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Forebrain glycine transporter 1 deletion enhances sensitivity to CS–US discontiguity in classical conditioning
Neurobiology of Learning and Memory 110 (2014) 47–54
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Forebrain glycine transporter 1 deletion enhances sensitivity to CS–US discontiguity in classical conditioning Philipp Singer a,b,1, Sylvain Dubroqua a,b,1, Benjamin K. Yee a,b,⇑ a b
Laboratory of Behavioural Neurobiology, Swiss Federal Institute of Technology (ETH) Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland Legacy Research Institute, 1225 NE 2nd Avenue, Portland, OR 97232, United States
a r t i c l e
i n f o
Article history: Received 3 January 2013 Revised 15 January 2014 Accepted 20 January 2014 Available online 27 January 2014 Keywords: Conditioned freezing GlyT1 Memory NMDA receptor SLC6A9 Trace conditioning
a b s t r a c t The deletion of glycine transporter 1 (GlyT1) in forebrain neurons can apparently strengthen Pavlovian aversive conditioning, but this phenotype is not expressed if conditioning followed non-reinforced preexposures of the to-be-conditioned stimulus (CS). To examine whether GlyT1 disruption may only enhance aversive associative learning under conditions that most favour the formation of CS–US excitatory link, we evaluated the impact of GlyT1 disruption on the trace conditioning procedure whereby a trace interval between a tone CS and a shock US was introduced during conditioning. CS and US occurrences were thus rendered discontiguous, which was expected to impede conditioning compared with contiguous CS–US pairing. Conditioned freezing to the CS was measured in a retention test conducted 48 h after conditioning. The genetic disruption significantly modified the temporal dynamics of the freezing response over the course of the 8-min presentation of the CS, although the immediate conditioned response to the CS was unaffected. The separation between ‘‘trace’’ and ‘‘no-trace’’ conditions was augmented in the mutant mice, but this only became apparent in mid-session; and the augmentation can be attributed to the combined effects of (i) weaker conditioned freezing in the mutant relative to control subjects 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 discontiguity further highlights the importance of GlyT1-dependent mechanisms in the regulation of associative learning. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Disruption or blockade of glycine transporter 1 (GlyT1) is an effective way to elevate extracellular glycine levels in the vicinity of N-methyl-D-aspartate receptors (NMDARs) and thereby to potentiate the activation of NMDARs upon presynaptic release of glutamate (Bergeron, Meyer, Coyle, & Greene, 1998). It is because the binding of glycine (or D-serine) to the glycine-B site on the NR1 subunit is required for NMDAR channel activation by glutamate stimulation (Kleckner & Dingledine, 1988). Importantly, glycine-B site occupancy is normally regulated by GlyT1s (coexpressed with NMDARs, Smith, Borden, Hartig, Branchek, & Weinshank, 1992), which maintain synaptic glycine concentration at sub-saturation levels (Berger, Dieudonne, & Ascher, 1998; Bergeron et al., 1998; Supplisson & Bergman, 1997). GlyT1 is therefore a possible drug target for diseases in which a functional deficiency ⇑ Corresponding author at: Legacy Research Institute, 1225 NE 2nd Avenue, Portland, OR 97232, USA. Fax: +1 503 413 5465. E-mail addresses: [email protected], [email protected] (B.K. Yee). 1 Contributed equally. http://dx.doi.org/10.1016/j.nlm.2014.01.014 1074-7427/Ó 2014 Elsevier Inc. All rights reserved.
of NMDAR is implicated. GlyT1 inhibition has been attempted as a therapy against the negative and cognitive symptoms of schizophrenia, which do not respond to current medication targeting dopamine D2 receptors (Javitt, 2009, 2012). Yet, the efficacy of GlyT1-inhibiting drugs against the cognitive symptoms of schizophrenia remains controversial (Harvey & Yee, 2013) despite the promise of negative symptoms alleviation in early clinical trials (Pinard et al., 2010). Preclinical models are instrumental in defining the scope of possible treatment efficacy (Möhler et al., 2011). The most relevant mouse model to date involves the conditional deletion of GlyT1 after birth by introduction of CaMKII-driven expression of Cre recombinase to delete the GlyT1 gene preferentially in forebrain neurons (Yee et al., 2006). A number of cognitive phenotypes have been reported in the GlyT1fl/fl:CaMKII-Cre+/ mutant mice across different tests of learning and memory (Möhler et al., 2011). Evidence for enhanced learning has been reported across different Pavlovian paradigms (Yee et al., 2006) in which the mutants mice consistently showed a stronger conditioned response (CR) to a conditioned stimulus (CS) that had previously been paired with an aversive unconditioned stimulus (US). This led to the authors’
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speculation that the genetic disruption enhanced the acquisition and/or the memory strength of learned CS–US association. However, this interpretation is unsatisfactory because it is not able to account for the lack of enhanced CR when conditioning was preceded by repeated non-reinforced exposures to the to-be-conditioned CS (Yee et al., 2006). Hence, whether Pavlovian conditioning was enhanced or not is critically dependent on the conditions in which learning occur. Indeed, the mutant mice were more sensitive to CS pre-exposure – exhibiting weaker CR compared to mutant mice without CS pre-exposure (i.e., giving rise to latent inhibition, Lubow & Moore, 1959), even though the number of CS pre-exposures was insufficient to weaken CR expression in the controls (i.e., they did not show latent inhibition). Instead of referring to the conditioned freezing phenotype as a broad strengthening of CS–US associative links, a more fitting description may involve modifications to the cognitive processes that govern the selective acquisition and/or expression of Pavlovian associations. Selectivity of learning is central to all theories of associative learning (e.g. Mackintosh, 1975; Pearce & Hall, 1980; Wagner, 1981), without which the adaptive value of associative learning would be seriously undermined. Here, we tested whether the conditional GlyT1 deletion would enhance the animals’ sensitivity to another critical parameter of CS–US associability, namely, the temporal proximity between the CS and the subsequent US. Pavlov (1927) was the first to describe that CR is weaker when CS and US are interspersed by a temporal gap, referred to as the ‘trace’ interval, during conditioning. By contrast, CS–US learning is favoured when the US follows the CS closely in time (i.e., without any CS–US trace interval). A linkage between the expression of latent inhibition and the trace conditioning effect has been justified on theoretical grounds (Ayres, Albert, & Bombace, 1987; DeVietti, Bauste, Nutt, & Barrett, 1987) and supported by experimental evidence. Specifically, latent inhibition and the trace conditioning effect are similarly disrupted by systemic amphetamine (Norman & Cassaday, 2003; Weiner, Lubow, & Feldon, 1988) and selective disruption of hippocampal a5 GABA-A receptors (Gerdjikov et al., 2008; Yee et al., 2004). The present study tested if GlyT1fl/fl:CaMKII-Cre+/ mutant mice with a phenotype of enhanced sensitivity to the non-reinforced history of a potential CS (Yee et al., 2006) might also express a phenotype of increased sensitivity to CS–US temporal discontiguity. If so, the differential expression of CR acquired under the trace and no-trace conditions would be larger in the mutant than control mice.
2. Materials and methods 2.1. Subjects A homozygous Glyt1tm1.2 fl/fl colony was established and maintained on a pure C57BL/6 background as described before (Yee et al., 2006). Forebrain neuron specific deletion of GlyT1 was achieved by CamKIIaCre-mediated recombination of the floxed GlyT1 allele. Appropriate heterozygous Cre mice were mated with Glyt1tm1.2 fl/fl mice to generate the desired mutant and control littermates. Animals of both sexes were employed in the present study. The mice were weaned at 21 days old, and littermates of the same sex were kept in groups of four to six in Makrolon TypeIII cages (Techniplast, Milan, Italy). The subjects were housed in a temperature- and humidity-controlled (at 22 °C and 55% R.H.) animal vivarium under a reversed light-dark cycle with lights off from 07.00 to 19.00 h. Testing was always conducted in the dark phase of the cycle. The animals were maintained under ad libitum water and 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 Swiss Federal Act on Animal Protection (1978) and Swiss Animal Protection Ordinance (1981) in accordance with the European Council Directive 86/609/EEC (1986). All efforts had been made to alleviate animal 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 measured using the conditioned freezing paradigm based on established parameters known to induce a robust trace conditioning effect in mice (Yee et al., 2004). The apparatus consisted of two sets of four conditioning chambers. The two sets were distinct from each other, and were installed in separate testing rooms, providing two distinct contexts as fully described before (Yee et al., 2006). The first set of chambers (context ‘A’) comprised four Coulbourn Instruments (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 centre, and through which scrambled electric shocks (the US, set at 0.3 mA) could be delivered (model E13-14; Coulbourn Instruments). A transparent Plexiglas enclosure confined the animals to a rectangular region (17.5 13 cm). The inside of the chambers was illuminated by a house light (2.8 W) positioned on the panel wall, 21 cm above the grid floor. The second set of chambers (context ‘B’) comprised four cylindrical (19 cm in diameter) enclosures made of clear Plexiglas resting on a metal mesh floor. Illumination inside the chamber was provided by an infrared light source instead of visible light. The CS was an 86-dBA tone provided by a sonalert (model SC628; Mallory, Indianapolis, IN, USA). Each of the eight chambers contained a miniature digital camera mounted 30 cm directly above the centre of the area of interest. The algorithm of the freezing response detection procedure has been validated and fully described before (Richmond et al., 1998). A detailed description of the trace conditioning procedure has been published elsewhere (Yee et al., 2004). The subjects of each group were randomly allocated into one of two conditions of CS–US pairing differing in the (trace) interval between CS offset and US onset: either 0 or 20 s (‘no-trace’ and ‘trace’ conditions, respectively). The respective 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, the animals were given three trials of CS–US pairings (CS: 30 s, US: 1 s) in context A. Each trial was preceded and followed by a 120 s inter-trial interval. 24 h later, the CR to the conditioning context was evaluated by returning the animals to context A for a period of 8 min. Another 24 h later, conditioned freezing to the tone CS was assessed in the neutral context B. The test session began with a 2 min acclimatization period, followed by the presentation of the CS for 8 min; and then the CS was turned off and the animals left in the chamber for an additional 2 min. 2.3. Statistical analysis All data were analysed by parametric analysis of variance (ANOVA) 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 with the inclusion of the within-subject factor time bins was performed in the analysis of data obtained on the CS-test day to provide a temporal profile of the phenotype. Fisher’s Least Significant Difference (LSD) post hoc pair-wise comparisons and planned contrast analysis were performed to assist interpretation of statistically significant effects emerged from an overall ANOVA. In addition, supplementary restricted ANOVAs and one-sample t-tests were
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applied to subsets of data. All statistical analyses were carried out using 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 seconds the animals were observed as immobile and expressed as percent time freezing within specific periods: CS, pre-CS and trace. The three variables were separately analysed. Supplementary analyses restricted to animals exposed either to the ‘no-trace’ or the ‘trace’ condition on day 1 were also performed to allow the comparison of freezing between distinct periods. 3.1.1. CS periods The level of freezing during the tone-CS presentation increased progressively across the three CS–US trials (Fig. 1A), but this increment was noticeably weaker in the ‘trace’ relative to the ‘no-trace’ condition, reaching a maximal difference on the third CS presentation. These impressions were confirmed by a 2 2 2 3 (genotype trace sex trials) split-plot ANOVA of percent time freezing, which revealed a significant main effect of trials [F(2,100) = 35.38, p < .01], trace [F(1,50) = 10.39, p < .01], and their 2-way interaction [F(2,100) = 8.03, p < .01]. Post hoc pair-wise comparison confirm that the difference between trace and no-trace conditions (collapsed across genotypes and sex) only achieve statistical significance on the third trial [t(100) = 5.66, p < .01]. Incidentally, male mice generally exhibited stronger freezing than female mice regardless of genotype and training condition, yielding a statistically significant sex effect [F(1,50) = 12.41, p < .01] but not any of its interactions. The average time of freezing in the presence of the CS on day 1 was 7.3 ± 1.4% in the female and 14.4 ± 1.5% in the male. 3.1.2. Pre-CS periods Freezing levels during the 30 s preceding the CS similarly increased across the three Pre-CS periods (Fig. 1B) in a manner that was essentially independent of genotype, training condition or sex. The increase in Pre-CS freezing likely reflected a general increase in fear due to the shock US rather than being specifically attributable to the CS itself. A 2 2 2 3 (genotype trace sex Pre-CS periods) ANOVA of percent time freezing only yielded a significant main effect of Pre-CS periods [F(2,100) = 73.71, p < .01]. 3.1.3. Trace intervals This variable refers to the freezing levels recorded during the 20 s trace intervals, and was therefore only available in animals tested under the trace condition. Again, a monotonic increase in freezing was observed across the three trace intervals in animals tested under the trace condition (Fig. 1C). Similar to the analysis of freezing recorded in the CS-periods above, a sex difference was observed with the male freezing at 24.5 ± 2.8% vs. female at 15.5 ± 2.5% on average. These led to the emergence of a significant main 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 trace intervals) ANOVA of percent time freezing. 3.1.4. Comparison between variables In the no-trace condition, a 2 2 3 2 (genotype sex trials dependent variables) ANOVA comparing CS and Pre-CS freezing variables only yielded an effect of trials as expected [F(2,48) = 53.79, p < .001]. And, there was no statistical indication
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that the animals froze differentially between the pre-CS and CS periods (see Fig. 1A and B). In the trace condition, a 2 2 3 3 (genotype sex trials dependent variables) ANOVA yielded a significant effect of variables [F(2,52) = 20.35, p < .001] as well as its interaction with trials [F(4,104) = 10.75, p < .001] besides the expected main effect of trials [F(2,52) = 62.97, p < .001]. Freezing during the CS period exhibited by animals trained under the trace condition became visibly lower than that observed in the Pre-CS and/or trace periods as conditioning progressed from trials 1 to 3 (Fig. 2). Notably, CSfreezing increased at the slowest pace, leading to a clear divergence from Pre-CS and trace interval freezing by the third trial. In trials 2 and 3, therefore, the transition from the Pre-CS to the CS periods was associated with a drop in freezing [F(1,26) = 16.99, p < .001 based on a restricted ANOVA] whereas the transition from the CS period to the succeeding trace interval corresponded to a rebound in freezing [F(1,26) = 65.46, p < .001 based on a restricted ANOVA]. The CS thus appeared to disrupt the on-going expression of freezing (indexed by Pre-CS freezing) on trials 2 and 3 on day 1, suggesting that the animals undergoing trace conditioning might have learned to perceive the CS as a safety signal. Although the average time freezing was numerically the highest during the trace intervals, a separate restricted ANOVA did not reveal a significant difference between the freezing levels obtained in the Pre-CS periods and trace intervals across trials 2–3 [p = .071]. Hence, the animals seemingly did not substantially differentiate between the 30 s before the CS from the 20 s following the CS (when the physical conditions of the test chambers were essentially identical) – i.e., the animals did not perceive the 20-s trace interval as a better predictor of the shock US compared with the pre-CS period.
3.2. Conditioned context freezing (day 2) The expression of conditioned freezing developed towards the training context was evaluated 24 h after conditioning by reexposing the animals to the training context in the absence of any discrete CS (Fig. 3). No difference was apparent between genotypes, sexes or training conditions. A 2 2 2 (genotype sex trace) ANOVA of percent time freezing over the 8-min test 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 of freezing was measured in a neutral context. The animals were acclimatized to the new context for 2 min (i.e., Pre-CS period) before the CS was presented continuously for a period of 8 min (i.e., CS-period). The test session was concluded with another 2-min in which the CS was absent (i.e., post-CS period).
3.3.1. Pre-CS period As shown in Fig. 4A, the general level of spontaneous freezing (immobility) was initially low but a gradual increase over time was visible. This increase was similarly observed in both genotypes regardless of training conditions (i.e., trace vs. no-trace). However, a sex difference was detected as male mice generally exhibited a higher level of spontaneous freezing (mean ± SE: male = 14.7 ± 1.7% vs. female = 9.6 ± 1.6%) regardless of genotype and training conditions. These impressions were confirmed by a 2 2 2 4 (genotype sex trace 30-s bins) ANOVA of percent time freezing during the Pre-CS period, which yielded a significant 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.
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3.3.2. CS Period Presentation of the CS predictably led to a rapid increase in freezing observed in animals previously conditioned under the no-trace condition. By contrast, the increase was modest in animals conditioned under the trace condition (Fig. 4B). This difference constitutes the trace conditioning effect; and its expression was visibly comparable between mutants and controls over the first 2 min. The data were examined with a finer temporal resolution across 10-s bins over the first minute of the CS as shown in Fig. 4D. This shows that 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 a time 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 into the CS presentation (Fig. 4B). The size of the trace effect, as illustrated by the stronger freezing in the no-trace condition relative to the weaker freezing in the trace condition, was significantly larger in the mutant mice. This genotype effect remained detectable at 7 min into the CS period. A 2 2 2 16 (genotype sex trace 30-s bins) ANOVA revealed a significant genotype by trace interaction [F(1,50) = 4.60, p < .05] and the temporal dependency of this effect [genotype trace bins: F(15,750) = 1.84, p < .05]. These genotypes effects were accompanied by the significant 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 investigated and characterized by pair-wise comparisons and planned contrast analysis (see Fig. 4B). First, a significant trace effect was detectable in the mutants throughout the 8-min CS-period. Second, the trace conditioning effect in the controls became weak in the middle of the CS-period. Pairwise comparisons between control/trace and control/no-trace animals failed to yield a significant difference in CS 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 apparent in the middle of the CS-period. The interaction of interest is identical to the contrast [(control/trace – control/no-trace animals) – (mutant/trace – mutant/no-trace animals)], i.e., with contrast coefficients (+1, 1, 1, +1). This specific contrast was significant from the third to the seventh minutes into the CS period except at bin 12 (see Fig. 4B). The temporal dependent profile of this phenotype is consistent with the lack of a genotype by trace interaction when the analysis was restricted to the first one [F = .001, ns] or two minutes [F = .16, ns] of the CS-period. Last, planned comparisons were performed to examine whether the phenotype on trace conditioning was associated with bidirectional effects in
freezing between the trace and the no-trace conditions. Suggestions for stronger freezing in the mutant in the no-trace condition was obtained in bins 7–8 and 10–11, and for weaker freezing in the mutant in the trace condition was obtained in bins 5–6 and 8–10 (see Fig. 4B). 3.3.3. Post-CS Period Examination of the post-CS period clearly showed a sharp increase in freezing that was most notable in animals trained under the trace condition. Although an increase was visible also in the animals trained under the no-trace condition, it was substantially weaker. The immediate response to the CS-offset was examined by an analysis of a difference score indexing the elevation of freezing levels from the last 30-s bin of the CS period to the first 30-s bin of the post-CS period (Fig. 4E). A 2 2 2 (genotype sex trace) ANOVA of the difference score revealed a main effect trace, consistent with the impression that animals trained under the trace condition responded more strongly to the termination of the CS [F(1,50) = 5.60, p < .05]. One-sample t-tests further indicated that only the mean difference score obtained from the two trace groups differed significantly from zero. The levels of freezing observed in the initial 30 s of the post-CS period (i.e., first post-CS bin in Fig. 4C) did not differ substantially between groups. Subsequently, the animals trained under the trace condition maintained a level of freezing that was substantially higher than animals trained under the no-trace condition (i.e., over the last 3 bins in Fig. 4C). In other words, the separation between trace and no-trace conditions now took a form that was the reverse of that seen in the CS-period. This may be expected if the trace animals rather than the no-trace animals had perceived the withdrawal of CS itself as a predictor of shock US. A 2 2 2 4 (genotype sex trace 30-s bins) ANOVA confirmed the presence of a highly significant trace effect [F(1,50) = 8.19, p < .01] as well as its interaction with bins [F(3,150) = 3.06, p < .05] and the main effect of bins [F(3,150) = 29.04, p < .001]. 4. Discussion The present study clearly demonstrated that the expression of the trace conditioning effect over time was modified by forebrain neuronal deletion of GlyT1. This phenotype revealed in the GlyT1fl/fl:CaMKII-Cre+/ mutant mice was specific to the extinction test of CS freezing, because neither acquisition performance (on day 1) nor the expression of context-freezing (on day 2) was significantly modified by the genetic disruption (Figs. 1–3). The 2-min
Fig. 1. Development of conditioned freezing across three CS–US pairings: (A) freezing behaviour is indexed by the percentage of time freezing during the 30 s of tone-CS presentation and is depicted for the no-trace (open symbols) and the trace (filled symbols) conditions separately. Freezing during the CS observed in the trace condition was weaker than in the no-trace condition – an effect that emerged over the three conditioning trials – and was comparable between genotypes. (B) Expression of freezing behaviour during the 30 s immediately preceding each CS presentation is shown as a function of trials. This was comparable between test conditions and genotypes. (C) For animals tested under the trace condition only, freezing levels during the 20-s trace intervals are depicted separately. Error bars refer to ±SEM.
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Fig. 2. A general increase in freezing was observed in all three measures (Pre-CS, CS and trace periods) in animals trained under the trace conditioning procedure on day 1. The rate of increase across conditioning trials 1–3, however, differed between the three variables. Notably, CS-freezing progressed at the slowest pace, leading to a clear divergence from Pre-CS and trace interval freezing by the third trial. In trials 2 and 3, therefore, we saw a drop of freezing from Pre-CS to the CS period, followed by a rebound when the CS had ended and the trace interval began. Error bars refer to ±SEM.
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described as stemming from a retardation of extinction. A similar suggestion has been made in the initial report of enhanced conditioned freezing in the GlyT1fl/fl:CaMKII-Cre+/ mice (Yee et al., 2006), which underwent Pavlovian conditioning procedures identical to the no-trace condition here. Yet, two subsequent reports that replicated the enhanced conditioned freezing phenotype in GlyT1fl/fl: CaMKII-Cre+/ mice did not obtain any evidence for a concomitant resistance to extinction (Dubroqua, Boison, Feldon, Mohler, & Yee, 2011; Dubroqua et al., 2010). This might undermine the interpretation that disruption of forebrain neuronal GlyT1 primarily affects the rate of extinction rather than the strength of the CS–US association. Nevertheless, equally crucial to the interpretation of the trace conditioning phenotype here is that the extinction perspective would view the weaker freezing in the mutant/trace group (relative to the control/trace group) as a facilitation of extinction. Hence, CS–US contiguity during acquisition can critically decide whether the rate of extinction would be slowed down or speeded up by the conditional deletion of GlyT1 in the forebrain. Here, the mutation effectively reversed the effect of CS–US contiguity on the rate of extinction seen in control mice (Fig. 4B). 4.2. The temporal perception of the CS
Fig. 3. Test of context freezing: The freezing response induced by the training context 24 h after conditioning was comparable amongst groups. Error bars refer to ±SEM.
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) emerged as soon as the CS was presented (Fig. 4B), and the separation between trace and no-trace groups was highly comparable between genotypes over the first two minutes in the presence of the CS. Essentially, no phenotype was evident in this period because the freezing behaviour observed was indistinguishable between mutant and control mice belonging to the same training condition during these two minutes. The observation that GlyT1fl/fl:CaMKIICre+/ mutant mice displayed a stronger trace conditioning effect only became detectable afterwards. Hence, the trace conditioning phenotype emerged over time, even though the average performance over the 8-min CS period gave an impression that the trace conditioning was significantly enhanced in the mutant mice. Post-hoc analyses confirmed that this phenotype was only robustly expressed in the mid-session, and indicated that the temporal dependency of this novel phenotype must be addressed. 4.1. The contribution of extinction over the extended CS presentation in the CS-test Because the phenotype did not emerge immediately as the CS was presented, it is important to consider the potential contribution of extinction learning over the extended period of the CS presentation. From this perspective, the enhanced freezing in the mutants relative to the controls in the no-trace group may be
Common to most conditioned freezing experiments (e.g. Zelikowsky, Bissiere, & Fanselow, 2012), the expression of the conditioned response (i.e., freezing) was measured in a novel context in which the CS was presented continuously for a period well beyond the duration of the CS presented during acquisition. One consequence of this procedure is that the CS might not be perceived as identical to the training CS when it was extended beyond 30 s in the test. Indeed, generalization decrement is predicted when the CS differs in duration between acquisition and test (Gallistel & Gibbon, 2000; Pavlov, 1927). One may therefore prefer to focus on the first 30 s of the CS presentation in the test session, because the data would be free from the confounding impact of generalization decrement as well as of extinction learning. As shown in Fig. 4B, there was no evidence to suspect that the expression of the trace conditioning effect had been modified by the GlyT1 disruption. Indeed, the mutant and controls mice (within either the no-trace or trace group) were highly comparable when the initial rise of the conditioned freezing response was tracked across 10-s bins (Fig. 4D). However, any attempt to explain the emergence of the phenotype later in the test as a consequence of an effect on generalization decrement resulting from the protraction of the CS is questionable. First, it cannot predict the abrupt expression of the phenotype at three minutes into the CS. Second, the phenotype did not become stronger gradually as the CS continued to extend over time; and if anything the phenotype was weaker by the end of the CS period (Fig. 4B). Hence, the temporal dynamics of the genotype effects undermine the possibility that the phenotype primarily reflects the effects of the mutation on generalization decrement 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 a safety signal on the conditioning day (day 1) in animals exposed to the trace conditioning procedure. By the third and final conditioning trial (but before shock US delivery), mutant and control mice in the trace group appeared to respond to the CS with a reduction in freezing by comparison to the pre-CS period immediately before, and to the trace interval that immediately followed the CS (Fig. 2). The rapid fall and rise of the freezing response is consistent with the interpretation that the CS was perceived as a safety signal. Although this effect was not significantly stronger
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in the mutants on the conditioning day, its possible contribution to the trace conditioning phenotype observed on the retention test on day 3 of the experiment is still worth considering. As a safety signal, the CS might therefore function as an inhibitor (see Bouton, 2007, p. 89) that prevents or reduces the expression of conditioned freezing in the trace groups recorded on the retention test. Consequently, the stronger trace conditioning effect seen in the mutants might stem from a stronger inhibitory CS–US link acquired by animals in the trace groups on the conditioning day. The possibility that conditional GlyT1 disruption promoted inhibitory as well as excitatory conditioning (Dubroqua et al., 2011; Yee et al., 2006) certainly warrants further investigation. Further experiments using paradigms that are specifically designed to measure the development of conditioned inhibition and safety signal, such as [A+/AB] (Rescorla, 1969) and [AX+/BX] (Kazama, Schauder, McKinnon, Bachevalier, & Davis, 2013; Myers & Davis, 2004) procedures, could clarify this interesting speculation.
1927), the animals trained under such conditions could also learn about the contingency between the trace itself and US. Indeed, freezing in the trace groups showed a rapid increase as soon as the CS was switched off (Fig. 4C), suggesting that they responded to the offset of the CS. This response was well-above that expressed by the animals in the no-trace groups that had never experienced a CS–US trace interval (Fig. 4E). Although freezing generally decreased over the rest of the post-CS period, the trace and no-trace groups clearly diverged, such that the trace groups exhibited stronger freezing than the no-trace control groups regardless of genotype. Indeed, the trace groups exhibited during the post-CS period their highest levels of freezing across the entire test session. To the extent that this analysis has shown that animals trained under the trace conditions had responded to the CS–US trace interval as a potential predictor of the shock US, there was no evidence that the disruption of GlyT1 had significantly altered the learning about the trace interval.
4.4. Learning about the trace
4.5. Comparing the trace conditioning and latent inhibition phenotypes of GlyT1 disruption
Although trace conditioning is operationally defined as weaker conditioned response to the CS when the pairing between CS and US during acquisition was interspersed by a trace interval (Pavlov,
Importantly, we qualify here that Pavlovian learning is not always enhanced (defined as a stronger CR specific to the CS) by
Fig. 4. Test of CS freezing: The freezing response recorded during the CS-test session was subjected to separate analyses: the Pre-CS (A), CS-period (B) and the post-CS periods (C). The data were presented as a function of successive 30-s bins. The significant three-way interaction observed in the CS period was further investigated and characterized by planned contrast analysis and pair-wise comparisons based on the error variance associated with the three-way interaction in the overall ANOVA. The contrast specifically compares between the mutants and controls the size of the trace conditioning effect at each successive bin. Those time bins in which this contrast achieved statistical significance [p < 0.05] are highlighted by the green underlay in B. Bins in which a significant pair-wise difference between control/no-trace and mutant/no-trace was detected are marked by ‘a’; and those in which a significant pair-wise difference between control/trace and mutant/trace was detected are marked by ‘b’. The first minute of the CSperiod is depicted as a function of 10-s bins to illustrate the evolution of the conditioned response to the CS at a higher temporal resolution (D). The increase of freezing levels from the last 30-s bin of the CS period to the first 30-s bin of the post-CS period is separately depicted (E). # Indicates a significant difference from zero (which indicates no change from the end of CS-period to the beginning of the post-CS period) based on one-sample tests. All data shown refer to mean ±SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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conditional GlyT1 disruption in forebrain neurons. Indeed, a reversed effect can be induced by specific changes in training conditions, but the psychological significance of this qualification remains to be fully appreciated and clarified. Under conditions that favour maximally the formation of the CS–US association with the CS being perceived unambiguously as a reliable and accurate predictor of the eminent occurrence of the US, the magnitude of the CR tends to be stronger in the mutants. On the other hand, deviation from such ideal conditions that corrupts this positive CS–US predictive link and thereby reduces CR, is seemingly more impactful in our mutant mice. This description fits not only the trace conditioning phenotype demonstrated here but also the latent inhibition phenotype previously reported (Yee et al., 2006). In latent inhibition, non-reinforced CS pre-exposure (presentation of CS alone, in the absence of any US) impedes the efficacy of subsequent CS–US pairing to produce a CR to the CS (Lubow and Moore, 1959). Mice with forebrain neuronal GlyT1 deletion are more sensitive to this CS pre-exposure effect: Latent inhibition was evident in the mutants when the amount of CS pre-exposure was insufficient to generate latent inhibition in wild type controls (Yee et al., 2006). According to DeVietti et al. (1987), the similar sensitivity to the latent inhibition (i.e., CS pre-exposure effect) and trace conditioning procedures may stem from a common psychological mechanism. They suggest that CS pre-exposures preferentially reduce attention to later segments of individual CS’s, which then effectively act as a trace interval between initial CS segments and the US during subsequent CS–US pairings. The perceived CS–US discontiguity thereby weakens conditioning and yields the LI effect (also see Ayres et al., 1987). Systemic amphetamine (Norman and Cassaday, 2003; Weiner et al., 1988) as well as selective disruption of hippocampal a5 GABA-A receptors (Gerdjikov et al., 2008; Yee et al., 2004) have been shown to reduce sensitivity to CS pre-exposure effect and CS–US discontiguity. Here, we extended these findings by showing that disruption of forebrain neuronal GlyT1 could enhance both the sensitivity to CS pre-exposure effect and CS–US discontiguity. It remains to be determined whether these parallel or complementary effects of these three distinct brain manipulations may be mediated through a common neural circuitry. 4.6. Conclusion Although further experiments are necessary to delineate the precise psychological and molecular mechanisms underlying the novel phenotype of GlyT1fl/fl:CaMKII-Cre+/ mutant mice, our experiment shows that the CS–US contiguity at the time of conditioning can critically determine how the genetic disruption might modify the expression of the acquired conditioned response. The present study thus reveals important insights into the role of forebrain neuronal GlyT1 in determining how past experience may shape current behaviour through Pavlovian learning mechanism. Acknowledgments The present study was supported by Swiss National Science Foundation Grant (3100–066855) with additional support from the Swiss Federal Institute of Technology Zurich. BKY received additional support from the National Institutes of Health (MH083973). SD and PS were recipients of a studentship from the Neural Plasticity & Repair – National Centre for Competence in Research (NCCR) funded by the Swiss National Science Foundation. We thank Prof. Hanns Möhler and Dr. Detlev Boison of Zurich University for providing the breeding pairs for the generation of the GlyT1fl/fl conditioned mouse line. The software development and maintenance of the behavioural equipment were provided by the late Peter Schmid, to whom we would like to express our
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deep gratitude for his excellent service over two decades. We are also indebted to the animal husbandry staffs and to Dr. Joram Feldon for making available the animal facilities and behavioural equipment necessary for the reported experiments.
References Ayres, J. J., Albert, M., & Bombace, J. C. (1987). Extending conditioned stimuli before versus after unconditioned stimuli: Implications for realtime models of conditioning. Journal of Experimental Psychology: Animal Behavioural Process, 13, 168–181. Berger, A. J., Dieudonne, S., & Ascher, P. (1998). Glycine uptake governs glycine site occupancy at NMDA receptors of excitatory synapses. Journal of Neurophysiology, 80, 3336–3340. Bergeron, R., Meyer, T. M., Coyle, J. T., & Greene, R. W. (1998). Modulation of Nmethyl-D-aspartate receptor function by glycine transport. Proceedings of the National Academy of Sciences of United States, 95, 15730–15734. Bouton, M. E. (2007). Learning and behavior: A contemporary synthesis. Sunderland, MA: Sinauer Associates Inc.. DeVietti, T. L., Bauste, R. L., Nutt, G., & Barrett, O. V. (1987). Latent inhibition: A trace conditioning phenomenon? Learning and Motivation, 67, 416–422. Dubroqua, S., Boison, D., Feldon, J., Mohler, H., & Yee, B. K. (2011). Examining the sex- and circadian dependency of a learning phenotype in mice with glycine transporter 1 deletion in two Pavlovian conditioning paradigms. Neurobiology of Learning and Memory, 96, 218–229. Dubroqua, S., Singer, P., Boison, D., Feldon, J., Mohler, H., & Yee, B. K. (2010). Impacts of forebrain neuronal glycine transporter 1 disruption in the senescent brain: Evidence for age-dependent phenotypes in Pavlovian learning. Behavioral Neuroscience, 124, 839–850. Gallistel, C. R., & Gibbon, J. (2000). Time, rate, and conditioning. Psychological Review, 107, 289–344. Gerdjikov, T. V., Rudolph, U., Keist, R., Mohler, H., Feldon, J., & Yee, B. K. (2008). Hippocampal alpha 5 subunit-containing GABA A receptors are involved in the development of the latent inhibition effect. Neurobiology of Learning and Memory, 89, 87–94. Harvey, R. J., & Yee, B. K. (2013). Glycine transporters as novel therapeutic targets in schizophrenia, alcohol dependence and pain. Nature Reviews Drug Discovery, 12, 866–885. Javitt, D. C. (2009). Glycine transport inhibitors for the treatment of schizophrenia: Symptom and disease modification. Current Opinion in Drug Discovery & Development, 12, 468–478. Javitt, D. C. (2012). Glycine transport inhibitors in the treatment of schizophrenia. Handbook of Experimental Pharmacology, 213, 367–399. Kazama, A. M., Schauder, K. B., McKinnon, M., Bachevalier, J., & Davis, M. (2013). A novel AX+/BX paradigm to assess fear learning and safety-signal processing with repeated-measure designs. Journal of Neuroscience Methods, 214(2), 177–183. http://dx.doi.org/10.1016/j.jneumeth.2013.01.022. Kleckner, N. W., & Dingledine, R. (1988). Requirement for glycine in activation of NMDA receptors expresses in Xenopus oocytes. Science, 214, 835–837. Lubow, R. E., & Moore, A. U. (1959). Latent inhibition: The effect of nonreinforced pre-exposure to the conditional stimulus. Journal of Comparative Physiological Psychology, 52, 415–419. Mackintosh, N. J. (1975). Theory of attention – Variations in associability of stimuli with reinforcement. Psychological Review, 82, 276–298. Möhler, H., Boison, D., Singer, P., Feldon, J., Pauly-Evers, M., & Yee, B. K. (2011). Glycine transporter 1 as a potential therapeutic target for schizophrenia-related symptoms: Evidence from genetically modified mouse models and pharmacological inhibition. Biochemical Pharmacology, 81, 1065–1077. Myers, K. M., & Davis, M. (2004). AX+, BX discrimination learning in the fearpotentiated startle paradigm: Possible relevance to inhibitory fear learning in extinction. Learning & Memory, 11, 464–475. Norman, C., & Cassaday, H. J. (2003). Amphetamine increases aversive conditioning to diffuse contextual stimuli and to a discrete trace stimulus when conditioned at higher footshock intensity. Journal of Psychopharmacology, 17, 67–76. Pavlov, I. P. (1927). Conditioned reflexes: An investigation of the physiological activity of the cerebral cortex. London: Oxford University Press. Pearce, J. M., & Hall, G. (1980). A model for Pavlovian learning: Variations in the effectiveness of conditioned but not of unconditioned stimuli. Psychological Review, 87, 532–552. Pinard, E., Alanine, A., Alberati, D., Bender, M., Borroni, E., Bourdeaux, P., & Zimmerli, D. (2010). Selective GlyT1 inhibitors: Discovery of [4-(3-fluoro-5trifluoromethylpyridin-2-yl)piperazin-1-yl][5-methanesulfonyl-2-((S)-2,2,2trifluoro-1-methylethoxy)phenyl]methanone (RG1678), a promising novel medicine to treat schizophrenia. Journal of Medicinal Chemistry, 53, 4603–4614. Rescorla, R. A. (1969). Conditioned inhibition of fear resulting from negative CS–US contingencies. Journal of Comparative Physiological Psychology, 67, 504–509. Richmond, M. A., Murphy, C. A., Pouzet, B., Schmid, P., Rawlins, J. N., & Feldon, J. (1998). A computer controlled analysis of freezing behaviour. Journal of Neuroscience Methods, 86, 91–99. Smith, K. E., Borden, L. A., Hartig, P. R., Branchek, T., & Weinshank, R. L. (1992). Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors. Neuron, 8, 927–935.
54
P. Singer et al. / Neurobiology of Learning and Memory 110 (2014) 47–54
Supplisson, S., & Bergman, C. (1997). Control of NMDA receptor activation by a glycine transporter co-expressed in Xenopus oocytes. Journal of Neuroscience, 17, 4580–4590. Wagner, A. R. (1981). SOP: A model of automatic memory processing in animal behavior. In N. E. Spear & R. R. Miller (Eds.), Information processing in animals: Memory mechanisms (pp. 5–47). Hillsdale, NJ: Erlbaum. Weiner, I., Lubow, R. E., & Feldon, J. (1988). Disruption of latent inhibition by acute administration of low doses of amphetamine. Pharmacology, Biochemistry and Behavior, 30, 871–878. Yee, B. K., Balic, E., Singer, P., Schwerdel, C., Grampp, T., Gabernet, L., et al. (2006). Disruption of glycine transporter 1 restricted to forebrain neurons is associated
with a procognitive and antipsychotic phenotypic profile. Journal of Neuroscience, 26, 3169–3181. Yee, B. K., Hauser, J., Dolgov, V. V., Keist, R., Mohler, H., Rudolph, U., et al. (2004). GABA receptors containing the alpha5 subunit mediate the trace effect in aversive and appetitive conditioning and extinction of conditioned fear. European Journal of Neuroscience, 20, 1928–1936. Zelikowsky, M., Bissiere, S., & Fanselow, M. S. (2012). Contextual fear memories formed in the absence of the dorsal hippocampus decay across time. Journal of Neuroscience, 32, 3393–3397.
Contents lists available at ScienceDirect
Neurobiology of Learning and Memory journal homepage: www.elsevier.com/locate/ynlme
Forebrain glycine transporter 1 deletion enhances sensitivity to CS–US discontiguity in classical conditioning Philipp Singer a,b,1, Sylvain Dubroqua a,b,1, Benjamin K. Yee a,b,⇑ a b
Laboratory of Behavioural Neurobiology, Swiss Federal Institute of Technology (ETH) Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland Legacy Research Institute, 1225 NE 2nd Avenue, Portland, OR 97232, United States
a r t i c l e
i n f o
Article history: Received 3 January 2013 Revised 15 January 2014 Accepted 20 January 2014 Available online 27 January 2014 Keywords: Conditioned freezing GlyT1 Memory NMDA receptor SLC6A9 Trace conditioning
a b s t r a c t The deletion of glycine transporter 1 (GlyT1) in forebrain neurons can apparently strengthen Pavlovian aversive conditioning, but this phenotype is not expressed if conditioning followed non-reinforced preexposures of the to-be-conditioned stimulus (CS). To examine whether GlyT1 disruption may only enhance aversive associative learning under conditions that most favour the formation of CS–US excitatory link, we evaluated the impact of GlyT1 disruption on the trace conditioning procedure whereby a trace interval between a tone CS and a shock US was introduced during conditioning. CS and US occurrences were thus rendered discontiguous, which was expected to impede conditioning compared with contiguous CS–US pairing. Conditioned freezing to the CS was measured in a retention test conducted 48 h after conditioning. The genetic disruption significantly modified the temporal dynamics of the freezing response over the course of the 8-min presentation of the CS, although the immediate conditioned response to the CS was unaffected. The separation between ‘‘trace’’ and ‘‘no-trace’’ conditions was augmented in the mutant mice, but this only became apparent in mid-session; and the augmentation can be attributed to the combined effects of (i) weaker conditioned freezing in the mutant relative to control subjects 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 discontiguity further highlights the importance of GlyT1-dependent mechanisms in the regulation of associative learning. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Disruption or blockade of glycine transporter 1 (GlyT1) is an effective way to elevate extracellular glycine levels in the vicinity of N-methyl-D-aspartate receptors (NMDARs) and thereby to potentiate the activation of NMDARs upon presynaptic release of glutamate (Bergeron, Meyer, Coyle, & Greene, 1998). It is because the binding of glycine (or D-serine) to the glycine-B site on the NR1 subunit is required for NMDAR channel activation by glutamate stimulation (Kleckner & Dingledine, 1988). Importantly, glycine-B site occupancy is normally regulated by GlyT1s (coexpressed with NMDARs, Smith, Borden, Hartig, Branchek, & Weinshank, 1992), which maintain synaptic glycine concentration at sub-saturation levels (Berger, Dieudonne, & Ascher, 1998; Bergeron et al., 1998; Supplisson & Bergman, 1997). GlyT1 is therefore a possible drug target for diseases in which a functional deficiency ⇑ Corresponding author at: Legacy Research Institute, 1225 NE 2nd Avenue, Portland, OR 97232, USA. Fax: +1 503 413 5465. E-mail addresses: [email protected], [email protected] (B.K. Yee). 1 Contributed equally. http://dx.doi.org/10.1016/j.nlm.2014.01.014 1074-7427/Ó 2014 Elsevier Inc. All rights reserved.
of NMDAR is implicated. GlyT1 inhibition has been attempted as a therapy against the negative and cognitive symptoms of schizophrenia, which do not respond to current medication targeting dopamine D2 receptors (Javitt, 2009, 2012). Yet, the efficacy of GlyT1-inhibiting drugs against the cognitive symptoms of schizophrenia remains controversial (Harvey & Yee, 2013) despite the promise of negative symptoms alleviation in early clinical trials (Pinard et al., 2010). Preclinical models are instrumental in defining the scope of possible treatment efficacy (Möhler et al., 2011). The most relevant mouse model to date involves the conditional deletion of GlyT1 after birth by introduction of CaMKII-driven expression of Cre recombinase to delete the GlyT1 gene preferentially in forebrain neurons (Yee et al., 2006). A number of cognitive phenotypes have been reported in the GlyT1fl/fl:CaMKII-Cre+/ mutant mice across different tests of learning and memory (Möhler et al., 2011). Evidence for enhanced learning has been reported across different Pavlovian paradigms (Yee et al., 2006) in which the mutants mice consistently showed a stronger conditioned response (CR) to a conditioned stimulus (CS) that had previously been paired with an aversive unconditioned stimulus (US). This led to the authors’
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speculation that the genetic disruption enhanced the acquisition and/or the memory strength of learned CS–US association. However, this interpretation is unsatisfactory because it is not able to account for the lack of enhanced CR when conditioning was preceded by repeated non-reinforced exposures to the to-be-conditioned CS (Yee et al., 2006). Hence, whether Pavlovian conditioning was enhanced or not is critically dependent on the conditions in which learning occur. Indeed, the mutant mice were more sensitive to CS pre-exposure – exhibiting weaker CR compared to mutant mice without CS pre-exposure (i.e., giving rise to latent inhibition, Lubow & Moore, 1959), even though the number of CS pre-exposures was insufficient to weaken CR expression in the controls (i.e., they did not show latent inhibition). Instead of referring to the conditioned freezing phenotype as a broad strengthening of CS–US associative links, a more fitting description may involve modifications to the cognitive processes that govern the selective acquisition and/or expression of Pavlovian associations. Selectivity of learning is central to all theories of associative learning (e.g. Mackintosh, 1975; Pearce & Hall, 1980; Wagner, 1981), without which the adaptive value of associative learning would be seriously undermined. Here, we tested whether the conditional GlyT1 deletion would enhance the animals’ sensitivity to another critical parameter of CS–US associability, namely, the temporal proximity between the CS and the subsequent US. Pavlov (1927) was the first to describe that CR is weaker when CS and US are interspersed by a temporal gap, referred to as the ‘trace’ interval, during conditioning. By contrast, CS–US learning is favoured when the US follows the CS closely in time (i.e., without any CS–US trace interval). A linkage between the expression of latent inhibition and the trace conditioning effect has been justified on theoretical grounds (Ayres, Albert, & Bombace, 1987; DeVietti, Bauste, Nutt, & Barrett, 1987) and supported by experimental evidence. Specifically, latent inhibition and the trace conditioning effect are similarly disrupted by systemic amphetamine (Norman & Cassaday, 2003; Weiner, Lubow, & Feldon, 1988) and selective disruption of hippocampal a5 GABA-A receptors (Gerdjikov et al., 2008; Yee et al., 2004). The present study tested if GlyT1fl/fl:CaMKII-Cre+/ mutant mice with a phenotype of enhanced sensitivity to the non-reinforced history of a potential CS (Yee et al., 2006) might also express a phenotype of increased sensitivity to CS–US temporal discontiguity. If so, the differential expression of CR acquired under the trace and no-trace conditions would be larger in the mutant than control mice.
2. Materials and methods 2.1. Subjects A homozygous Glyt1tm1.2 fl/fl colony was established and maintained on a pure C57BL/6 background as described before (Yee et al., 2006). Forebrain neuron specific deletion of GlyT1 was achieved by CamKIIaCre-mediated recombination of the floxed GlyT1 allele. Appropriate heterozygous Cre mice were mated with Glyt1tm1.2 fl/fl mice to generate the desired mutant and control littermates. Animals of both sexes were employed in the present study. The mice were weaned at 21 days old, and littermates of the same sex were kept in groups of four to six in Makrolon TypeIII cages (Techniplast, Milan, Italy). The subjects were housed in a temperature- and humidity-controlled (at 22 °C and 55% R.H.) animal vivarium under a reversed light-dark cycle with lights off from 07.00 to 19.00 h. Testing was always conducted in the dark phase of the cycle. The animals were maintained under ad libitum water and 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 Swiss Federal Act on Animal Protection (1978) and Swiss Animal Protection Ordinance (1981) in accordance with the European Council Directive 86/609/EEC (1986). All efforts had been made to alleviate animal 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 measured using the conditioned freezing paradigm based on established parameters known to induce a robust trace conditioning effect in mice (Yee et al., 2004). The apparatus consisted of two sets of four conditioning chambers. The two sets were distinct from each other, and were installed in separate testing rooms, providing two distinct contexts as fully described before (Yee et al., 2006). The first set of chambers (context ‘A’) comprised four Coulbourn Instruments (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 centre, and through which scrambled electric shocks (the US, set at 0.3 mA) could be delivered (model E13-14; Coulbourn Instruments). A transparent Plexiglas enclosure confined the animals to a rectangular region (17.5 13 cm). The inside of the chambers was illuminated by a house light (2.8 W) positioned on the panel wall, 21 cm above the grid floor. The second set of chambers (context ‘B’) comprised four cylindrical (19 cm in diameter) enclosures made of clear Plexiglas resting on a metal mesh floor. Illumination inside the chamber was provided by an infrared light source instead of visible light. The CS was an 86-dBA tone provided by a sonalert (model SC628; Mallory, Indianapolis, IN, USA). Each of the eight chambers contained a miniature digital camera mounted 30 cm directly above the centre of the area of interest. The algorithm of the freezing response detection procedure has been validated and fully described before (Richmond et al., 1998). A detailed description of the trace conditioning procedure has been published elsewhere (Yee et al., 2004). The subjects of each group were randomly allocated into one of two conditions of CS–US pairing differing in the (trace) interval between CS offset and US onset: either 0 or 20 s (‘no-trace’ and ‘trace’ conditions, respectively). The respective 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, the animals were given three trials of CS–US pairings (CS: 30 s, US: 1 s) in context A. Each trial was preceded and followed by a 120 s inter-trial interval. 24 h later, the CR to the conditioning context was evaluated by returning the animals to context A for a period of 8 min. Another 24 h later, conditioned freezing to the tone CS was assessed in the neutral context B. The test session began with a 2 min acclimatization period, followed by the presentation of the CS for 8 min; and then the CS was turned off and the animals left in the chamber for an additional 2 min. 2.3. Statistical analysis All data were analysed by parametric analysis of variance (ANOVA) 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 with the inclusion of the within-subject factor time bins was performed in the analysis of data obtained on the CS-test day to provide a temporal profile of the phenotype. Fisher’s Least Significant Difference (LSD) post hoc pair-wise comparisons and planned contrast analysis were performed to assist interpretation of statistically significant effects emerged from an overall ANOVA. In addition, supplementary restricted ANOVAs and one-sample t-tests were
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applied to subsets of data. All statistical analyses were carried out using 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 seconds the animals were observed as immobile and expressed as percent time freezing within specific periods: CS, pre-CS and trace. The three variables were separately analysed. Supplementary analyses restricted to animals exposed either to the ‘no-trace’ or the ‘trace’ condition on day 1 were also performed to allow the comparison of freezing between distinct periods. 3.1.1. CS periods The level of freezing during the tone-CS presentation increased progressively across the three CS–US trials (Fig. 1A), but this increment was noticeably weaker in the ‘trace’ relative to the ‘no-trace’ condition, reaching a maximal difference on the third CS presentation. These impressions were confirmed by a 2 2 2 3 (genotype trace sex trials) split-plot ANOVA of percent time freezing, which revealed a significant main effect of trials [F(2,100) = 35.38, p < .01], trace [F(1,50) = 10.39, p < .01], and their 2-way interaction [F(2,100) = 8.03, p < .01]. Post hoc pair-wise comparison confirm that the difference between trace and no-trace conditions (collapsed across genotypes and sex) only achieve statistical significance on the third trial [t(100) = 5.66, p < .01]. Incidentally, male mice generally exhibited stronger freezing than female mice regardless of genotype and training condition, yielding a statistically significant sex effect [F(1,50) = 12.41, p < .01] but not any of its interactions. The average time of freezing in the presence of the CS on day 1 was 7.3 ± 1.4% in the female and 14.4 ± 1.5% in the male. 3.1.2. Pre-CS periods Freezing levels during the 30 s preceding the CS similarly increased across the three Pre-CS periods (Fig. 1B) in a manner that was essentially independent of genotype, training condition or sex. The increase in Pre-CS freezing likely reflected a general increase in fear due to the shock US rather than being specifically attributable to the CS itself. A 2 2 2 3 (genotype trace sex Pre-CS periods) ANOVA of percent time freezing only yielded a significant main effect of Pre-CS periods [F(2,100) = 73.71, p < .01]. 3.1.3. Trace intervals This variable refers to the freezing levels recorded during the 20 s trace intervals, and was therefore only available in animals tested under the trace condition. Again, a monotonic increase in freezing was observed across the three trace intervals in animals tested under the trace condition (Fig. 1C). Similar to the analysis of freezing recorded in the CS-periods above, a sex difference was observed with the male freezing at 24.5 ± 2.8% vs. female at 15.5 ± 2.5% on average. These led to the emergence of a significant main 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 trace intervals) ANOVA of percent time freezing. 3.1.4. Comparison between variables In the no-trace condition, a 2 2 3 2 (genotype sex trials dependent variables) ANOVA comparing CS and Pre-CS freezing variables only yielded an effect of trials as expected [F(2,48) = 53.79, p < .001]. And, there was no statistical indication
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that the animals froze differentially between the pre-CS and CS periods (see Fig. 1A and B). In the trace condition, a 2 2 3 3 (genotype sex trials dependent variables) ANOVA yielded a significant effect of variables [F(2,52) = 20.35, p < .001] as well as its interaction with trials [F(4,104) = 10.75, p < .001] besides the expected main effect of trials [F(2,52) = 62.97, p < .001]. Freezing during the CS period exhibited by animals trained under the trace condition became visibly lower than that observed in the Pre-CS and/or trace periods as conditioning progressed from trials 1 to 3 (Fig. 2). Notably, CSfreezing increased at the slowest pace, leading to a clear divergence from Pre-CS and trace interval freezing by the third trial. In trials 2 and 3, therefore, the transition from the Pre-CS to the CS periods was associated with a drop in freezing [F(1,26) = 16.99, p < .001 based on a restricted ANOVA] whereas the transition from the CS period to the succeeding trace interval corresponded to a rebound in freezing [F(1,26) = 65.46, p < .001 based on a restricted ANOVA]. The CS thus appeared to disrupt the on-going expression of freezing (indexed by Pre-CS freezing) on trials 2 and 3 on day 1, suggesting that the animals undergoing trace conditioning might have learned to perceive the CS as a safety signal. Although the average time freezing was numerically the highest during the trace intervals, a separate restricted ANOVA did not reveal a significant difference between the freezing levels obtained in the Pre-CS periods and trace intervals across trials 2–3 [p = .071]. Hence, the animals seemingly did not substantially differentiate between the 30 s before the CS from the 20 s following the CS (when the physical conditions of the test chambers were essentially identical) – i.e., the animals did not perceive the 20-s trace interval as a better predictor of the shock US compared with the pre-CS period.
3.2. Conditioned context freezing (day 2) The expression of conditioned freezing developed towards the training context was evaluated 24 h after conditioning by reexposing the animals to the training context in the absence of any discrete CS (Fig. 3). No difference was apparent between genotypes, sexes or training conditions. A 2 2 2 (genotype sex trace) ANOVA of percent time freezing over the 8-min test 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 of freezing was measured in a neutral context. The animals were acclimatized to the new context for 2 min (i.e., Pre-CS period) before the CS was presented continuously for a period of 8 min (i.e., CS-period). The test session was concluded with another 2-min in which the CS was absent (i.e., post-CS period).
3.3.1. Pre-CS period As shown in Fig. 4A, the general level of spontaneous freezing (immobility) was initially low but a gradual increase over time was visible. This increase was similarly observed in both genotypes regardless of training conditions (i.e., trace vs. no-trace). However, a sex difference was detected as male mice generally exhibited a higher level of spontaneous freezing (mean ± SE: male = 14.7 ± 1.7% vs. female = 9.6 ± 1.6%) regardless of genotype and training conditions. These impressions were confirmed by a 2 2 2 4 (genotype sex trace 30-s bins) ANOVA of percent time freezing during the Pre-CS period, which yielded a significant 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.
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3.3.2. CS Period Presentation of the CS predictably led to a rapid increase in freezing observed in animals previously conditioned under the no-trace condition. By contrast, the increase was modest in animals conditioned under the trace condition (Fig. 4B). This difference constitutes the trace conditioning effect; and its expression was visibly comparable between mutants and controls over the first 2 min. The data were examined with a finer temporal resolution across 10-s bins over the first minute of the CS as shown in Fig. 4D. This shows that 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 a time 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 into the CS presentation (Fig. 4B). The size of the trace effect, as illustrated by the stronger freezing in the no-trace condition relative to the weaker freezing in the trace condition, was significantly larger in the mutant mice. This genotype effect remained detectable at 7 min into the CS period. A 2 2 2 16 (genotype sex trace 30-s bins) ANOVA revealed a significant genotype by trace interaction [F(1,50) = 4.60, p < .05] and the temporal dependency of this effect [genotype trace bins: F(15,750) = 1.84, p < .05]. These genotypes effects were accompanied by the significant 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 investigated and characterized by pair-wise comparisons and planned contrast analysis (see Fig. 4B). First, a significant trace effect was detectable in the mutants throughout the 8-min CS-period. Second, the trace conditioning effect in the controls became weak in the middle of the CS-period. Pairwise comparisons between control/trace and control/no-trace animals failed to yield a significant difference in CS 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 apparent in the middle of the CS-period. The interaction of interest is identical to the contrast [(control/trace – control/no-trace animals) – (mutant/trace – mutant/no-trace animals)], i.e., with contrast coefficients (+1, 1, 1, +1). This specific contrast was significant from the third to the seventh minutes into the CS period except at bin 12 (see Fig. 4B). The temporal dependent profile of this phenotype is consistent with the lack of a genotype by trace interaction when the analysis was restricted to the first one [F = .001, ns] or two minutes [F = .16, ns] of the CS-period. Last, planned comparisons were performed to examine whether the phenotype on trace conditioning was associated with bidirectional effects in
freezing between the trace and the no-trace conditions. Suggestions for stronger freezing in the mutant in the no-trace condition was obtained in bins 7–8 and 10–11, and for weaker freezing in the mutant in the trace condition was obtained in bins 5–6 and 8–10 (see Fig. 4B). 3.3.3. Post-CS Period Examination of the post-CS period clearly showed a sharp increase in freezing that was most notable in animals trained under the trace condition. Although an increase was visible also in the animals trained under the no-trace condition, it was substantially weaker. The immediate response to the CS-offset was examined by an analysis of a difference score indexing the elevation of freezing levels from the last 30-s bin of the CS period to the first 30-s bin of the post-CS period (Fig. 4E). A 2 2 2 (genotype sex trace) ANOVA of the difference score revealed a main effect trace, consistent with the impression that animals trained under the trace condition responded more strongly to the termination of the CS [F(1,50) = 5.60, p < .05]. One-sample t-tests further indicated that only the mean difference score obtained from the two trace groups differed significantly from zero. The levels of freezing observed in the initial 30 s of the post-CS period (i.e., first post-CS bin in Fig. 4C) did not differ substantially between groups. Subsequently, the animals trained under the trace condition maintained a level of freezing that was substantially higher than animals trained under the no-trace condition (i.e., over the last 3 bins in Fig. 4C). In other words, the separation between trace and no-trace conditions now took a form that was the reverse of that seen in the CS-period. This may be expected if the trace animals rather than the no-trace animals had perceived the withdrawal of CS itself as a predictor of shock US. A 2 2 2 4 (genotype sex trace 30-s bins) ANOVA confirmed the presence of a highly significant trace effect [F(1,50) = 8.19, p < .01] as well as its interaction with bins [F(3,150) = 3.06, p < .05] and the main effect of bins [F(3,150) = 29.04, p < .001]. 4. Discussion The present study clearly demonstrated that the expression of the trace conditioning effect over time was modified by forebrain neuronal deletion of GlyT1. This phenotype revealed in the GlyT1fl/fl:CaMKII-Cre+/ mutant mice was specific to the extinction test of CS freezing, because neither acquisition performance (on day 1) nor the expression of context-freezing (on day 2) was significantly modified by the genetic disruption (Figs. 1–3). The 2-min
Fig. 1. Development of conditioned freezing across three CS–US pairings: (A) freezing behaviour is indexed by the percentage of time freezing during the 30 s of tone-CS presentation and is depicted for the no-trace (open symbols) and the trace (filled symbols) conditions separately. Freezing during the CS observed in the trace condition was weaker than in the no-trace condition – an effect that emerged over the three conditioning trials – and was comparable between genotypes. (B) Expression of freezing behaviour during the 30 s immediately preceding each CS presentation is shown as a function of trials. This was comparable between test conditions and genotypes. (C) For animals tested under the trace condition only, freezing levels during the 20-s trace intervals are depicted separately. Error bars refer to ±SEM.
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Fig. 2. A general increase in freezing was observed in all three measures (Pre-CS, CS and trace periods) in animals trained under the trace conditioning procedure on day 1. The rate of increase across conditioning trials 1–3, however, differed between the three variables. Notably, CS-freezing progressed at the slowest pace, leading to a clear divergence from Pre-CS and trace interval freezing by the third trial. In trials 2 and 3, therefore, we saw a drop of freezing from Pre-CS to the CS period, followed by a rebound when the CS had ended and the trace interval began. Error bars refer to ±SEM.
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described as stemming from a retardation of extinction. A similar suggestion has been made in the initial report of enhanced conditioned freezing in the GlyT1fl/fl:CaMKII-Cre+/ mice (Yee et al., 2006), which underwent Pavlovian conditioning procedures identical to the no-trace condition here. Yet, two subsequent reports that replicated the enhanced conditioned freezing phenotype in GlyT1fl/fl: CaMKII-Cre+/ mice did not obtain any evidence for a concomitant resistance to extinction (Dubroqua, Boison, Feldon, Mohler, & Yee, 2011; Dubroqua et al., 2010). This might undermine the interpretation that disruption of forebrain neuronal GlyT1 primarily affects the rate of extinction rather than the strength of the CS–US association. Nevertheless, equally crucial to the interpretation of the trace conditioning phenotype here is that the extinction perspective would view the weaker freezing in the mutant/trace group (relative to the control/trace group) as a facilitation of extinction. Hence, CS–US contiguity during acquisition can critically decide whether the rate of extinction would be slowed down or speeded up by the conditional deletion of GlyT1 in the forebrain. Here, the mutation effectively reversed the effect of CS–US contiguity on the rate of extinction seen in control mice (Fig. 4B). 4.2. The temporal perception of the CS
Fig. 3. Test of context freezing: The freezing response induced by the training context 24 h after conditioning was comparable amongst groups. Error bars refer to ±SEM.
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) emerged as soon as the CS was presented (Fig. 4B), and the separation between trace and no-trace groups was highly comparable between genotypes over the first two minutes in the presence of the CS. Essentially, no phenotype was evident in this period because the freezing behaviour observed was indistinguishable between mutant and control mice belonging to the same training condition during these two minutes. The observation that GlyT1fl/fl:CaMKIICre+/ mutant mice displayed a stronger trace conditioning effect only became detectable afterwards. Hence, the trace conditioning phenotype emerged over time, even though the average performance over the 8-min CS period gave an impression that the trace conditioning was significantly enhanced in the mutant mice. Post-hoc analyses confirmed that this phenotype was only robustly expressed in the mid-session, and indicated that the temporal dependency of this novel phenotype must be addressed. 4.1. The contribution of extinction over the extended CS presentation in the CS-test Because the phenotype did not emerge immediately as the CS was presented, it is important to consider the potential contribution of extinction learning over the extended period of the CS presentation. From this perspective, the enhanced freezing in the mutants relative to the controls in the no-trace group may be
Common to most conditioned freezing experiments (e.g. Zelikowsky, Bissiere, & Fanselow, 2012), the expression of the conditioned response (i.e., freezing) was measured in a novel context in which the CS was presented continuously for a period well beyond the duration of the CS presented during acquisition. One consequence of this procedure is that the CS might not be perceived as identical to the training CS when it was extended beyond 30 s in the test. Indeed, generalization decrement is predicted when the CS differs in duration between acquisition and test (Gallistel & Gibbon, 2000; Pavlov, 1927). One may therefore prefer to focus on the first 30 s of the CS presentation in the test session, because the data would be free from the confounding impact of generalization decrement as well as of extinction learning. As shown in Fig. 4B, there was no evidence to suspect that the expression of the trace conditioning effect had been modified by the GlyT1 disruption. Indeed, the mutant and controls mice (within either the no-trace or trace group) were highly comparable when the initial rise of the conditioned freezing response was tracked across 10-s bins (Fig. 4D). However, any attempt to explain the emergence of the phenotype later in the test as a consequence of an effect on generalization decrement resulting from the protraction of the CS is questionable. First, it cannot predict the abrupt expression of the phenotype at three minutes into the CS. Second, the phenotype did not become stronger gradually as the CS continued to extend over time; and if anything the phenotype was weaker by the end of the CS period (Fig. 4B). Hence, the temporal dynamics of the genotype effects undermine the possibility that the phenotype primarily reflects the effects of the mutation on generalization decrement 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 a safety signal on the conditioning day (day 1) in animals exposed to the trace conditioning procedure. By the third and final conditioning trial (but before shock US delivery), mutant and control mice in the trace group appeared to respond to the CS with a reduction in freezing by comparison to the pre-CS period immediately before, and to the trace interval that immediately followed the CS (Fig. 2). The rapid fall and rise of the freezing response is consistent with the interpretation that the CS was perceived as a safety signal. Although this effect was not significantly stronger
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in the mutants on the conditioning day, its possible contribution to the trace conditioning phenotype observed on the retention test on day 3 of the experiment is still worth considering. As a safety signal, the CS might therefore function as an inhibitor (see Bouton, 2007, p. 89) that prevents or reduces the expression of conditioned freezing in the trace groups recorded on the retention test. Consequently, the stronger trace conditioning effect seen in the mutants might stem from a stronger inhibitory CS–US link acquired by animals in the trace groups on the conditioning day. The possibility that conditional GlyT1 disruption promoted inhibitory as well as excitatory conditioning (Dubroqua et al., 2011; Yee et al., 2006) certainly warrants further investigation. Further experiments using paradigms that are specifically designed to measure the development of conditioned inhibition and safety signal, such as [A+/AB] (Rescorla, 1969) and [AX+/BX] (Kazama, Schauder, McKinnon, Bachevalier, & Davis, 2013; Myers & Davis, 2004) procedures, could clarify this interesting speculation.
1927), the animals trained under such conditions could also learn about the contingency between the trace itself and US. Indeed, freezing in the trace groups showed a rapid increase as soon as the CS was switched off (Fig. 4C), suggesting that they responded to the offset of the CS. This response was well-above that expressed by the animals in the no-trace groups that had never experienced a CS–US trace interval (Fig. 4E). Although freezing generally decreased over the rest of the post-CS period, the trace and no-trace groups clearly diverged, such that the trace groups exhibited stronger freezing than the no-trace control groups regardless of genotype. Indeed, the trace groups exhibited during the post-CS period their highest levels of freezing across the entire test session. To the extent that this analysis has shown that animals trained under the trace conditions had responded to the CS–US trace interval as a potential predictor of the shock US, there was no evidence that the disruption of GlyT1 had significantly altered the learning about the trace interval.
4.4. Learning about the trace
4.5. Comparing the trace conditioning and latent inhibition phenotypes of GlyT1 disruption
Although trace conditioning is operationally defined as weaker conditioned response to the CS when the pairing between CS and US during acquisition was interspersed by a trace interval (Pavlov,
Importantly, we qualify here that Pavlovian learning is not always enhanced (defined as a stronger CR specific to the CS) by
Fig. 4. Test of CS freezing: The freezing response recorded during the CS-test session was subjected to separate analyses: the Pre-CS (A), CS-period (B) and the post-CS periods (C). The data were presented as a function of successive 30-s bins. The significant three-way interaction observed in the CS period was further investigated and characterized by planned contrast analysis and pair-wise comparisons based on the error variance associated with the three-way interaction in the overall ANOVA. The contrast specifically compares between the mutants and controls the size of the trace conditioning effect at each successive bin. Those time bins in which this contrast achieved statistical significance [p < 0.05] are highlighted by the green underlay in B. Bins in which a significant pair-wise difference between control/no-trace and mutant/no-trace was detected are marked by ‘a’; and those in which a significant pair-wise difference between control/trace and mutant/trace was detected are marked by ‘b’. The first minute of the CSperiod is depicted as a function of 10-s bins to illustrate the evolution of the conditioned response to the CS at a higher temporal resolution (D). The increase of freezing levels from the last 30-s bin of the CS period to the first 30-s bin of the post-CS period is separately depicted (E). # Indicates a significant difference from zero (which indicates no change from the end of CS-period to the beginning of the post-CS period) based on one-sample tests. All data shown refer to mean ±SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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conditional GlyT1 disruption in forebrain neurons. Indeed, a reversed effect can be induced by specific changes in training conditions, but the psychological significance of this qualification remains to be fully appreciated and clarified. Under conditions that favour maximally the formation of the CS–US association with the CS being perceived unambiguously as a reliable and accurate predictor of the eminent occurrence of the US, the magnitude of the CR tends to be stronger in the mutants. On the other hand, deviation from such ideal conditions that corrupts this positive CS–US predictive link and thereby reduces CR, is seemingly more impactful in our mutant mice. This description fits not only the trace conditioning phenotype demonstrated here but also the latent inhibition phenotype previously reported (Yee et al., 2006). In latent inhibition, non-reinforced CS pre-exposure (presentation of CS alone, in the absence of any US) impedes the efficacy of subsequent CS–US pairing to produce a CR to the CS (Lubow and Moore, 1959). Mice with forebrain neuronal GlyT1 deletion are more sensitive to this CS pre-exposure effect: Latent inhibition was evident in the mutants when the amount of CS pre-exposure was insufficient to generate latent inhibition in wild type controls (Yee et al., 2006). According to DeVietti et al. (1987), the similar sensitivity to the latent inhibition (i.e., CS pre-exposure effect) and trace conditioning procedures may stem from a common psychological mechanism. They suggest that CS pre-exposures preferentially reduce attention to later segments of individual CS’s, which then effectively act as a trace interval between initial CS segments and the US during subsequent CS–US pairings. The perceived CS–US discontiguity thereby weakens conditioning and yields the LI effect (also see Ayres et al., 1987). Systemic amphetamine (Norman and Cassaday, 2003; Weiner et al., 1988) as well as selective disruption of hippocampal a5 GABA-A receptors (Gerdjikov et al., 2008; Yee et al., 2004) have been shown to reduce sensitivity to CS pre-exposure effect and CS–US discontiguity. Here, we extended these findings by showing that disruption of forebrain neuronal GlyT1 could enhance both the sensitivity to CS pre-exposure effect and CS–US discontiguity. It remains to be determined whether these parallel or complementary effects of these three distinct brain manipulations may be mediated through a common neural circuitry. 4.6. Conclusion Although further experiments are necessary to delineate the precise psychological and molecular mechanisms underlying the novel phenotype of GlyT1fl/fl:CaMKII-Cre+/ mutant mice, our experiment shows that the CS–US contiguity at the time of conditioning can critically determine how the genetic disruption might modify the expression of the acquired conditioned response. The present study thus reveals important insights into the role of forebrain neuronal GlyT1 in determining how past experience may shape current behaviour through Pavlovian learning mechanism. Acknowledgments The present study was supported by Swiss National Science Foundation Grant (3100–066855) with additional support from the Swiss Federal Institute of Technology Zurich. BKY received additional support from the National Institutes of Health (MH083973). SD and PS were recipients of a studentship from the Neural Plasticity & Repair – National Centre for Competence in Research (NCCR) funded by the Swiss National Science Foundation. We thank Prof. Hanns Möhler and Dr. Detlev Boison of Zurich University for providing the breeding pairs for the generation of the GlyT1fl/fl conditioned mouse line. The software development and maintenance of the behavioural equipment were provided by the late Peter Schmid, to whom we would like to express our
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deep gratitude for his excellent service over two decades. We are also indebted to the animal husbandry staffs and to Dr. Joram Feldon for making available the animal facilities and behavioural equipment necessary for the reported experiments.
References Ayres, J. J., Albert, M., & Bombace, J. C. (1987). Extending conditioned stimuli before versus after unconditioned stimuli: Implications for realtime models of conditioning. Journal of Experimental Psychology: Animal Behavioural Process, 13, 168–181. Berger, A. J., Dieudonne, S., & Ascher, P. (1998). Glycine uptake governs glycine site occupancy at NMDA receptors of excitatory synapses. Journal of Neurophysiology, 80, 3336–3340. Bergeron, R., Meyer, T. M., Coyle, J. T., & Greene, R. W. (1998). Modulation of Nmethyl-D-aspartate receptor function by glycine transport. Proceedings of the National Academy of Sciences of United States, 95, 15730–15734. Bouton, M. E. (2007). Learning and behavior: A contemporary synthesis. Sunderland, MA: Sinauer Associates Inc.. DeVietti, T. L., Bauste, R. L., Nutt, G., & Barrett, O. V. (1987). Latent inhibition: A trace conditioning phenomenon? Learning and Motivation, 67, 416–422. Dubroqua, S., Boison, D., Feldon, J., Mohler, H., & Yee, B. K. (2011). Examining the sex- and circadian dependency of a learning phenotype in mice with glycine transporter 1 deletion in two Pavlovian conditioning paradigms. Neurobiology of Learning and Memory, 96, 218–229. Dubroqua, S., Singer, P., Boison, D., Feldon, J., Mohler, H., & Yee, B. K. (2010). Impacts of forebrain neuronal glycine transporter 1 disruption in the senescent brain: Evidence for age-dependent phenotypes in Pavlovian learning. Behavioral Neuroscience, 124, 839–850. Gallistel, C. R., & Gibbon, J. (2000). Time, rate, and conditioning. Psychological Review, 107, 289–344. Gerdjikov, T. V., Rudolph, U., Keist, R., Mohler, H., Feldon, J., & Yee, B. K. (2008). Hippocampal alpha 5 subunit-containing GABA A receptors are involved in the development of the latent inhibition effect. Neurobiology of Learning and Memory, 89, 87–94. Harvey, R. J., & Yee, B. K. (2013). Glycine transporters as novel therapeutic targets in schizophrenia, alcohol dependence and pain. Nature Reviews Drug Discovery, 12, 866–885. Javitt, D. C. (2009). Glycine transport inhibitors for the treatment of schizophrenia: Symptom and disease modification. Current Opinion in Drug Discovery & Development, 12, 468–478. Javitt, D. C. (2012). Glycine transport inhibitors in the treatment of schizophrenia. Handbook of Experimental Pharmacology, 213, 367–399. Kazama, A. M., Schauder, K. B., McKinnon, M., Bachevalier, J., & Davis, M. (2013). A novel AX+/BX paradigm to assess fear learning and safety-signal processing with repeated-measure designs. Journal of Neuroscience Methods, 214(2), 177–183. http://dx.doi.org/10.1016/j.jneumeth.2013.01.022. Kleckner, N. W., & Dingledine, R. (1988). Requirement for glycine in activation of NMDA receptors expresses in Xenopus oocytes. Science, 214, 835–837. Lubow, R. E., & Moore, A. U. (1959). Latent inhibition: The effect of nonreinforced pre-exposure to the conditional stimulus. Journal of Comparative Physiological Psychology, 52, 415–419. Mackintosh, N. J. (1975). Theory of attention – Variations in associability of stimuli with reinforcement. Psychological Review, 82, 276–298. Möhler, H., Boison, D., Singer, P., Feldon, J., Pauly-Evers, M., & Yee, B. K. (2011). Glycine transporter 1 as a potential therapeutic target for schizophrenia-related symptoms: Evidence from genetically modified mouse models and pharmacological inhibition. Biochemical Pharmacology, 81, 1065–1077. Myers, K. M., & Davis, M. (2004). AX+, BX discrimination learning in the fearpotentiated startle paradigm: Possible relevance to inhibitory fear learning in extinction. Learning & Memory, 11, 464–475. Norman, C., & Cassaday, H. J. (2003). Amphetamine increases aversive conditioning to diffuse contextual stimuli and to a discrete trace stimulus when conditioned at higher footshock intensity. Journal of Psychopharmacology, 17, 67–76. Pavlov, I. P. (1927). Conditioned reflexes: An investigation of the physiological activity of the cerebral cortex. London: Oxford University Press. Pearce, J. M., & Hall, G. (1980). A model for Pavlovian learning: Variations in the effectiveness of conditioned but not of unconditioned stimuli. Psychological Review, 87, 532–552. Pinard, E., Alanine, A., Alberati, D., Bender, M., Borroni, E., Bourdeaux, P., & Zimmerli, D. (2010). Selective GlyT1 inhibitors: Discovery of [4-(3-fluoro-5trifluoromethylpyridin-2-yl)piperazin-1-yl][5-methanesulfonyl-2-((S)-2,2,2trifluoro-1-methylethoxy)phenyl]methanone (RG1678), a promising novel medicine to treat schizophrenia. Journal of Medicinal Chemistry, 53, 4603–4614. Rescorla, R. A. (1969). Conditioned inhibition of fear resulting from negative CS–US contingencies. Journal of Comparative Physiological Psychology, 67, 504–509. Richmond, M. A., Murphy, C. A., Pouzet, B., Schmid, P., Rawlins, J. N., & Feldon, J. (1998). A computer controlled analysis of freezing behaviour. Journal of Neuroscience Methods, 86, 91–99. Smith, K. E., Borden, L. A., Hartig, P. R., Branchek, T., & Weinshank, R. L. (1992). Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors. Neuron, 8, 927–935.
54
P. Singer et al. / Neurobiology of Learning and Memory 110 (2014) 47–54
Supplisson, S., & Bergman, C. (1997). Control of NMDA receptor activation by a glycine transporter co-expressed in Xenopus oocytes. Journal of Neuroscience, 17, 4580–4590. Wagner, A. R. (1981). SOP: A model of automatic memory processing in animal behavior. In N. E. Spear & R. R. Miller (Eds.), Information processing in animals: Memory mechanisms (pp. 5–47). Hillsdale, NJ: Erlbaum. Weiner, I., Lubow, R. E., & Feldon, J. (1988). Disruption of latent inhibition by acute administration of low doses of amphetamine. Pharmacology, Biochemistry and Behavior, 30, 871–878. Yee, B. K., Balic, E., Singer, P., Schwerdel, C., Grampp, T., Gabernet, L., et al. (2006). Disruption of glycine transporter 1 restricted to forebrain neurons is associated
with a procognitive and antipsychotic phenotypic profile. Journal of Neuroscience, 26, 3169–3181. Yee, B. K., Hauser, J., Dolgov, V. V., Keist, R., Mohler, H., Rudolph, U., et al. (2004). GABA receptors containing the alpha5 subunit mediate the trace effect in aversive and appetitive conditioning and extinction of conditioned fear. European Journal of Neuroscience, 20, 1928–1936. Zelikowsky, M., Bissiere, S., & Fanselow, M. S. (2012). Contextual fear memories formed in the absence of the dorsal hippocampus decay across time. Journal of Neuroscience, 32, 3393–3397.