe animals were observed as immobile and expressed as percenttime freezing within specific periods: CS, pre-CS and trace. Thethree variables were separately analysed. Supplementary analysesrestricted to animals exposed either to the ‘no-trace’ or the ‘trace’condition on day 1 were also performed to allow the comparison offreezing between distinct periods.

3.1.1. CS periodsThe level of freezing during the tone-CS presentation increased

progressively across the three CS–US trials (Fig. 1A), but this incre-ment was noticeably weaker in the ‘trace’ relative to the ‘no-trace’condition, reaching a maximal difference on the third CS presenta-tion. These impressions were confirmed by a 2 � 2 � 2 � 3 (geno-type � trace � sex � trials) split-plot ANOVA of percent timefreezing, which revealed a significant main effect of trials[F(2,100) = 35.38, p < .01], trace [F(1,50) = 10.39, p < .01], and their2-way interaction [F(2,100) = 8.03, p < .01]. Post hoc pair-wise com-parison confirm that the difference between trace and no-traceconditions (collapsed across genotypes and sex) only achievestatistical significance on the third trial [t(100) = 5.66, p < .01].Incidentally, male mice generally exhibited stronger freezing thanfemale mice regardless of genotype and training condition, yieldinga statistically significant sex effect [F(1,50) = 12.41, p < .01] but notany of its interactions. The average time of freezing in the presenceof the CS on day 1 was 7.3 ± 1.4% in the female and 14.4 ± 1.5% inthe male.

3.1.2. Pre-CS periodsFreezing levels during the 30 s preceding the CS similarly in-

creased across the three Pre-CS periods (Fig. 1B) in a manner thatwas essentially independent of genotype, training condition orsex. The increase in Pre-CS freezing likely reflected a general in-crease in fear due to the shock US rather than being specificallyattributable to the CS itself. A 2 � 2 � 2 � 3 (geno-type � trace � sex � Pre-CS periods) ANOVA of percent time freez-ing only yielded a significant main effect of Pre-CS periods[F(2,100) = 73.71, p < .01].

3.1.3. Trace intervalsThis variable refers to the freezing levels recorded during the

20 s trace intervals, and was therefore only available in animalstested under the trace condition. Again, a monotonic increase infreezing was observed across the three trace intervals in animalstested under the trace condition (Fig. 1C). Similar to the analysisof freezing recorded in the CS-periods above, a sex differencewas observed with the male freezing at 24.5 ± 2.8% vs. female at15.5 ± 2.5% on average. These led to the emergence of a significantmain effect of trace intervals [F(2,52) = 34.94, p < .01] and sex[F(1,26) = 5.79, p < .05] from the 2 � 2 � 3 (genotype � sex � traceintervals) ANOVA of percent time freezing.

3.1.4. Comparison between variablesIn the no-trace condition, a 2 � 2 � 3 � 2 (genotype � sex � tri-

als � dependent variables) ANOVA comparing CS and Pre-CS freez-ing variables only yielded an effect of trials as expected[F(2,48) = 53.79, p < .001]. And, there was no statistical indication

that the animals froze differentially between the pre-CS and CSperiods (see Fig. 1A and B).

In the trace condition, a 2 � 2 � 3 � 3 (genotype � sex � tri-als � dependent variables) ANOVA yielded a significant effect ofvariables [F(2,52) = 20.35, p < .001] as well as its interaction with tri-als [F(4,104) = 10.75, p < .001] besides the expected main effect of tri-als [F(2,52) = 62.97, p < .001]. Freezing during the CS periodexhibited by animals trained under the trace condition became vis-ibly lower than that observed in the Pre-CS and/or trace periods asconditioning progressed from trials 1 to 3 (Fig. 2). Notably, CS-freezing increased at the slowest pace, leading to a clear diver-gence from Pre-CS and trace interval freezing by the third trial.In trials 2 and 3, therefore, the transition from the Pre-CS to theCS periods was associated with a drop in freezing [F(1,26) = 16.99,p < .001 based on a restricted ANOVA] whereas the transition fromthe CS period to the succeeding trace interval corresponded to a re-bound in freezing [F(1,26) = 65.46, p < .001 based on a restricted AN-OVA]. The CS thus appeared to disrupt the on-going expression offreezing (indexed by Pre-CS freezing) on trials 2 and 3 on day 1,suggesting that the animals undergoing trace conditioning mighthave learned to perceive the CS as a safety signal. Although theaverage time freezing was numerically the highest during the traceintervals, a separate restricted ANOVA did not reveal a significantdifference between the freezing levels obtained in the Pre-CS peri-ods and trace intervals across trials 2–3 [p = .071]. Hence, the ani-mals seemingly did not substantially differentiate between the30 s before the CS from the 20 s following the CS (when the phys-ical conditions of the test chambers were essentially identical) –i.e., the animals did not perceive the 20-s trace interval as a betterpredictor of the shock US compared with the pre-CS period.

3.2. Conditioned context freezing (day 2)

The expression of conditioned freezing developed towards thetraining context was evaluated 24 h after conditioning by re-exposing the animals to the training context in the absence ofany discrete CS (Fig. 3). No difference was apparent between geno-types, sexes or training conditions. A 2 � 2 � 2 (geno-type� sex� trace) ANOVA of percent time freezing over the 8-mintest period did not reveal any significant outcomes.

3.3. Conditioned CS freezing (day 3)

Two days after conditioning, the CR to the tone CS in the form offreezing was measured in a neutral context. The animals wereacclimatized to the new context for 2 min (i.e., Pre-CS period) be-fore the CS was presented continuously for a period of 8 min (i.e.,CS-period). The test session was concluded with another 2-minin which the CS was absent (i.e., post-CS period).

3.3.1. Pre-CS periodAs shown in Fig. 4A, the general level of spontaneous freezing

(immobility) was initially low but a gradual increase over timewas visible. This increase was similarly observed in both genotypesregardless of training conditions (i.e., trace vs. no-trace). However,a sex difference was detected as male mice generally exhibiteda higher level of spontaneous freezing (mean ± SE: male =14.7 ± 1.7% vs. female = 9.6 ± 1.6%) regardless of genotype andtraining conditions. These impressions were confirmed by a2 � 2 � 2 � 4 (genotype � sex � trace � 30-s bins) ANOVA of per-cent time freezing during the Pre-CS period, which yielded a signif-icant effect of bins [F(3,150) = 19.71, p < .001] and sex [F(1,50) = 4.78,p < .05]. No other effects achieved statistical significance.

50 P. Singer et al. / Neurobiology of Learning and Memory 110 (2014) 47–54

3.3.2. CS PeriodPresentation of the CS predictably led to a rapid increase in

freezing observed in animals previously conditioned under theno-trace condition. By contrast, the increase was modest in animalsconditioned under the trace condition (Fig. 4B). This difference con-stitutes the trace conditioning effect; and its expression was visiblycomparable between mutants and controls over the first 2 min. Thedata were examined with a finer temporal resolution across 10-sbins over the first minute of the CS as shown in Fig. 4D. This showsthat the most rapid rise in freezing was seen in the no-trace groups,and it was completed over the course of the first 30 s, peaking at atime that corresponded to the onset of the US (i.e., 30 s into the CS)on the conditioning day.

Next, mutants and controls began to diverge three minutes intothe CS presentation (Fig. 4B). The size of the trace effect, as illus-trated by the stronger freezing in the no-trace condition relativeto the weaker freezing in the trace condition, was significantly lar-ger in the mutant mice. This genotype effect remained detectableat 7 min into the CS period. A 2 � 2 � 2 � 16 (geno-type � sex � trace � 30-s bins) ANOVA revealed a significant geno-type by trace interaction [F(1,50) = 4.60, p < .05] and the temporaldependency of this effect [genotype � trace � bins: F(15,750) = 1.84,p < .05]. These genotypes effects were accompanied by the signifi-cant effect of trace [F(1,50) = 33.22, p < .01] and bins [F(15,750) = 4.73,p < .05].

The significant three-way interaction was further investigatedand characterized by pair-wise comparisons and planned contrastanalysis (see Fig. 4B). First, a significant trace effect was detectablein the mutants throughout the 8-min CS-period. Second, the traceconditioning effect in the controls became weak in the middle ofthe CS-period. Pairwise comparisons between control/trace andcontrol/no-trace animals failed to yield a significant difference inCS bins 7, 9–11 and 13–14. Third, as a result, the genotype effect(in the form of the genotype by trace interaction) was only appar-ent in the middle of the CS-period. The interaction of interest isidentical to the contrast [(control/trace – control/no-trace animals)– (mutant/trace – mutant/no-trace animals)], i.e., with contrastcoefficients (+1, �1, �1, +1). This specific contrast was significantfrom the third to the seventh minutes into the CS period exceptat bin 12 (see Fig. 4B). The temporal dependent profile of this phe-notype is consistent with the lack of a genotype by trace interac-tion when the analysis was restricted to the first one [F = .001,ns] or two minutes [F = .16, ns] of the CS-period. Last, plannedcomparisons were performed to examine whether the phenotypeon trace conditioning was associated with bidirectional effects in

Fig. 1. Development of conditioned freezing across three CS–US pairings: (A) freezing bpresentation and is depicted for the no-trace (open symbols) and the trace (filled symbolweaker than in the no-trace condition – an effect that emerged over the three conditibehaviour during the 30 s immediately preceding each CS presentation is shown as a funanimals tested under the trace condition only, freezing levels during the 20-s trace inte

freezing between the trace and the no-trace conditions. Sugges-tions for stronger freezing in the mutant in the no-trace conditionwas obtained in bins 7–8 and 10–11, and for weaker freezing in themutant in the trace condition was obtained in bins 5–6 and 8–10(see Fig. 4B).

3.3.3. Post-CS PeriodExamination of the post-CS period clearly showed a sharp in-

crease in freezing that was most notable in animals trained underthe trace condition. Although an increase was visible also in theanimals trained under the no-trace condition, it was substantiallyweaker. The immediate response to the CS-offset was examinedby an analysis of a difference score indexing the elevation of freez-ing levels from the last 30-s bin of the CS period to the first 30-s binof the post-CS period (Fig. 4E). A 2 � 2 � 2 (genotype � sex � trace)ANOVA of the difference score revealed a main effect trace, consis-tent with the impression that animals trained under the trace con-dition responded more strongly to the termination of the CS[F(1,50) = 5.60, p < .05]. One-sample t-tests further indicated thatonly the mean difference score obtained from the two trace groupsdiffered significantly from zero.

The levels of freezing observed in the initial 30 s of the post-CSperiod (i.e., first post-CS bin in Fig. 4C) did not differ substantiallybetween groups. Subsequently, the animals trained under the tracecondition maintained a level of freezing that was substantiallyhigher than animals trained under the no-trace condition (i.e., overthe last 3 bins in Fig. 4C). In other words, the separation betweentrace and no-trace conditions now took a form that was the reverseof that seen in the CS-period. This may be expected if the trace ani-mals rather than the no-trace animals had perceived the with-drawal of CS itself as a predictor of shock US. A 2 � 2 � 2 � 4(genotype � sex � trace � 30-s bins) ANOVA confirmed the pres-ence of a highly significant trace effect [F(1,50) = 8.19, p < .01] aswell as its interaction with bins [F(3,150) = 3.06, p < .05] and themain effect of bins [F(3,150) = 29.04, p < .001].

4. Discussion

The present study clearly demonstrated that the expression ofthe trace conditioning effect over time was modified by forebrainneuronal deletion of GlyT1. This phenotype revealed in theGlyT1fl/fl:CaMKII-Cre+/� mutant mice was specific to the extinctiontest of CS freezing, because neither acquisition performance (onday 1) nor the expression of context-freezing (on day 2) was signif-icantly modified by the genetic disruption (Figs. 1–3). The 2-min

ehaviour is indexed by the percentage of time freezing during the 30 s of tone-CSs) conditions separately. Freezing during the CS observed in the trace condition wasoning trials – and was comparable between genotypes. (B) Expression of freezingction of trials. This was comparable between test conditions and genotypes. (C) Forrvals are depicted separately. Error bars refer to ±SEM.

Fig. 2. A general increase in freezing was observed in all three measures (Pre-CS, CSand trace periods) in animals trained under the trace conditioning procedure on day1. The rate of increase across conditioning trials 1–3, however, differed between thethree variables. Notably, CS-freezing progressed at the slowest pace, leading to aclear divergence from Pre-CS and trace interval freezing by the third trial. In trials 2and 3, therefore, we saw a drop of freezing from Pre-CS to the CS period, followed bya rebound when the CS had ended and the trace interval began. Error bars refer to±SEM.

Fig. 3. Test of context freezing: The freezing response induced by the trainingcontext 24 h after conditioning was comparable amongst groups. Error bars refer to±SEM.

P. Singer et al. / Neurobiology of Learning and Memory 110 (2014) 47–54 51

acclimatization at the beginning of the test session of CS-freezing(on day 3), that preceded the onset of the CS, was uneventful.The impact of CS–US discontiguity (i.e., the trace effect) emergedas soon as the CS was presented (Fig. 4B), and the separation be-tween trace and no-trace groups was highly comparable betweengenotypes over the first two minutes in the presence of the CS.Essentially, no phenotype was evident in this period because thefreezing behaviour observed was indistinguishable between mu-tant and control mice belonging to the same training conditionduring these two minutes. The observation that GlyT1fl/fl:CaMKII-Cre+/� mutant mice displayed a stronger trace conditioning effectonly became detectable afterwards. Hence, the trace conditioningphenotype emerged over time, even though the average perfor-mance over the 8-min CS period gave an impression that the traceconditioning was significantly enhanced in the mutant mice.Post-hoc analyses confirmed that this phenotype was only robustlyexpressed in the mid-session, and indicated that the temporaldependency of this novel phenotype must be addressed.

4.1. The contribution of extinction over the extended CS presentationin the CS-test

Because the phenotype did not emerge immediately as the CSwas presented, it is important to consider the potential contribu-tion of extinction learning over the extended period of the CS pre-sentation. From this perspective, the enhanced freezing in themutants relative to the controls in the no-trace group may be

described as stemming from a retardation of extinction. A similarsuggestion has been made in the initial report of enhanced condi-tioned freezing in the GlyT1fl/fl:CaMKII-Cre+/� mice (Yee et al.,2006), which underwent Pavlovian conditioning procedures identi-cal to the no-trace condition here. Yet, two subsequent reports thatreplicated the enhanced conditioned freezing phenotype in GlyT1fl/fl:CaMKII-Cre+/� mice did not obtain any evidence for a concomitantresistance to extinction (Dubroqua, Boison, Feldon, Mohler, & Yee,2011; Dubroqua et al., 2010). This might undermine the interpre-tation that disruption of forebrain neuronal GlyT1 primarily affectsthe rate of extinction rather than the strength of the CS–USassociation.

Nevertheless, equally crucial to the interpretation of the traceconditioning phenotype here is that the extinction perspectivewould view the weaker freezing in the mutant/trace group (rela-tive to the control/trace group) as a facilitation of extinction.Hence, CS–US contiguity during acquisition can critically decidewhether the rate of extinction would be slowed down or speededup by the conditional deletion of GlyT1 in the forebrain. Here,the mutation effectively reversed the effect of CS–US contiguityon the rate of extinction seen in control mice (Fig. 4B).

4.2. The temporal perception of the CS

Common to most conditioned freezing experiments (e.g. Zeli-kowsky, Bissiere, & Fanselow, 2012), the expression of the condi-tioned response (i.e., freezing) was measured in a novel contextin which the CS was presented continuously for a period well be-yond the duration of the CS presented during acquisition. One con-sequence of this procedure is that the CS might not be perceived asidentical to the training CS when it was extended beyond 30 s inthe test. Indeed, generalization decrement is predicted when theCS differs in duration between acquisition and test (Gallistel & Gib-bon, 2000; Pavlov, 1927). One may therefore prefer to focus on thefirst 30 s of the CS presentation in the test session, because the datawould be free from the confounding impact of generalization dec-rement as well as of extinction learning. As shown in Fig. 4B, therewas no evidence to suspect that the expression of the trace condi-tioning effect had been modified by the GlyT1 disruption. Indeed,the mutant and controls mice (within either the no-trace or tracegroup) were highly comparable when the initial rise of the condi-tioned freezing response was tracked across 10-s bins (Fig. 4D).

However, any attempt to explain the emergence of the pheno-type later in the test as a consequence of an effect on generaliza-tion decrement resulting from the protraction of the CS isquestionable. First, it cannot predict the abrupt expression of thephenotype at three minutes into the CS. Second, the phenotypedid not become stronger gradually as the CS continued to extendover time; and if anything the phenotype was weaker by the endof the CS period (Fig. 4B). Hence, the temporal dynamics of thegenotype effects undermine the possibility that the phenotype pri-marily reflects the effects of the mutation on generalization decre-ment related to the temporal perception of the CS.

4.3. CS as a safety signal in trace conditioning

There was also evidence that the CS might be perceived as asafety signal on the conditioning day (day 1) in animals exposedto the trace conditioning procedure. By the third and final condi-tioning trial (but before shock US delivery), mutant and controlmice in the trace group appeared to respond to the CS with areduction in freezing by comparison to the pre-CS period immedi-ately before, and to the trace interval that immediately followedthe CS (Fig. 2). The rapid fall and rise of the freezing response isconsistent with the interpretation that the CS was perceived as asafety signal. Although this effect was not significantly stronger

52 P. Singer et al. / Neurobiology of Learning and Memory 110 (2014) 47–54

in the mutants on the conditioning day, its possible contribution tothe trace conditioning phenotype observed on the retention test onday 3 of the experiment is still worth considering.

As a safety signal, the CS might therefore function as an inhib-itor (see Bouton, 2007, p. 89) that prevents or reduces the expres-sion of conditioned freezing in the trace groups recorded on theretention test. Consequently, the stronger trace conditioning effectseen in the mutants might stem from a stronger inhibitory CS–USlink acquired by animals in the trace groups on the conditioningday. The possibility that conditional GlyT1 disruption promotedinhibitory as well as excitatory conditioning (Dubroqua et al.,2011; Yee et al., 2006) certainly warrants further investigation.Further experiments using paradigms that are specifically designedto measure the development of conditioned inhibition and safetysignal, such as [A+/AB�] (Rescorla, 1969) and [AX+/BX�] (Kazama,Schauder, McKinnon, Bachevalier, & Davis, 2013; Myers & Davis,2004) procedures, could clarify this interesting speculation.

4.4. Learning about the trace

Although trace conditioning is operationally defined as weakerconditioned response to the CS when the pairing between CS andUS during acquisition was interspersed by a trace interval (Pavlov,

Fig. 4. Test of CS freezing: The freezing response recorded during the CS-test session was(C). The data were presented as a function of successive 30-s bins. The significant three-wby planned contrast analysis and pair-wise comparisons based on the error variance assocompares between the mutants and controls the size of the trace conditioning effectsignificance [p < 0.05] are highlighted by the green underlay in B. Bins in which a significaare marked by ‘a’; and those in which a significant pair-wise difference between controlperiod is depicted as a function of 10-s bins to illustrate the evolution of the conditioned rfrom the last 30-s bin of the CS period to the first 30-s bin of the post-CS period is separchange from the end of CS-period to the beginning of the post-CS period) based on one-sato colour in this figure legend, the reader is referred to the web version of this article.)

1927), the animals trained under such conditions could also learnabout the contingency between the trace itself and US. Indeed,freezing in the trace groups showed a rapid increase as soon asthe CS was switched off (Fig. 4C), suggesting that they respondedto the offset of the CS. This response was well-above that expressedby the animals in the no-trace groups that had never experienced aCS–US trace interval (Fig. 4E). Although freezing generally de-creased over the rest of the post-CS period, the trace and no-tracegroups clearly diverged, such that the trace groups exhibited stron-ger freezing than the no-trace control groups regardless of geno-type. Indeed, the trace groups exhibited during the post-CSperiod their highest levels of freezing across the entire test session.

To the extent that this analysis has shown that animals trainedunder the trace conditions had responded to the CS–US trace inter-val as a potential predictor of the shock US, there was no evidencethat the disruption of GlyT1 had significantly altered the learningabout the trace interval.

4.5. Comparing the trace conditioning and latent inhibitionphenotypes of GlyT1 disruption

Importantly, we qualify here that Pavlovian learning is notalways enhanced (defined as a stronger CR specific to the CS) by

subjected to separate analyses: the Pre-CS (A), CS-period (B) and the post-CS periodsay interaction observed in the CS period was further investigated and characterized

ciated with the three-way interaction in the overall ANOVA. The contrast specificallyat each successive bin. Those time bins in which this contrast achieved statisticalnt pair-wise difference between control/no-trace and mutant/no-trace was detected/trace and mutant/trace was detected are marked by ‘b’. The first minute of the CS-esponse to the CS at a higher temporal resolution (D). The increase of freezing levelsately depicted (E). # Indicates a significant difference from zero (which indicates nomple tests. All data shown refer to mean ±SEM. (For interpretation of the references

P. Singer et al. / Neurobiology of Learning and Memory 110 (2014) 47–54 53

conditional GlyT1 disruption in forebrain neurons. Indeed, a re-versed effect can be induced by specific changes in training condi-tions, but the psychological significance of this qualificationremains to be fully appreciated and clarified. Under conditions thatfavour maximally the formation of the CS–US association with theCS being perceived unambiguously as a reliable and accurate pre-dictor of the eminent occurrence of the US, the magnitude of the CRtends to be stronger in the mutants. On the other hand, deviationfrom such ideal conditions that corrupts this positive CS–US pre-dictive link and thereby reduces CR, is seemingly more impactfulin our mutant mice. This description fits not only the trace condi-tioning phenotype demonstrated here but also the latent inhibitionphenotype previously reported (Yee et al., 2006).

In latent inhibition, non-reinforced CS pre-exposure (presenta-tion of CS alone, in the absence of any US) impedes the efficacyof subsequent CS–US pairing to produce a CR to the CS (Lubowand Moore, 1959). Mice with forebrain neuronal GlyT1 deletionare more sensitive to this CS pre-exposure effect: Latent inhibitionwas evident in the mutants when the amount of CS pre-exposurewas insufficient to generate latent inhibition in wild type controls(Yee et al., 2006). According to DeVietti et al. (1987), the similarsensitivity to the latent inhibition (i.e., CS pre-exposure effect)and trace conditioning procedures may stem from a common psy-chological mechanism. They suggest that CS pre-exposures prefer-entially reduce attention to later segments of individual CS’s,which then effectively act as a trace interval between initial CS seg-ments and the US during subsequent CS–US pairings. The per-ceived CS–US discontiguity thereby weakens conditioning andyields the LI effect (also see Ayres et al., 1987). Systemic amphet-amine (Norman and Cassaday, 2003; Weiner et al., 1988) as wellas selective disruption of hippocampal a5 GABA-A receptors (Ger-djikov et al., 2008; Yee et al., 2004) have been shown to reduce sen-sitivity to CS pre-exposure effect and CS–US discontiguity. Here,we extended these findings by showing that disruption of fore-brain neuronal GlyT1 could enhance both the sensitivity to CSpre-exposure effect and CS–US discontiguity. It remains to bedetermined whether these parallel or complementary effects ofthese three distinct brain manipulations may be mediated througha common neural circuitry.

4.6. Conclusion

Although further experiments are necessary to delineate theprecise psychological and molecular mechanisms underlying thenovel phenotype of GlyT1fl/fl:CaMKII-Cre+/� mutant mice, ourexperiment shows that the CS–US contiguity at the time of condi-tioning can critically determine how the genetic disruption mightmodify the expression of the acquired conditioned response. Thepresent study thus reveals important insights into the role of fore-brain neuronal GlyT1 in determining how past experience mayshape current behaviour through Pavlovian learning mechanism.

Acknowledgments

The present study was supported by Swiss National ScienceFoundation Grant (3100–066855) with additional support fromthe Swiss Federal Institute of Technology Zurich. BKY receivedadditional support from the National Institutes of Health(MH083973). SD and PS were recipients of a studentship fromthe Neural Plasticity & Repair – National Centre for Competencein Research (NCCR) funded by the Swiss National Science Founda-tion. We thank Prof. Hanns Möhler and Dr. Detlev Boison of ZurichUniversity for providing the breeding pairs for the generation ofthe GlyT1fl/fl conditioned mouse line. The software developmentand maintenance of the behavioural equipment were providedby the late Peter Schmid, to whom we would like to express our

deep gratitude for his excellent service over two decades. We arealso indebted to the animal husbandry staffs and to Dr. Joram Fel-don for making available the animal facilities and behaviouralequipment necessary for the reported experiments.

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