Conditional loss of GluN2B in cortex and hippocampus impairs attentional set formation

Abstract

The ability to attend to appropriate stimuli, to plan actions and then alter those actions when environmental conditions change, is essential for an organism to thrive. There is increasing evidence that these executive control processes are mediated in part by N-methyl-D-aspartate receptors (NMDAR). NMDAR subunits confer different physiological properties to the receptor, interact with distinct intracellular postsynaptic scaffolding and signaling molecules and are differentially expressed during development. Recent findings have suggested that the GluN2B subunit may play a unique role in both the acquisition of adaptive choice and the behavioral flexibility required to shift between choices. Here we investigated the role of GluN2B containing NMDARs in the ability to learn, reverse and shift between stimulus dimensions. Mutant mice (floxed-GluN2B x CaMKII-Cre) lacking GluN2B in the dorsal CA1 of the hippocampus and throughout the cortex were tested on an attentional set-shifting task. To explore the role that alterations in motor behavior may have on these behaviors, gross and fine motor behaviors were analyzed in mutant and floxed-control mice. Results show that corticohippocampal loss of GluN2B selectively impaired an initial reversal in a stimulus specific manner and impaired the ability of mutant mice to form an attentional set. Further, GluN2B mice showed normal motor behavior in both overall movement and individual limb behaviors. Together, these results further support the role of NMDAR, and GluN2B in particular, in aspects of executive control including behavioral flexibility and attentional processes.

Introduction

Alterations in executive control domains that accompany numerous neuropsychiatric, developmental, and substance abuse disorders have come into increasing focus due to consistent reports of severe quality of life impact in clinical populations. The range of cognitive processes included in executive control includes planning, attention, behavioral flexibility and working memory. Although not as readily apparent as more global deficits in cognition, alterations in executive control can lead to difficulty thriving in complex environments (Brennan and Arnsten, 2008, Evans et al., 2013). In particular, deficits in attention can cause problems maintaining focus on salient environmental stimuli and an ability to effectively shift attention from one task to another.

Attentional processes across species are mediated by distinct cortical subregions that support different aspects of learning and behavioral flexibility. In rodents, distinct functional regions within the ventromedial prefrontal cortex (vmPFC) are involved in supporting these different roles in attention (Floresco and Jentsch, 2011, Hamilton and Brigman, 2015). Of particular note, the anterior cingulate cortex (ACC) has been shown to be involved in mediating associative learning by directing attention to stimulus features, while the prelimbic cortex (PrL) is involved in the maintenance of attentional strategy once it is established (Bussey et al., 1997, Oualian and Gisquet-Verrier, 2010). These and other subregions of the vmPFC also support the ability to shift attention when the cognitive demands of task change. For example, there is evidence that the infralimbic cortex (IL) drives top-down control of attention required to initiate shifts between stimulus dimensions during attentional set-shifting (Bussey et al., 1997a, Ragozzino et al., 1999, Birrell and Brown, 2000a, Bissonette et al., 2008, Floresco et al., 2008, Oualian and Gisquet-Verrier, 2010). This type of behavioral flexibility is complementary but functionally distinct from reversal learning, which requires a shift in response to a previously present but unrewarded stimulus, and is mediated by vmPFC subregions, as well as the orbitofrontal cortex (OFC) (Dias et al., 1996, Chudasama and Robbins, 2003, Moore et al., 2009, Rudebeck and Murray, 2011, Rudebeck et al., 2013).

Both the ability to focus attentional processes to obtain desired outcomes, and quickly shift those responses as environmental demands change, requires the induction of plasticity within cortical circuits. The N-methyl-D-aspartate receptor (NMDAR) is posited to be an important molecular mechanism subserving such plasticity, given its critical role in learning and certain forms of synaptic plasticity (Cotman et al., 1988, Malenka and Bear, 2004, Bannerman et al., 2006). More specifically, NMDARs containing GluN2B, the primary subunit expressed in the cortex and hippocampus throughout development (Cull-Candy et al., 2001, Kohr et al., 2003, Marquardt et al., 2014) has been proposed to play a key role in behavioral flexibility. In support of this, pharmacological blockade of GluN2B via Ro25-6981 or age-related loss of GluN2B impairs hippocampal and cortical long-term potentiation (LTP), and associated learning processes (Clayton et al., 2002, Takehara et al., 2004, Zhao et al., 2005, Nakazawa et al., 2006, Gardoni et al., 2009). Moreover, and of particular relevance to the current study, systemic administration of a GluN2B antagonist has been found to impair operant reversal learning and set-shifting in rats (Dalton et al., 2011b). However, the role of GluN2B in specific brain regions, including the vmPFC, in mediating cognitive flexibility remains unclear.

Early genetic models of global GluN2B loss were unsuitable for behavioral phenotyping due to neonatal lethality produced by constitutive deletion of the subunit, though neonates were found to have deficient long-term depression (LTD) (Kutsuwada et al., 1996). As an alternative approach using a Cre/LoxP-based conditional model, we have previously shown that post-developmental loss of GluN2B in the cortex and the dorsal CA1 subregion of the hippocampus impairs spatial and contextual learning and hippocampal LTP (Brigman et al., 2010). These GluN2B mutants were also found to be impaired on tasks dependent upon cortically-mediated behavioral flexibility, in the form of reversal learning, a finding replicated by pharmacological GluN2B blockade (via Ro 25-6981) in the OFC. To extend this data and further elucidate the role of corticohippocampal GluN2B in cognitive flexibility, we tested GluN2B^NULL mice on an attentional set-shifting task (ASST) that entails elements of discriminative learning, reversal, and set-shifting in species-specific stimulus domains (Birrell and Brown, 2000b, Young et al., 2010). In addition, given the motor demands required for successful performance in the ASST and the relative lack of information regarding motor behavior in this model, we also phenotyped the mutants for fine motor and movement patterns using an advanced gait analysis system.

Materials and Methods

Subjects

GluN2B mutant mice were generated as previously described (Brigman et al., 2010, Brigman et al., 2013). Briefly, the GluN2B gene was disrupted by inserting a loxP site downstream of the 599 bp exon 3 or exon 5 (depending on transcript) and a neomycin resistance gene cassette flanked by 2 loxP sites upstream of this exon. A 129/SvEvTac was used as the embryonic stem cell donor and C57BL/6J was used for blastocysts and as the genetic background for backcrossing. Analysis of 150 single nucleotide polymorphism markers at 15–20 Mb intervals estimated the genetic background of the mutant cross to be 95% C57BL/6J (Brigman et al., 2010). Floxed mutant mice were crossed with (C57BL/6J-congenic) transgenic mice expressing Cre recombinase driven by the CaMKII promoter (T29-1 line) (Fukaya et al., 2003) to produce mutant mice with excision of GluN2B (GluN2B^NULL) and non-excised floxed littermate controls (GluN2B^FLOX).

Mice were bred at the University of New Mexico Health Sciences Center and housed in same sex groupings of 2–4 per cage in a temperature- and humidity- controlled vivarium. Lighting was on a reverse 12 h light/dark cycle (lights off 0800 h) and testing was performed during the dark phase. Male and female mice (aged 8 weeks at onset of testing, n= 6–7 per sex/genotype) were slowly reduced and then maintained at ~85% free-feeding body weight to ensure motivation to work for food reward. All experimental procedures were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the University of New Mexico Health Sciences Center Institutional Animal Care and Use Committee.

Attentional Set-Shifting Task (ASST)

Prior to testing, mice were acclimated to the 14 mg pellet food reward by provision of 10 pellets per mouse in the home cage for 7 days while in the testing room. Testing was conducted in an acrylic chamber measuring 30 × 18 × 12 cm, divided into a start box and 2 choice chambers. Two ceramic digging bowls (4.5 × 2.5 cm) were placed on platforms (11 × 5 cm) in each choice chamber and were separated from the start box by a clear acrylic panel. Access to digging bowls was limited by a removable divider. Scented medium was made by mixing 150 g of cob bedding with 20 crushed 14 mg dustless precision pellets (#F0568, BioServ, Frenchtown, NJ) and 3 g of commercially available powdered spices: ginger, nutmeg, garlic, coriander, thyme and cinnamon (McCormick & Company, Sparks, MD, USA). Approach platforms were manufactured to size from commercially available materials in-house and included sandpaper, wood, neoprene, metal wire, tile and a plastic fiber sponge.

The ASST was conducted as previously described (Young et al., 2010, Marquardt et al., 2014). On day 1, mice were acclimated to the testing chamber and trained to dig in unscented cob medium for food reward by slowly increasing the digging requirement to receive pellets. First, pellets were available on the chamber floor and in the bottom of each empty digging bowl. Mice were allowed to explore the testing chamber until all pellets were eaten. Next, mice were required to retrieve pellets available in each digging bowl, placed on top of ~0.5 cm of digging medium. Following consumption of pellets on top of medium, 1 pellet was buried under ~0.5 cm of digging medium and 2 placed on top in each digging bowl. Across the next 5 consecutive trials, pellet placement was gradually shifted, digging medium increased, number of baited cups reduced, and pellets reduced until mice were trained to retrieve 1 pellet buried under ~2.5 cm in 1 of the 2 bowls. Mice were then given 6 more repetitions of this problem (1 pellet under ~2.5 cm of medium in 1 bowl) to reinforce task behavior. A total of 50 pellets were made available across all trial types of the first training session. Trials were timed from divider lifting until all pellets were consumed. Mice that ceased digging for pellets were returned to their home cage for 10 to 45 min before resuming their last attempted trial.

On Day 2, training introduced mice to each odor and platform combination (Table 1). A single food pellet was placed ~¾ beneath the surface of the digging medium in 1 bowl, which was assigned randomly between trials. The non-rewarded bowl was sham-baited to prevent food-odor confounds. There were a total of 24 trials to demonstrate each odor and platform combination with a ~15 sec inter-trial interval.

Table 1. Example of testing stages and stimulus combinations for the Attentional Set-Shifting Task.

Starting dimension (odor vs tactile) was counterbalanced across mice.

	Dimensions		Exemplars
	Relevant	Irrelevant	S+	S−
Simple Discrimination (SD)	Odor	n/a	O1	O2
Compound Discrimination (CD)	Odor	Platform	O1/P1	O2/P2
			O1/P2	O2/P1
Compound Discrimination Reversal (CDR)	Odor	Platform	O2/P1	O1/P2
			O2/P2	O1/P1
Intra-dimensional Shift (IDS)	Odor	Platform	O3/P3	O4/P4
			O3/P4	O4/P3
Intra-dimensional Shift Reversal (IDR)	Odor	Platform	O4/P3	O3/P4
			O4/P4	O3/P3
Extra-dimensional Shift (EDS)	Platform	Odor	P5/O5	P6/O6
			P5/O6	P6/O5
Extra-dimensional Shift Reversal (EDR)	Platform	Odor	P6/O5	P5/O6
			P6/O6	P5/O5

On day 3, mice were tested in succession on each of 7 problems (Table 1). For each problem, criterion was 6 consecutive correct trials and mice were discontinued if they required 60 trials on any 1 problem or 150 trials total. In the simple discrimination (SD), mice were trained to discriminate 2 exemplars in either the odor or platform dimension (counterbalanced across genotypes, n= 12–13 dimension/genotype). Upon reaching criterion mice were moved to the compound discrimination (CD), in which the second, non-rewarded dimension, was added. Mice were required to respond in accordance to the learned SD, and ignore the second dimension. For the first 4 trials of the SD and CD stages, mice were allowed to dig in the incorrect bowl without consequence, although an error was recorded. In subsequent trials, if an error was made the opposite choice chamber was blocked to prevent reward collection. If a correct response was made, the mouse was allowed to collect the pellet. If the mouse did not dig in either bowl within 2 min, the trial was recorded as ‘no decision.’ Upon completion of the CD, the rewarded exemplar was reversed to test for a compound discrimination reversal (CDR).

Following the CDR, a novel set of exemplars in each dimension was introduced and mice were rewarded for responding to 1 exemplar in the initially learned dimension (intra-dimensional shift (IDS)). Next, in an intra-dimensional reversal (IDR), there was a new correct stimulus, in the same dimension as in the IDS. Following IDR, a second novel set of exemplars in both dimensions was introduced to test for the extra-dimensional shift (EDS). Here, the rewarded exemplar was in the previously irrelevant dimension. Finally, the correct exemplar within the newly learned dimension was reversed to form an extra-dimensional reversal (EDR) problem.

Trials to criterion and errors were recorded for each stage. Trial latencies to respond were measured from the time the barrier was raised until digging was initiated. A dig was defined as the moment when the mouse’s nose or paw broke the surface of the cob-digging medium. Three GluN2B^NULL and 2 GluN2B^FLOX mice that either stopped digging or required > 150 trials to complete the ASST were omitted from the analysis.

Gait analysis

Following completion of the ASST, mice were tested for fine motor movement and gait using the Catwalk XT system (Noldus Information Technology, Wageningen, Netherlands), as previously described (Neumann et al., 2009, Huehnchen et al., 2013). The mouse was placed in a start box at one end of a 1.3 m long runway consisting of a glass platform covered by a removable black tunnel. The mouse was allowed to walk freely across the runway to reach their home-cage at the opposite end. The runway was illuminated from one side by reflected green fluorescent light. A high-speed camera recorded footprints on the glass from light reflected by downward pressure of individual footfalls. Three runway trials were recorded for each mouse, with a trial being regarded as compliant by the software if the mouse crossed the recording area in under 5 sec and did not show a maximum speed variation > 60%. All trials marked as compliant were reviewed manually, if the mouse stopped or turned in mid-run the trials were excluded.

Analysis was performed using Catwalk XT 8.1 Software. Gait and movement were analyzed for right fore (RF), left fore (LF), right hind (RH) and left hind (LH) paws on the following variables: paw print area (size of paw print area during a full stance), stride length (distance between 2 consecutive paw placements of the same paw), swing (time interval between 2 consecutive paw placements of the same paw), and swing speed (velocity of an individual paw between 2 consecutive placements). The percentage of step patterns categorized as cruciate, alternate or rotary was analyzed as previously described (Neumann et al., 2009).

Statistical analysis

For the ASST, trials to criterion were analyzed using multivariate analysis of variance (ANOVA) with stage (SD, CD, CDR, IDS, IDR, EDS, and EDR) as a within-subjects factor and sex, genotype and initial dimension as between-subjects factors. There were no significant main effects or interactions for sex, so males and females were combined. Since genotype and stage did not significantly interact with initial dimension, stage was not treated as an independent variable in subsequent analyses. Within-subjects ANOVAs were conducted to compare performance, within genotype, across the discrimination (SD, CD, IDS), reversal (CDR, IDR, EDR) and set-shifting (EDS) (Birrell and Brown, 2000). Attentional set formation was analyzed using a paired t-test. For gait analysis, ANOVA was calculated for each gait analysis category, with paw and genotype or step category and genotype as between-subject factors.

Results

ASST

All mice showed a significant reduction in trial latency across the 2 training sessions (F1,1=58.77; p<.01) with no significant differences between genotypes on either session of training (Fig 1a). No significant differences were found between genotypes on trial latencies across the entire problem series (ANOVA: p>.05, Fig 1b). No significant main effect (ANOVA: p>.05) of genotype, sex or initial dimension was seen for the problem series. However, there was a main effect of ASST stage (F1,6=2.97; p<.01) for all animals and a significant interaction (F1,6=4.25; p<.01) for stage x dimension. Newman-Keuls post-hoc tests revealed that all mice trained with odor as the salient dimension learned significantly faster than those that were trained on platform (F1,23=5.48; p<.05), an effect that was lost by the compound reversal stage.

Fig. 1. GluN2B^NULL mice are normal on measures of motivation on the Attentional Set-Shifting Task.

(a) Mice showed a significant reduction in trial latency across the 2 sessions of training, regardless of genotype. (b) No differences were seen between genotypes on motivation to perform any stage of the task, as measured by average trial latency. Task stages: T1= Training Session 1; T2= Training Session 2; SD= Simple Discrimination; CD= Compound Discrimination; CDR= Compound Reversal; IDS= Intra-dimensional Shift; IDR= Intra-dimensional Reversal; EDS= Extra-dimensional Shift; EDR= Extra-dimensional Reversal. n=12–13/genotype. *P<.01 T1 vs T2.

Analysis of stages that required a reversal of the previous association revealed a significant genotype x dimension interaction for the first reversal only (CDR) whereby GluN2B^NULL required significantly more trials to learn a compound reversal when platform, but not odor, was the salient dimension (F1,1=4.48; p<.05; Fig 2). No main effect of genotype or problem and no interaction were seen on the subsequent 2 (IDR, EDR) reversals. Newman-Keuls post-hoc tests revealed that all genotypes required significantly more trials to complete both the CDR and IDR versus the proceeding CD and IDS problem respectively (p<.05).

Fig. 2. GluN2B^NULL mice show a stimulus-specific impairment on an initial reversal.

GluN2B^NULL mice were no different from GluN2B^FLOX controls on an initial reversal of compound stimuli using odor stimuli, but required significantly more trials than GluN2B^FLOX controls to reverse when tactile stimuli were used. No significant differences between genotypes or stimulus dimensions were seen on either of the 2 subsequent reversals. CDR= Compound Reversal; IDR= Intra-dimensional Reversal; EDR= Extra-dimensional Reversal. n=12–13/genotype. *P<.01 interaction effect.

Analysis of stages that required only pairwise discrimination found no difference in trials to criterion by genotype or stage and no significant stage x genotype (ANOVA: p>.05). A comparison of IDS versus EDS performance revealed that GluN2B^FLOX (t(11)=−2.27; p<0.05) but not GluN2B^NULL (t(11)=−1.50; p=.16) required significantly more trials on the EDS task, confirming the formation of an attentional set in floxed controls (Fig 3). There were no significant differences in trial latencies by genotype or discrimination stage. Comparison of EDS stage performance revealed no significant main effect of genotype (ANOVA: p>.05).

Fig. 3. GluN2B^NULL mice show impairment in the formation of an attentional set.

No significant differences between genotypes or stimulus dimensions were seen on problems that required discrimination (SD, CD, IDS). Regardless of genotype, mice required significantly more trials to complete the CDR and IDR, relative to the preceding CD and IDS problem. However, while GluN2B^FLOX controls showed formation of attentional set, as measured by significantly increased trials to perform the EDS regardless of starting dimension, GluN2B^NULL mice did not. SD= Simple Discrimination; CD= Compound Discrimination; CDR= Compound Reversal; IDS= Intra-dimensional Shift; IDR= Intra-dimensional Reversal; EDS= Extra-dimensional Shift; EDR= Extra-dimensional Reversal. n=12–13/genotype. *P<.01 IDS vs EDS in GluN2B^FLOX.

Gait analysis

All mice from both genotypes were able to complete sufficient compliant runs to be included in gait analysis (Fig 4a). Examination of print area for left and right fore- and hind-paws revealed no main effect of genotype or paw and no significant interaction (ANOVA: p>.05; Fig 4b). Stride length analysis revealed a significant main effect of paw (F1,3=3.75; p<.05): Newman-Keuls post-hoc tests revealed that both left and right fore-paws had significantly longer stride length than hind-paws (Fig 4c). However, no significant main effect of genotype and no interaction was found (ANOVA: p>.05). Similarly, there was a main effect of paw with significantly increased swing for fore-paws versus hind-paws (F1,1=7.08; p<.01) but no main effect of genotype or interaction (ANOVA: p>.05, Fig 4d).

Fig. 4. GluN2B^NULL mice show normal gait and motor coordination.

(a) Representative compliant runs depicting automatically-detected left hind (LH), right hind (RH), left front (LF), and right front (RF) paws. (b) No significant differences were seen between genotypes for total print area for any paw. Genotypes did not differ in stride length (c), swing time (d), or swing speed (e). Alternate step patterns (f) were the dominate step sequences regardless of genotype. n=12–13/genotype. Aa, Ab=alternate step patterns; Ca, Cb=cruciate step patterns; Ra, Rb=rotary step patterns. n=12–13/genotype.

No main effect of paw or genotype and no interaction were seen for the swing speed variable (ANOVA: p>.05, Fig 4e). Finally, step pattern was examined by the 6 primary sequences within alternate (Aa: RF-RH-LF-LH; Ab: LF-RH-RF-LH), cruciate (Ca: RF-LF-RH-LH; Cb: LF-RF-LH-RH) and rotary (Ra: RF-LF-LH-RH; Rb: LF-RF-RH-LH) sequences. There was a significant main effect of sequence type with both alternate patterns (Aa and Ab) being the dominate sequence (Fig 4f). However, there was no main effect of genotype on step pattern and no interaction found (ANOVA: p>.05).

Discussion

The main finding of the current study was that mice with mutant loss of GluN2B containing NMDARs on cortical and CA1 hippocampal principal neurons showed a selective and sensory-specific deficit during an initial reversal problem and a failure to form an attentional set. Learning of both olfactory and tactile discriminations was normal in the mutants compared to floxed-controls, as was gait and fine motor function.

The current findings confirm and extend earlier work implicating GluN2B in cognitive flexibility. Prior pharmacological studies found that systemic administration of the GluN2B-selective antagonist, Ro25-6981, impaired reversal of a previously learned cued operant response and response-shifting to an egocentric strategy (Dalton et al., 2011). In addition, we have previously reported that the same GluN2B mutant mouse did not affect learning of a operant-based pairwise visual discrimination, but did impair the ability to reverse the learned choice – an effect that manifested during the early, cortically-mediated, phase of reversal learning (Brigman et al., 2013). Complementary pharmacological experiments showed that antagonism (via Ro 25-6981) of GluN2B in the lateral OFC replicated a reversal deficit in non-mutant mice, suggesting this loss of GluN2B in this region may have been responsible for the mutant phenotype (Brigman et al., 2013). The current findings demonstrate that cognitive inflexibility produced by corticohippocampal GluN2B loss generalizes across different experimental settings, by showing that the mutants were impaired on the first of 3 reversal problems in the ASST digging-task. Importantly, advanced gait analysis revealed no significant effects on limb control as measured by total step area, stride length, swing and swing speed or step patterns. This suggests that individual limb movements, overall gait and coordination are not altered by GluN2B loss. Given the high motor demands required to make choices and dig for appetitive reward in the ASST, it is essential to ensure that genetic or pharmacological manipulations do not alter either the motivation to perform the tasks or the motor systems required for efficient performance. Particularly as alterations in NMDAR function in the cortex may alter motor and motivational processes (Bannerman et al., 2006). This data also supports and compliments previous findings showing that alterations in GluN2B lead to deficits in cognitive processes while sparing general motor behavior (Brigman et al., 2010, Brigman et al., 2013).

Interestingly, however, we found that the impairment was specific to the stimulus modality needed to solve the initial reversal problem. Specifically, the mutants required more trials than controls to reverse when the dimension was tactile, but not olfactory. It was also notable that, relative to the olfactory dimension, the tactile dimension was initially more difficult to discriminate for all mice. Thus, the deficit in the mutants may relate to the relatively greater difficulty of reversing in the tactile dimension. Though this remains to be formally tested, it is reminiscent of recent data showing that lesions of the PFC subregions or gene deletion of GluN2A produces deficits in associative learning and reversal when the cognitive demand of tasks is high (Bussey et al., 1997, Brigman and Rothblat, 2008, Marquardt et al., 2014).

Another novel finding from the current work was that the GluN2B mutants were unable to develop an attentional set for an initially trained dimension. In contrast to this phenotype, a prior pharmacological study found that systemic administration of the GluN2B-selective antagonist, Ro 25-6981, improved set-shifting performance in mice tested on an ASST similar to that used in the current study (Kos et al., 2011). These differences may be explained by timing of the loss of GluN2B function relative to behavioral testing. Kos et al, and another study by Dalton and colleagues, found that attentional set formation was facilitated if GluN2B was antagonized as the set was formed, whereas GluN2B blockade after a set had already been formed produced perseveration on later set-shifts (Dalton et al., 2011, Kos et al., 2011). Given the GluN2B deletion is not restricted to any specific testing phase, the current findings show that the dominant effect of subunit loss throughout testing is one of impairment.

Though the current data begin to link GluN2B effects on set-shifting to specific brain regions, namely the cortex and CA1 hippocampus, the precise subregions involved remain to be elucidated. Various PFC subregions represent good candidates based on previous work (Hamilton and Brigman, In press). For instance, lesions of the PrL or IL have been shown to disrupt performance on set shifting tasks (Ng et al., 2007, Oualian and Gisquet-Verrier, 2010). A key role for these regions is further supported by in vivo electrophysiological recordings showing task-related neuronal activity in IL and PrL during initiation and maintenance of attention-set formation (Rich and Shapiro, 2009). Furthermore, NMDARs localized to these regions are implicated in set-shifting by the findings that microinfusion of NMDAR antagonist (via MK801) into the PrL and IL impair set-shifting in operant and odor/tactile paradigms in rats (Stefani et al., 2003, Stefani and Moghaddam, 2005). These data suggest that impaired set-shifting in the GluN2B mutant mice may stem from loss of subunit function in the IL or PrL, though it is interesting that the nature of the subunit mutation was more specific (i.e., limited to the initial set) than the general NMDAR block produced by the drug infusions. Future work using, for example, viral-mediated, knockdown of GluN2B in the IL and PrL will be important to dissecting the critical role of these regions.

In summary, the current study found that conditional deletion of the NMDAR GluN2B subunit on corticohippocampal pyramidal neurons produced impairments in measures of cognitive flexibility and attentional set formation, without concurrent deficits in discrimination learning or motor function. These findings add to a growing literature linking glutamatergic and NMDAR dysfunction to cognitive-executive deficits in schizophrenia and other neuropsychiatric conditions (Davies et al., 2013, Jimenez-Sanchez et al., 2014), and suggest targeting GluN2B as a novel approach to treating these disorders.