RAPID COMMUNICATION

Contribution of the Retrosplenial Cortex to Temporal DiscriminationLearning

Travis P. Todd,* Heidi C. Meyer, and David J. Bucci

ABSTRACT: The retrosplenial cortex (RSC) has an important role incontextual learning and memory. While the majority of experimentshave focused on the physical context, the present study asked whetherthe RSC is involved in processing the temporal context. Rats weretrained in a temporal discrimination procedure where the duration ofthe intertrial interval (ITI) signaled whether or not the next tone condi-tioned stimulus would be paired with food pellet reinforcement. Whenthe tone was presented after a 16-min ITI it was reinforced, but when itwas presented after a 4-min ITI it was not. Rats demonstrated successfuldiscrimination in this procedure by responding more to the tone onreinforced trials than on non-reinforced trials. Pre-training electrolyticlesions of the RSC attenuated acquisition of the temporal discrimina-tion. The results are the first to demonstrate a role for the RSC in proc-essing temporal information and in turn extend the role of the RSCbeyond the physical context to now include the temporal context.VC 2014 Wiley Periodicals, Inc.

KEY WORDS: retrosplenial; temporal context; episodic memory

The retrosplenial cortex (RSC) is situated at the interface betweenprimary cortical sensory areas and parahippocampal and hippocampalregions (van Strein et al., 2009; Sugar et al., 2011). Specifically, theRSC receives visuo-spatial input and has strong reciprocal connectionswith the postrhinal cortex, postsubiculum, medial entorhinal cortex, aswell as direct connections with the hippocampus. Thus, the RSC is wellpositioned to influence learning and memory processes that are medi-ated by medial temporal lobe structures.

Behaviorally, the RSC has an important role in contextual learningand memory (for a review see Bucci and Robinson, in press). Permanentlesions of the RSC disrupt contextual fear conditioning regardless ofwhether those lesions are made prior to, or after, fear conditioning (e.g.,Keene and Bucci, 2008). Furthermore, temporary blockade of NMDAreceptors in the RSC impairs retrieval of contextual fear memories(Corcoran et al., 2011). In these experiments, “context” typically refers tothe background stimuli provided by the physical apparatus (e.g., operant

chamber). Thus, these findings indicate that the RSCis involved in processing the physical context.

Both external and internal cues can act as contexts(Bouton, 2002, 2010). Just as physical features of theenvironment (external) can provide a source of con-textual information, so too can the passage of time(internal). In the present study, we tested the hypothe-sis that the role of the RSC in processing contextsextends beyond physical stimuli. We used a temporaldiscrimination learning paradigm to determine theinvolvement of the RSC in processing temporal con-text (Bouton and Garc�ıa-Guti�errez, 2006; Todd et al.,2010; Bouton and Hendrix, 2011). While there is evi-dence that the hippocampus is sometimes involved inprocessing temporal information (e.g., Iordanovaet al., 2009; Campese and Delamater, 2014; cf. Kydet al., 2008), to our knowledge this has never beenstudied in the RSC or other parahippocampal regions.

Seventeen adult male Long Evans rats, �60-daysold, were obtained from Harlan Laboratories (India-napolis, IN). Rats were housed individually andallowed at least 6 days to acclimate to the vivariumprior to surgery with food available ad libitum (Purinastandard rat chow; Nestle Purina, St. Louis, MO). Allsurgeries took place over the course of a four-dayperiod that immediately followed the acclimationperiod. Eight rats received bilateral electrolytic lesions(2.5 mA, 15 sec at each site) of the RSC (see Table 1for coordinates) prior to the behavioral training usingsurgical procedures previously described (e.g., Robin-son et al., 2011). Electrolytic lesions were chosen toprovide a high degree of control over the extent ofdamage (Ross and Eichenbaum, 2006), which was animportant factor in this study given the close proxim-ity of the RSC to related cortico-hippocampal regions(e.g., posterior parietal cortex, postsubiculum; Burwelland Amaral, 1998). Control rats (n 5 8) receivedsham lesions consisting of a craniotomy and shallow,non-puncturing burr holes to minimize damage tounderlying cortex. Rats were allowed to recover for atleast 2 weeks before beginning behavioral training.During that period they were allowed to return topre-surgical weight before being subsequently food-restricted to 85% of their baseline weight. One shamlesioned rat developed a head-tilt after surgery and

Psychological and Brain Sciences, Dartmouth College, Hanover, NHGrant sponsor: National Science Foundation Grant; Grant number:IOS0922075; Grant sponsor: National Institutes of Health; Grant number:MH105125.*Correspondence to: Travis P. Todd, Department of Psychological andBrain Sciences, Dartmouth College, Hanover, NH 03755, USA.E-mail: travis.p.todd@dartmouth.eduAccepted for publication 21 October 2014.DOI 10.1002/hipo.22385Published online 00 Month 2014 in Wiley Online Library(wileyonlinelibrary.com).

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was removed from the experiment. This rat was replaced withan un-operated control rat from the same shipment.

Following recovery, rats were trained in a temporal discrimi-nation procedure (Bouton and Hendrix, 2011). The behavioralprocedures were carried out in standard conditioning chambers(Med Associates) previously described (Robinson et al., 2011).The chambers were illuminated by a house light that wasmounted 15 cm above the grid floor on the back wall of thechamber. A speaker was located 15 cm above and to the rightof the food cup (recessed in the center of the front wall) andwas used to present the auditory stimulus (1,500 Hz, 78 dB).A pair of infrared photocells was mounted just inside the foodcup to detect head entries into the cup. The unconditionedstimulus was the presentation of two 45-mg grain-based rodentfood pellets (Bio-Serv).

On the first day of the experiment, all rats received a single30-min session of magazine training during which food pelletswere delivered freely on a random time 30 sec (RT 30s) sched-ule resulting in 60 pellets being delivered on average. For thenext 20 consecutive days, rats received a single daily session(�85 min) in which the tone conditioned stimulus (CS) waspresented eight times. For all rats, the tone co-terminated withdelivery of the unconditioned stimulus (US) when it followeda 16-min intertrial interval (ITI), but not when it followed the4-min ITI. Both groups received four reinforced (R) and fournon-reinforced (N) trials every session. Rats demonstrate suc-cessful discrimination in this procedure by responding more tothe tone after a 16-min ITI relative to a 4-min ITI. Therewere two, double alternating sequences of trials. On odd daysthe trials were RRNNRRNN and on even days the trials wereNNRRNNRR (e.g., Bouton and Hendrix, 2011).

The computer recorded the amount of time spent in thefood cup during three 10-sec periods: the 10-sec period priorto CS presentation of each trial (pre-CS), the 10-sec periodduring CS presentation, and the 10-sec period immediately fol-lowing CS presentation (post-CS).

After the behavioral procedures were completed, lesions wereverified and analyzed using methods previously described (e.g.,Keene and Bucci, 2008; Robinson et al., 2011). The results are

presented in Figure 1. Bilateral damage of the RSC wasobserved in all rats and the average area of the RSC damagedon each of the five sections analyzed from each rat was68 6 14%. A representative lesion is illustrated in Figure 1a.Damage to the RSC was present on 98 6 4% of the sectionscollected from each subject, indicating that damage extendedthroughout the rostro-caudal extent of the RSC (Fig. 1b).Minor damage to cortical regions outside of the RSC (anteriorcingulate, secondary motor cortex, secondary visual cortex andforceps major corpus callosum) was observed in all of the rats.However, extra-RSC damage was present on only 39 6 14% ofthe sections analyzed. In addition, 5 of the 8 rats exhibitedminor damage to the cingulum bundle.

Acquisition of the temporal discrimination is presented inFigure 2. The time spent in the food cup during the CS on R(16-min ITI) and N (4-min ITI) trials is presented for bothcontrol (upper panel) and RSC-lesioned (lower panel) rats.Overall, RSC-lesioned rats were impaired in this form of tem-poral discrimination learning. At the completion of the firsthalf of training (10 sessions), control rats demonstrated a cleardiscrimination, responding more to the tone on the R trialsrelative to the N trials. In contrast, RSC-lesioned ratsresponded non-differentially to both trial types. However, withcontinued training, the RSC-lesioned rats were indeed capableof discriminating trial types. To confirm these impressions,each 10-session block was analyzed with a 2 (Group: RSC vs.Control) 3 2 (Trial Type: R vs. N) 3 10 (Session) ANOVA.For the first half of training (sessions 1–10), this analysisrevealed a main effect of session, F(9, 126) 5 5.161, P< 0.01,and trial type, F(1, 14) 5 8.82, P< 0.05, as well as an interac-tion between session and trial type, F(9, 126) 5 6.64, P< 0.01.Importantly, there was a significant interaction between groupand trial type, F(1, 14) 5 4.55, P 5 0.05. Neither the maineffect of group, nor any other interactions were significant,largest F(9, 126) 5 1.7, P 5 0.1. Simple effect analyses revealedthat the effect of trial type was significant for control rats, F(1,14) 5 13.02, P< 0.01, but not RSC-lesioned rats, F< 1. Thus,after the first 10 sessions, control rats had acquired the temporaldiscrimination but RSC-lesioned rats had not. More so, theeffect of group was not significant within either trial type, largestF(1, 14) 5 1.38, P 5 0.26, suggesting that the overall level ofresponding between groups was not different during this period.

Over the second half of training, both groups correctly dis-criminated trial types. For sessions 11–20 there was a maineffect of trial type, F(1, 14) 5 25.86, P< 0.01, and an interac-tion between trial type and session, F(9, 126) 5 3.82, P< 0.01.No other main effects or interactions were significant, largestF(9, 126) 5 1.42, p 5 .2. The lack of a significant main effectof group or interaction between group and other factors indi-cates that RSC-lesioned and control rats were not significantlydifferent during the second half of training.

There were no substantial differences in pre-CS (i.e., base-line) behavior. Separate ANOVAs on session 1–10 and 11–20did not reveal any significant main effects or interactions larg-est F(1, 14) 5 1.9, p 5 .2. Averaged over sessions 1–10, controlrats spent 0.41 6 0.10 (SEM) and 0.35 6 0.08 sec in the food

TABLE 1.

Stereotaxic Coordinates for Restrosplenial Cortex (RSC) Lesions

AP ML DV

22.0 6 0.3 22.0 and 22.7

23.5 6 0.4 22.0 and 22.7

25.0 6 0.4 and 6 0.1 22.0 and 22.7 (medial site)

and 22.0 (lateral site)

26.5 6 0.8 and 6 1.5 22.0 and 22.8 (medial site)

and 23.4 (lateral site)

28.0 6 1.6 and 6 2.4 22.5 (medial site) and 23.1 (lateral site)

29.0 6 3.4 24.0

All anterior–posterior (AP), medial–lateral (ML), and dorsal–ventral (DV)measurements are derived from bregma, midline, and skull surface, respectively(measurements are in mm).

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cup R and N trials, respectively, and RSC-lesioned rats spent0.27 6 0.07 and 0.40 6 0.09 sec in the food cup for R and Ntrials respectively. Averaged over sessions 11–20, control ratsspent 0.43 6 0.11 and 0.35 6 0.10 sec in the food cup R andN trials, respectively, and RSC-lesioned rats spent 0.23 6 0.15and 0.16 6 0.09 sec in the food cup for R and N trialsrespectively.

As training progressed, both groups responded more in thepost-CS period after R trials (due to the presence of food) thanN trials. However, there were no systematic group (RSC vs.Control) differences during the first or second half of training.For sessions 1–10, there was a main effect of session, F(9,126) 5 8.02, P< 0.01, trial type, F(1, 14) 5 147.39, P< 0.01and an interaction between session and trial type, F(9,

FIGURE 1. (a) Photomicrograph of a representative RSC lesion at 3.0 mm posterior tobregma. The dotted line indicates the boundaries of the RSC. (b) Schematic diagram indicatingthe rostro-caudal extent and the largest (gray) and smallest (black) lesions of the RSC. Abbrevi-ations: RSC, retrosplenial cortex; M2, secondary motor cortex; V2, secondary visual cortex;fmj, forceps major corpus callosum. Figure 1(b) is adapted from The Rat Brain in StereotaxicCoordinates (6th edition), G. Paxinos and C. Watson, 2007. Copyright 2007, with permissionfrom Elsevier.

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126) 5 4.55, P< 0.01. For sessions 11–20 there was a maineffect of trial type, F(1, 14) 5 76.01, P< 0.01. No other maineffects or interactions were significant during either half oftraining, largest F(9, 126) 5 1.67, p 5 .10. Averaged over ses-sions 1–10, control rats spent 5.13 6 0.62 (SEM) and1.63 6 0.45 sec in the food cup R and N trials, respectively,and RSC-lesioned rats spent 4.36 6 0.43 and 1.34 6 0.45 secin the food cup for R and N trials respectively. Averaged oversessions 11–20, control rats spent 6.11 6 0.58 and 1.66 6 0.34sec in the food cup R and N trials, respectively, and RSC-lesioned rats spent 5.53 6 0.72 and 1.78 6 0.51 sec in thefood cup for R and N trials respectively.

In summary, permanent lesions of the RSC attenuated, butdid not completely abolish, temporal discrimination learning.Importantly, the deficit produced by the RSC lesions was spe-cific to discriminating between trial types (i.e., temporal inter-vals); lesions did not produce a general inability to respond.For example, there were no group differences (RSC vs. Con-trol) in baseline behavior (pre-CS) or food related consumptionbehavior (post-CS). Furthermore, although in the first half oftraining the RSC lesioned rats did not discriminate betweentrial types, the overall level of responding did not differbetween lesions and controls. Taken together, these findingssuggest that damage to the RSC produces a specific deficit inthe ability to differentiate between temporal intervals.

It is already known that the RSC is involved in processingphysical contexts (e.g., Keene and Bucci, 2008; Corcoran et al.,2011). However, interoceptive cues, such as the passage of timecan also act as contexts (e.g., Bouton, 2010). The fact thatlesions of RSC disrupt temporal discrimination learning sug-gests a potential role for the RSC in processing the temporalcontext, in addition to the physical context. This is consistentwith the notion that the RSC is part of the “where/when”pathway (Bucci and Robinson, in press). Until now, the RSC’srole in temporal processing has never been demonstrated.

It is also relevant to note the implications of these findingsfor a role of the RSC in episodic memory. Episodic memory isthought to include “what,” “where,” and “when” informationand is mediated by medial temporal lobe structures (e.g.,Eichenbaum, 2000, 2001, Tulving and Markowitsch, 1998).Studies of temporal sequence learning have demonstrated animportant role for the hippocampus in “when” processing(e.g., Fortin et al., 2002; Lehn et al., 2009). However, the pres-ent findings extend beyond the hippocampus, and demonstratea role for the RSC as well. It is possible that the RSC contrib-utes “when” information to episodic memories.

It remains to be determined the full extent of the RSC’s rolein temporal processing. In the current study, rats were requiredto discriminate between two intervals: 16 and 4 min. Futureexperiments might investigate whether the RSC is involved inprocessing temporal information that is shorter and/or longerin duration than the present intervals.

Nevertheless, the present data are the first to demonstrate arole for the RSC in temporal processing and thus extend therole of the RSC beyond processing the physical context to nowinclude the temporal context.

Acknowledgment

Authors thank Dr. Robert Leaton for valuable comments ona previous version of this manuscript.

REFERENCES

Bouton ME. 2002. Context, ambiguity, and unlearning: Sources ofrelapse after behavioral extinction. Biol Psychiatry 52:976–986.

Bouton ME. 2010. The multiple forms of “context” in associativelearning theory. In Mesquita B, Feldman Barrent L, Smith ER, edi-tors. The Mind in Context. New York: The Guilford press, pp.233–258.

Bouton ME, Garc�ıa-Guti�errez A. 2006. Intertrial interval as a contex-tual stimulus. Behav Process 71:307–317.

Bouton ME, Hendrix MC. 2011. Intertrial interval as a contextualstimulus: Further analysis of a novel asymmetry in temporal dis-crimination learning. J Exp Psychol Anim Behav Process 37:79–93.

Bucci DJ, Robinson S. Toward a conceptualizatin of retrohippocampalcontributions to learning and memory. Neurobiol Learn Mem (inpress).

FIGURE 2. Responding during the tone over 20 sessions forcontrols (upper panel) and RSC lesions (lower panel).R 5 reinforced (following 16 m ITI), N 5 nonreinforced (following4 m ITI). Dashed vertical line indicates first vs. second half oftraining. Data are mean 6 standard error.

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Burwell RD, Amaral DG. 1998. Cortical afferents of the perirhinal,postrhinal, and entorhinal cortices of the rat. J Comp Neurol 398:1–27.

Campese VD, Delamter AR. 2014. Dorsal hippocampus inactivationimpairs spontaneous recovery of Pavlovian magazine approachresponding in rats. Behav Brain Res 269:37–43.

Corcoran KA, Donnan MD, Tronson NC, Guzm�an YF, Gao C,Jovasevic V, Guedea AL, Radulovic J. 2011. NMDA receptors inretrosplenial cortex are necessary for retrieval of recent and remotecontext fear memory. J Neurosci 31:11655–11659.

Eichenbaum H. 2000. A cortical-hippocampal system for declarativememory. Nat Rev Neurosci 1:41–50.

Eichenbaum H. 2001. The hippocampus and declarative memory:Cognitive mechanisms and neural codes. Behav Brain Res 127:199–207.

Fortin NJ, Agster KL, Eichenbaum HB. 2002. Critical role for thehippocampus in memory for sequences of events. Nat Neurosci 5:458–462.

Keene CS, Bucci DJ. 2008. Contributions of the retrosplenial andposterior parietal cortices to cue-specific and contextual fear condi-tioning. Behav Neurosci 122:89–97.

Iordanova MD, Burnett DJ, Aggleton JP, Good M, Honey RC. 2009.The role of the hippocampus in mnemonic integration andretrieval: Complementary evidence from lesion and inactivationstudies. Eur J Neurosci 30:2177–2189.

Kyd RJ, Pearce JM, Haselgrove M, Amin E, Aggleton JP. 2008. Theeffects of hippocampal system lesions on a novel temporal discrim-ination in rats. Behav Brain Res 187:159–171.

Lehn H, Steffenach H, van Stein NM, Veltman DJ, Witter MP,Haberg AK. 2009. A specific role of the human hippocampus inrecall of temporal sequences. J Neurosci 29:3475–3484.

Robinson S, Keene CS, Iaccarino HF, Duan D, Bucci DJ. 2011.Involvement of retrosplenial cortex in forming associations betweenmultiple sensory stimuli. Behav Neurosci 125:578–587.

Ross RS, Eichenbaum H. 2006. Dynamics of hippocampal and corti-cal activation during consolidation of nonspatial memory.J Neurosci 26:4852–4859.

Sugar J, Witter MP, van Strein NM, Cappaert NL. 2011. The retro-splenial cortex: Intrinsic connectivity and connections with the(para)hippocampal region in the rat. An interactive connectome.Front Neuroinform 5:7.

Todd TP, Winterbauer NE, Bouton ME. 2010. Interstimulus intervalas a discriminative cue; evidence of the generality of a novel asym-metry in temporal discrimination learning. Behav Process 62:27–48.

Tulving E, Markowitsch HJ. 1998. Episodic and declarative memory:Role of the hippocampus. Hippocampus 8:198–204.

van Strein NM, Cappaert NL, Witter MP. 2009. The anatomy ofmemory: An interactive overview of the parahippocampal-hippocampal network. Nat Rev Neurosci 10:272–282.

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