Stefan Köhler, 1* Stacey Danckert, 1 Joseph S. Gati, 2 and Ravi S. Menon 2 INTRODUCTION HIPPOCAMPUS 15: (2005)

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1 HIPPOCAMPUS 15: (2005) Novelty Responses to Relational and Non-relational Information in the Hippocampus and the Parahippocampal Region: A Comparison Based on Event-Related fmri Stefan Köhler, 1* Stacey Danckert, 1 Joseph S. Gati, 2 and Ravi S. Menon 2 ABSTRACT: We conducted two functional magnetic resonance imaging (fmri) experiments that examined novelty responses in the human medial temporal lobe (MTL) to determine whether the hippocampus makes contributions to memory processing that differ from those of structures in the adjacent parahippocampal region. In light of proposals that such differential contributions may pertain to relational processing demands, we assessed event-related fmri responses in the MTL for novel single objects and for novel spatial and non-spatial object relationships; subjects were asked to detect these different types of novelties among previously studied items, and they successfully performed this task during scanning. A double dissociation that emerged from the response pattern of regions in the hippocampus and perirhinal cortex provided the strongest support for functional specialization in the MTL. A region in the right middle hippocampus responded to the novelty of spatial and non-spatial relationships but not to the novelty of individual objects. By contrast, a region in right perirhinal cortex, situated in the anterior collateral sulcus, responded to the novelty of individual objects but not to that of either type of relationship. Other MTL regions that responded to novelty in the present study showed no reliable difference in their response to the various novelty types; these regions included anterior parts of the hippocampus and posterior aspects of parahippocampal cortex. Together, our findings indicate that relational processing demands are a critical determinant of functional specialization in the human MTL. They also suggest, however, that a neuroanatomical framework that only distinguishes between the hippocampus and the parahippocampal region is not sufficiently refined to account for all functional differences and similarities observed with respect to relational processes in the human MTL. VC 2005 Wiley-Liss, Inc. KEY WORDS: medial temporal lobe; perirhinal cortex; parahippocampal cortex; neuroimaging; recognition memory; episodic memory INTRODUCTION Starting with the description of dense global amnesia in patient H.M. in the 1950s (Scoville and Milner, 1957), research in neuropsychology and cognitive neuroscience has firmly established that the primate medial temporal lobe (MTL) plays a key role in episodic long-term 1 Department of Psychology, University of Western Ontario, London, Ontario, Canada; 2 Laboratory for Functional Magnetic Resonance Research, Robarts Research Institute, London, Ontario, Canada Grant sponsors: Natural Sciences and Engineering Research Council of Canada and Canadian Institutes for Health Research. *Correspondence to: Dr. Stefan Köhler, Department of Psychology, University of Western Ontario, London, Ontario, Canada, N6A 5C2. stefank@uwo.ca. Accepted for publication 9 May 2005 DOI /hipo Published online 5 July 2005 in Wiley InterScience ( wiley.com). memory. Neuroanatomical investigations have demonstrated that the MTL of human and nonhuman primates is not a homogeneous region but consists of several different structures that can be distinguished based on their cytoarchitectonic composition and patterns of connectivity (Amaral and Insausti, 1990; Suzuki, 1996; Lavenex and Amaral, 2000). In light of this neuroanatomical evidence, recent research has begun to address whether the cognitive functions of different MTL structures are also distinct; much of the current debate in the field centers around the question of whether the hippocampus (including fields CA 1 3, the dentate gurus, and the subiculum) supports memory processes that are different from those of adjacent structures in the parahippocampal region, in particular the perirhinal and parahippocampal cortices, or whether the functional contributions of the hippocampus and adjacent parahippocampal structures are indistinguishable in terms of the cognitive dichotomies proposed so far (for review, see Suzuki and Eichenbaum, 2000; Brown and Aggleton, 2001; Squire et al., 2004). In humans, studies conducted with functional MRI (fmri) in healthy individuals have assumed an important role in this debate (e.g., Gabrieli et al., 1997; Eldridge et al., 2000; Stark and Squire, 2001a; Yonelinas et al., 2001; Davachi and Wagner, 2002; Stark and Okado, 2003; Davachi et al., 2003; Ranganath et al., 2004). Several theoretical proposals have been put forward to distinguish the functional role of the hippocampus from that of adjacent MTL structures, in particular with respect to recognition memory (Eichenbaum et al., 1994; Aggleton and Brown, 1999; Mayes, 2002; Yonelinas, 2002; Norman and O Reilly, 2003). A thread that is common to these proposals is the notion that the hippocampus supports memory performance specifically on tasks that require the formation and recovery of relationships between the separate components of an episode or between multiple semantically unrelated stimuli (i.e., arbitrary associations). Perirhinal cortex in the adjacent parahippocampal region, by contrast, is thought to support performance on tasks that do not require processing of such relationships but can be completed based on assessing the familiarity of individual stimuli. Neural responses to novelty provide an important source of information that can be used to test hypoth- VC 2005 WILEY-LISS, INC.

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3 DIFFERENTIAL NOVELTY RESPONSES IN THE HUMAN MTL 765 eses about differential functional contributions of the hippocampus and adjacent parahippocampal structures to memory processing. Differential MTL responses to novel as compared with familiar stimuli have been observed across several species with different recording and imaging techniques, and have been instrumental in informing theoretical discussions about the MTL (Li et al., 1993; Knight, 1996; Tulving et al., 1996; Brown and Xiang, 1998; Grunwald et al., 1998; Martin, 1999; Wan et al., 1999; Dolan and Strange, 2002; Hunkin et al., 2002; Henson et al., 2003; O Kane et al., in press). Comparisons of neural responses to different types of novelties can provide important insight about the specific features of episodes that are processed by a given MTL structure. Numerous fmri investigations in humans have reported hippocampal activation when subjects view novel as compared with recently encountered complex visual scenes (e.g., Stern et al., 1996; Constable et al., 2000; Kirchhoff et al., 2000). Based on the idea that, in novel scenes, multiple objects are often encountered together in combinations or in locations in which they have never been seen before, these findings have been taken as support for the hypothesis that the hippocampus is involved in processing of relationships (e.g., Cohen et al., 1999; Eichenbaum, 1999; Schacter and Wagner, 1999). Novel scenes can, however, also contain novel individual objects that subjects may not have encountered recently or ever before. Consequently, these findings, although compatible with a relational processing account, do not reveal whether the hippocampal novelty response is indeed specifically tied to the processing of relationships. Furthermore, novelty activations for scenes are typically not limited to the hippocampus but extend into the parahippocampal region (e.g., Stern et al., 1996; Kirchhoff et al., 2000). It is possible that these parahippocampal activations, unlike those in the hippocampus, are driven by the novelty of individual objects in the scenes. Without further manipulations of the stimulus material or processing demands, however, such an interpretation remains speculative. Düzel and colleagues (2003) recently reported direct neuroimaging evidence showing that the human hippocampus responds to the novelty of stimulus relationships. In their eventrelated fmri study, subjects were scanned while making discriminations between previously studied face object pairings and FIGURE 1. Experimental tasks used in Experiment 1 (a c) and 2 (d e). (a) Sequence of trials used for familiarization of item pairs during prescanning session. (b) Sequence of trials in fmri runs that require detection of novel spatial (i.e., object location) relationships. The novel item is shown with a bold border. Note that there is no novelty in non-spatial (i.e., object object) relationships in this item. (c) Sequence of trials in fmri runs that require detection of novel non-spatial relationships. There is no novelty in spatial relationships in the novel item. (d) Sequence of trials used for familiarization of single objects during prescanning session. (e) Sequence of trials in fmri runs that require detection of novel single objects. Note that in the execution of these experiments, the order of trials in the familiarization phase was randomized and did not correspond to that in the test phase. novel ones; novel items differed from the studied ones either in terms of the specific grouping between a face and an object, or in terms of the location in which the object was presented. Novelty responses in the MTL were found to be confined to the hippocampus and were observed for both the spatial and the non-spatial relationships. Additional support for a hippocampal involvement in processing of novel spatial relationships comes from an fmri investigation reported by Pihlajamäki et al. (2004), who focused on MTL responses to novel objects and their spatial arrangements. Their results also hint that the hippocampal response to novelty may not be limited to item relationships. The block design employed in this study, however, poses limits to conclusions that can be drawn with respect to specific noveltyassessment processes. Given the central importance of the distinction between relational and single-item processing for current MTL theories, we conducted an event-related fmri study that further examined novelty responses in the MTL in order to determine whether there is functional specialization that maps onto this theoretical distinction. We compared novelty responses in the hippocampus with those in the parahippocampal region for single objects and for object relationships while subjects performed a novelty detection task. In light of the longstanding proposal that some MTL functions may be uniquely linked to the spatial domain (O Keefe and Nadel, 1978), we examined novelty responses to relationships of the spatial (i.e., object location) and of the non-spatial (i.e., object object) kind (see Fig. 1). General Overview MATERIALS AND METHODS The study consisted of two separate fmri experiments. Experiment 1 was designed to examine the response of MTL structures to novel spatial and non-spatial object relationships. For this purpose, subjects first studied a series of pictures of unique object pairings before scanning; the location of these objects in the displays varied across items in the series (Fig. 1a). During scanning, subjects were exposed to the group of previously studied items and a smaller number of novel items; they were asked to detect the novel items, which were systematically altered versions of the studied ones and were novel either in terms of object location (Fig. 1b) or object object relationships (Fig. 1c). fmri novelty responses in the MTL were determined by comparing activity for the novel with that for the corresponding previously studied items. Experiment 2 was designed to examine the response of MTL structures to novel individual objects in a situation in which processing of relationships was minimized; the design and procedure were identical to those in Experiment 1 except that the stimuli used were pictures of single objects always presented in the center of the screen. Such a stimulus set-up is critical in the context of the question addressed here because novel objects cannot be introduced in pairings without also

4 766 KÖHLER ET AL. creating novel object object relationships; moreover, the presentation of all objects in the same location ensures that the saliency of object location relationships is reduced to a minimum. To determine whether any novelty activation observed in the MTL in the two experiments was specific to a particular type of novelty, we conducted region-of-interest (ROI) analyses that compared the response of all activated MTL regions to all types of novelties across both experiments. Because these crossexperimental comparisons are central to the aim of the current study, Methods and Results are presented together for both experiments. Subjects Twelve right-handed subjects participated in each fmri experiment (Expt. 1: 6 women, 6 men; age range 21 31; mean age 24.3; Expt. 2: 5 women, 7 men; age range 21 32; mean age 25.8). They were screened for the absence of any neurological or psychiatric condition and were reimbursed for their participation. All subjects gave their written informed consent before participation. The study protocol was approved by the Ethics Board for Medical Research at the University of Western Ontario. Stimulus Materials The stimuli presented were line drawings of common objects taken from the work of Snodgrass and Vanderwart (1975) and a similar parallel set made available by the Laboratory of Experimental Psychology, Katholieke Universiteit Leuven (van Diepen and De Graef, 1994). The digitized drawings were presented as white lines on a black background. Forty-eight easily identifiable objects with distinct names (name agreement scores 80%) served as stimuli in each experiment. In Experiment 1, these objects were paired to create a list of 24 stimuli with the constraint that the objects in each pair be semantically unrelated. Using a total of 44 possible locations of an invisible 8 6 grid (excluding corners), these 24 object pairs were placed into 12 unique spatial configurations. Duplicating each spatial configuration in combination with two different object pairs allowed us to change object object relationships without a concurrent change in object location relationships. The resulting stimuli served as the items used for prefamiliarization before scanning (see Fig. 1a). To create 24 items with novel object location relationships, we switched the locations of the two objects within each pair (see Fig. 1b). To create 24 items with novel object object relationships, we replaced one of the objects in each pair with an object from another pair with the same spatial configuration and location (see Fig. 1c). In Experiment 2, the set of 48 objects was split randomly into halves to create two sets that could then be used for prefamiliarization or for novel-item presentation during scanning (see Figs. 1d,e). Experimental Tasks To prefamiliarize subjects with items that could later be used as old items in the novelty detection task, subjects were presented with a set of 24 stimuli 24 h before scanning in both experiments; they were asked to memorize these stimuli for a subsequent memory test. In Experiment 1, they were instructed to pay special attention to the object location and object object relationships embedded in each item. Stimuli were presented visually on a computer screen for 2 s each with an intertrial interval of 5 s during familiarization. The entire set of stimuli was presented four times in random order; multiple presentations were used to optimize performance in the subsequent novelty detection task. Immediately before scanning, subjects were reminded of these items with two additional presentations, combined with the same presentation parameters and instructions. During scanning, subjects were presented with the previously studied items that were intermixed with novel ones; they were instructed to detect the novel items by indicating for each stimulus whether it was new or whether it had previously been studied. Each experiment consisted of multiple fmri runs filled with this novelty detection task (Experiment 1: 6 runs; Experiment 2: 3 runs). The order of runs was counterbalanced across participants. Each run included the presentation of 24 previously studied and 8 novel items; these 32 items were placed in pseudorandom order with the constraint that no more than 6 old items be shown consecutively and that all novel items be separated by at least one old item. In each run, all novel items were of the same type and subjects were informed in advance about the type of novelty to be detected; practice trials were given to illustrate the different types of novelties. Each trial lasted for 12.5 s; it began with the presentation of the stimulus for 2.5 s, followed by a 10-s fixation period. This period was included to optimize separation of the fmri BOLD signals associated with neighboring trials (see Bandettini and Cox, 2000). In the last 2.5 s of this interval, the fixation cross changed its color to warn subjects about the upcoming start of the next trial. Stimuli were projected via an LCD projector onto a transparent screen at a fixed distance that allowed for easy object identification. Subjects viewed the projected stimuli through an overhead mirror and were asked to provide their behavioral responses manually, using an MR-compatible keypad, during the 2.5 s of stimulus presentation. fmri Data Acquisition and Image Analysis All imagings were performed on a 4 Tesla whole body MRI system (Varian, Palo Alto, CA; Siemens, Erlangen, Germany). The MTL is exceptionally difficult to image with standard fmri protocols because of large variations in magnetic properties of the structures surrounding it (i.e. air-tissue), in particular in its most anterior extent (Ojemann et al., 1997; Schacter and Wagner, 1999; Constable et al., 2000). To minimize artifacts and increase the signal-to-noise ratio in the MTL, we introduced a semiautomated, localized second-order shimming algorithm (Klassen and Menon, 2004) and combined it with a multi-shot, short echo-time imaging sequence. The plane of image acquisition was positioned perpendicular to the longitudinal axis of the hippocampus (determined based on initial scout images). For each subject, 19 contiguous 5-mm-thick slices were obtained covering the brain from the most anterior portion of the tempo-

5 DIFFERENTIAL NOVELTY RESPONSES IN THE HUMAN MTL 767 TABLE 1. Medial Temporal Lobe Regions Showing Significant Novelty Response in Critical Contrasts at P < (uncorrected) Region Peak coordinates (x y z) t value Expt. 1 Novel > old object location relationships Right middle hippocampus Novel > old object object relationships Right anterior hippocampus Right middle hippocampus Left middle hippocampus Expt. 2 Novel > old single objects Right perirhinal cortex Right parahippocampal cortex Note: Df ¼ 11 for all t tests. ral lobe in which a reliable echo-planar image signal could be obtained (generally at least 2 cm anterior to the rostral ending of the hippocampus) to the occipital pole. The field of view was 19.2 by 19.2 cm (64 64 matrix) providing for an in-plane resolution of 3 mm. T2*-weighted echo-planar image acquisition was used for all functional scans (TE ¼ 10 ms; TR ¼ 625 ms; volume acquisition time ¼ 2.5 s; flip angle ¼ 408; 4 segments per plane; navigator-corrected). Each run involved the acquisition of 185 image volumes (five volumes per trial in the behavoural task). The initial five volumes, which did not correspond to any particular trial, were acquired only for MR signal stabilization and excluded from all analyses. An additional T1-weighted high-resolution MRI volume was obtained for the display of neuroanatomy in the same experimental session, using a 3D MDEFT pulse sequence; it was acquired with the same plane orientation as the functional scans but covered the entire brain (160 slices; 1.25 mm thick; matrix; 0.75 mm in-plane resolution). Image postprocessing and event-related statistical analyses were performed using BrainVoyager 2000 software (Version 4.8). Functional images were inspected to ensure that no motion artifacts were present, and corrected for signal drift within runs using a high-pass filter. Functional data were resampled into isotropic voxels (3 3 3 mm), coregistered with the anatomical images, transformed into standardized space (Talairach and Tournoux, 1988), and overlaid on the anatomical images (resampled into isotropic voxels of mm). Following transformation, images were smoothed with a 3D Gaussian Kernel and a full-width at half maximum value of 6 mm. Statistical analyses were performed using the General Linear Model (GLM). We first computed estimates for the critical event-related predictors for each subject and then entered these estimates into a second-level analysis to assess their significance in a random-effects model (i.e., based on error variance across subjects). The time-course of the hemodynamic response was modeled with a gamma function in these analyses, and all data were corrected for temporal autocorrelation. To characterize the response of different MTL regions to different types of novelties, we followed a two-step procedure, relying on group-based statistical analyses in both steps. First, we identified all MTL regions that showed a response to any of the three types novelty investigated in our experiments. For this purpose, we computed, for each type of novelty separately, the contrast between correctly identified new and correctly identified old items across the three relevant runs. To allow for the tightest possible comparison, these contrasts specifically focused on only those old items that directly corresponded to the eight novel items in the same run (i.e., those eight old items to which the changes in relationships had been applied to create the novel items in that run). This selection also guaranteed a counterbalanced order in that the new items preceded the corresponding old items on half of the trials, while the reverse order held for the other half of the trials. Since there was no correspondence between specific novel and old items in Experiment 2, eight old items were sampled pseudorandomly from the larger set, such that they followed a run distribution comparable to that of the pertinent old items in Experiment 1. (Notably, when all old items, rather than just eight items per run, were included in the statistical analyses, the pattern of results was virtually unchanged, leading to the same conclusions as reported later.) Results from the three different novelty contrasts were expressed as t-maps, thresholded at P < (uncorrected), and examined for MTL activation, defined as at least 10 contiguous voxels (1 1 1 mm) above threshold, in the hippocampus and the parahippocampal region. In the second step of our analyses, we probed how the MTL regions that were identified with the three novelty contrasts described (listed in Table 1) responded to the other two types of novelties investigated. For this purpose, we centered ROIs around the peak voxels identified and sampled those voxels that were contiguously activated at the specified threshold, limiting their size to a maximum of mm. We then assessed the response of these ROIs for each novelty contrast, and statistically compared it across the different types of novelties using Analysis of Variance (ANOVA). The statistical significance criterion adopted for these ROI-based analyses was P < This lenient criterion was chosen so as to detect even subtle novelty effects in all contrasts. In turn, it allowed us to be con-

6 768 KÖHLER ET AL. servative when making inferences about the selectivity of the novelty responses observed. Given that the regions for the these follow-up ANOVAs were selected based on a novelty response in one of the relevant contrasts to begin with, the reported P values, however, should be considered to be more descriptive than exact (i.e., the analyses can be considered to be biased). RESULTS Behavioral accuracy was at virtually perfect levels in all three conditions (mean percentage of correct responses > 0.96 for each of them), indicating that participants were highly sensitive to all novelty types in their detection performance. Statistical analyses of the fmri data revealed activation in the MTL for all three types of novelties examined. The MTL regions that showed a significant response in each of the three critical novelty contrasts at the selected threshold of P < (uncorrected) are presented, together with their peak coordinates, in Table 1. Notably, we found no activation in the MTL for any of the reverse contrasts (i.e., higher activity for old than novel items). All MTL regions that showed a novelty response to spatial and nonspatial relationships in the first set of analyses for Experiment 1 were located in the hippocampus (see Table 1). A portion of the right hippocampus, approximately midway along its anterior posterior extent, showed an increased activation for novel as compared with that for familiar relationships of both types (see Fig. 2a). Additional activation was observed for novel object object relationships in a corresponding left hippocampal region and in a more anterior right hippocampal region. In these two areas, the novelty response for object location relationships was not significant at the selected threshold; however, it became evident as a weaker response when further examined in the subsequent ROI analyses (see Table 2). To determine directly whether the response in any of these activated hippocampal regions differed significantly for object object as compared with that for object location relationships, we performed further GLM-based analyses on these ROIs, in which the novelty response was coded as the difference between predictors (b weights) for the two different types of novel as compared to old items on a subject-by-subject basis (i.e., in a random-effects model). These analyses revealed a significantly larger response in the left hippocampus for object-object than for object-location relationships (F(1,11) ¼ , P < 0.005) but no difference in the response of the two right hippocampal regions (both F(1,11) < 1; P > 0.10). The first set of analyses for Experiment 2 revealed significant novelty activation for individual objects only in the parahippocampal region (see Table 1 and Fig. 2b). One area of activation was found in an anterior part of the collateral sulcus, most likely corresponding to perirhinal cortex (Amaral and Insausti, 1990; Insausti et al., 1998; Pruessner et al., 2002); another distinct area of activation was found more posteriorly in the collateral sulcus in parahippocampal cortex. When we created ROIs for these two areas to determine how they responded to the novel spatial and non-spatial relationships tested in Experiment 1, we found no significant novelty response to either type of relationship in the more anterior region corresponding to perirhinal cortex; the more posterior region in parahippocampal cortex, however, showed a significant response to both types of relationships (see Table 2). In turn, we also examined the hippocampal ROIs identified in Experiment 1 for their novelty response to the individual objects tested in Experiment 2. These analyses revealed a weak response in the left hippocampal ROI but no significant response in the right hippocampal ROIs to novel single objects (see Table 2). Taken together, the results of the analyses presented so far suggest that some but not all MTL regions show novelty responses that are selective and depend on whether the stimuli encountered are novel in terms of stimulus relationships or in terms of the identity of single objects. To assess this selectivity statistically, we performed direct comparisons of the novelty response in our ROIs across the two experiments, again using the differences in b weights for novel as compared with previously studied items as estimates for the different types of novelty responses. One-way ANOVAs with a between-subject design and novelty type as the factor of interest revealed significant differences in the novelty response in the right middle hippocampal ROI (F(2,33) ¼ 3.532; P < 0.05) and in the right perirhinal ROI (F(2,33) ¼ 3.945; P < 0.05) across novelty types. Planned comparisons confirmed that the right middle hippocampal region identified in Experiment 1 responded more strongly to the novelty of relationships than to that of individual objects (F(1,33) ¼ 7.831, P < 0.01; see Fig. 2a). The right perirhinal cortex identified in Experiment 2, by contrast, showed the opposite pattern of responses (F(1,33) ¼ 7.053, P < 0.05; see Fig. 2b). This differential response pattern across both regions was confirmed as a significant cross-over interaction (F(2,33) ¼ ; P < ) in a two-way ANOVA, in which the type of novelty served as a between-subject factor and the MTL region served as a within-subject factor. Notably, in all other MTL regions that showed any significant novelty response in Experiment 1 or 2 (listed in Table 1), this response was found to be comparable across both experiments, with no statistical evidence for selectivity. DISCUSSION In the present two event-related fmri experiments, we found that some but not all MTL regions show novelty responses that are selective and differ depending on whether the stimuli encountered are novel in terms of stimulus relationships or in terms of the identity of single objects. A double dissociation that emerged from the response pattern of regions in the hippocampus and perirhinal cortex across the two experiments provided the strongest support for functional specialization in the MTL. A region in the right middle hippocampus responded to the novelty of spatial and non-spatial relationships but not to the novelty of individual objects. By contrast, a region in right perirhinal cortex, situated in the anterior collateral sulcus, responded to the novelty

7 DIFFERENTIAL NOVELTY RESPONSES IN THE HUMAN MTL 769 FIGURE 2. MTL regions showing a double dissociation in their novelty response. (a) Coronal and sagittal view of the right hippocampal region showing a novelty response to spatial and non-spatial relationships but not to single objects. Activation map corresponds to contrast between items with novel spatial relationships vs. old ones in Experiment 1. (b) Coronal and sagittal view of right perirhinal cortex region showing a novelty response to single objects but not to spatial and non-spatial relationships. Activation map corresponds to contrast between items with single novel objects vs. old ones in Experiment 2. Both activation maps were thresholded at T > 4.0 for purposes of illustration and were superimposed on one of the subjects structural MR images. Note that the response profiles shown were derived from the ROI-based statistical analyses (corresponding to data presented in Table 2) and show the differences in regression weights for the three types of novel as compared with old items.

8 770 KÖHLER ET AL. TABLE 2. Comparison of Responses in Medial Temporal Lobe Regions to Different Types of Novelties Across Experiments 1 and 2 Based on ROI Analyses Expt. 1 Expt. 2 Novel > old object object relationships Novel > old single objects Novel > old object location relationships t value p value t value p value t value p value Region of interest Center coordinates (x y z) Right anterior hippocampus Right middle hippocampus Left middle hippocampus Right perirhinal cortex Right parahippocampal cortex Note: ROIs were derived from first set of analyses summarized in Table 1, and were centered around peaks identified. Df ¼ 11 for all t tests. of individual objects but not of either type of relationship. Other MTL regions that responded to novelty in the present study showed no reliable difference in their response to the various novelty types when probed across experiments; these regions included anterior parts of the hippocampus and posterior aspects of parahippocampal cortex. Together, our findings indicate that relational processing demands are a critical determinant of functional specialization in the human MTL. They also suggest, however, that a neuroanatomical framework that only distinguishes between the hippocampus and the parahippocampal region is not sufficiently refined to account for all functional differences and similarities observed with respect to relational processes in the human MTL. Novelty Responses in Perirhinal Cortex The novelty response for individual objects in perirhinal cortex, which we found under conditions in which relational processing demands were minimized, provides support for the notion, derived from lesion data and electrophysiological recordings in nonhuman primates, that this part of the MTL may contribute to recognition memory through the assessment of relative item familiarity (Li et al., 1993; Murray and Bussey, 1999; Brown and Aggleton, 2001). Although the mapping of hemodynamic to single-neuron novelty responses in the primate brain is not fully understood at present, and thus demands caution in interpretation (see Henson and Rugg, 2003), it is worth noting that our data are consistent with electrophysiological findings showing decrements in single-neuron responses to previously encountered as opposed to novel objects in perirhinal cortex (for review, see Brown and Xiang, 1998). Such decrements have been suggested to form the basis for the familiarity assessment subserved by perirhinal cortex. The present results are also in line with recent findings from three independent event-related fmri studies showing a decreased anterior MTL response for individual previously encountered items as compared with that for items not encountered before in the study context (Henson et al., 2003). As in the present study, this fmri novelty response was observed in the vicinity of right perirhinal cortex (see also Vandenberghe et al., 1995; Pihlajamäki et al., 2004). Findings from another fmri study may, at first glance, appear to be inconsistent with the selectivity of the novelty response for single items that we observed in perirhinal cortex. Pihlajamäki et al. (2003) reported higher fmri activity in perirhinal cortex when subjects memorized novel pairs of nameable objects in an encoding task than when they followed repeated presentations of two highly familiar pairs in a baseline condition. However, given that the relationships as well as the individual objects that made up the pairs were novel in the encoding condition, this finding can still be interpreted to reflect a perirhinal response to novel single items. Some evidence from electrophysiological recordings in nonhuman primates also point to an involvement of perirhinal cortex in processing stimulus stimulus associations on conditional memory tasks (e.g., Sakai and Miyashita, 1991; Naya et al., 2003). Murray and Bussey (1999) have convincingly argued, however, that the information carried by such pair-coding

9 DIFFERENTIAL NOVELTY RESPONSES IN THE HUMAN MTL 771 neurons, as a result of many training sessions, is more akin to context-independent semantic memory than to episodic memory, and may be most critical for item identification. The results of the present study, and other evidence reviewed by Brown and Aggleton (2001), suggest that the involvement of perirhinal cortex in assessing item familiarity is limited to individual objects, likely because it is based on information other than the kind carried by pair-coding neurons. Novelty Responses in the Hippocampus All hippocampal regions that responded to novelty in the present study did so in response to the novelty of relationships. Among them, a region in the right middle hippocampus was found to respond selectively to relationships (i.e., it did not respond to the novelty of individual objects). Our findings provide support for the proposal that the hippocampus is involved in processing of relationships between the separate components of an episode, or between multiple semantically unrelated stimuli (Eichenbaum et al., 1994; Cohen et al., 1999; Mayes, 2002; Norman and O Reilly, 2003). They are also in keeping with the evidence from several other neuroimaging studies showing hippocampal involvement in relational processes (Henke et al., 1999; Eldridge et al., 2000; Davachi and Wagner, 2002; Düzel et al., 2003; Sperling et al., 2003; Giovanello et al., 2004; Pihlajamäki et al., 2004; Preston et al., 2004; Prince et al., 2005), and showing that this involvement can be dissociated functionally from that of perirhinal cortex (Davachi et al., 2003; Ranganath et al., 2004). Notably, our findings extend this past research by revealing that not all parts of the hippocampus that are involved in processing relationships do so in a selective manner. The theoretical implications of this pattern of results for the functional organization of the MTL will be discussed in the final section of this article. Concerning the role of different types of relationships, it is worth noting that a part of the left hippocampus showed a significantly larger response to non-spatial than to spatial relationships in Experiment 1. To the extent that object object relationships can be verbalized more easily than object location relationships, this finding fits well with the widely accepted view of hemispheric specialization in the MTL along the verbal versus non-verbal processing dimension (Milner, 1974; Lee et al., 2002). Our hippocampal findings also support the notion that functions of the human hippocampus, even in the right hemisphere, are not limited to the spatial domain (Eichenbaum et al., 1999). Novelty Responses in Parahippocampal Cortex Another MTL region that responded to novelty in the current study, but not selectively to any particular kind, was a portion of right posterior parahippocampal cortex. We identified this region based on its above-threshold response to the novelty of individual objects in Experiment 2. When further examined in our subsequent ROI analyses, we found that its response to novel spatial and non-spatial relationships in Experiment 1, although below the conservative threshold of P < in the initial set of analyses, was not significantly different from that to novel single objects in Experiment 2. Findings from lesion studies in nonhuman primates, as well as neuroanatomical considerations pertaining to the distribution of parietal-lobe projections to different MTL structures, have led to the proposal that parahippocampal cortex (areas TH and TF) plays a critical role in memory for spatial relationships (Suzuki and Eichenbaum, 2000; Malkova and Mishkin, 2003). Some evidence from functional neuroimaging in human beings and neuropsychological patient studies provide additional support for this idea, especially in the context of navigational tasks (Habib and Sirigu, 1987; Aguirre et al., 1996; Bohbot et al., 1998; Maguire et al., 1998; Barrash et al., 2000). Recent research involving direct comparisons of parahippocampal contributions to the processing of different kinds of information suggests, however, as do the current results, that the memory functions of parahippocampal cortex may not be limited to the spatial domain (Köhler et al., 2002; Düzel et al., 2003; Pihlajamäki et al., 2004; Alvarado and Bachevalier, 2005). In fact, parahippocampal cortex may in itself be a functionally heterogeneous structure. A recent fmri study that examined novelty responses in parahippocampal cortex on a subject-by-subject basis, taking into consideration the variability in morphology across individuals, found that those portions of parahippocampal cortex that respond to the novelty of spatial relationships are situated more posteriorly than those that respond to novel objects introduced into familiar spatial arrangements of familiar objects (Pihlajamäki et al., 2004). The current study may not have been able to identify functional heterogeneity within parahippocampal cortex at this fine-grained level because the group-based approach of analyses involved averaging and standardization of the subjects anatomical MR images. Interpreting Novelty Responses in the MTL Novelty responses were investigated in the present study in the context of a behavioral task that required subjects to detect novel items within a set of familiar ones. The MTL signals examined reflected the corresponding difference in the event-related fmri BOLD response. Thus, the events serving as baseline for evaluation of the novelty response in our analyses entail the same task components and require the same mental set as the experimental events, except for the specific processes that lead to or immediately follow the detection of novel information. Relying on this type of comparison is advantageous in that the brain structures examined do not need to be assumed to be in a passive state or at resting level in the baseline condition (see Stark and Squire, 2001b). This approach does not imply, however, that those MTL regions that show a novelty response are not involved when subjects experience or recognize previously encountered items as familiar. Rather, such a comparison simply reveals relative differences in involvement for the two types of items. In the current study, old new differences were only found in the direction of increases for the novel items. In other research, however, differences in the opposite direction have sometimes also been reported in the hippocampus or the parahippocampal cortex (e.g., Eldridge et al., 2000; Düzel et al., 2003). One possibility is that increases for old items only become evident under conditions in which the nature of the recognition task or

10 772 KÖHLER ET AL. the stimuli at hand invoke strong recollective processes at the time of retrieval (see Eldridge et al., 2000; Sharot et al., 2004). These types of recollective processes quite likely did not play a significant role in the present study, because the task instructions emphasized the detection of novel items. Given that the number of novel items was smaller than that of previously presented items in our experimental paradigm, it could be argued that the novelty activation we observed reflects an attentional oddball response in the MTL. Although we cannot rule out that oddball responses contributed to the observed MTL activations, we maintain that they cannot account for the full pattern of results reported here. First, if the novelty activation simply reflected a nonspecific oddball response it should be comparable for all types of novelties examined, because the number of novel items was held constant across conditions. This is, however, not what we found in all MTL regions. Second, two independent studies have shown that oddball responses in the MTL adapt very quickly to items of the same type, and can typically be found only if analyses are restricted to the beginning of the study (Strange and Dolan, 2001; Yamaguchi et al., 2004). Third, Constable et al. (2000) reported direct evidence showing that the fmri response in the hippocampus to novel as compared with previously studied scenes is comparable when the novel items appear as the minority or as the majority of items in the study. Because the task during scanning in our experiments required subjects to make discriminations between novel and recently seen stimuli, it is tempting to conclude that the novelty activations observed reflect processes related to making recognition decisions. Evidence from other fmri studies suggest, however, that novelty responses in the MTL may also reflect encoding processes. It has been shown, for example, that activity in MTL regions that display a novelty response during initial stimulus exposure is correlated with performance on a subsequent memory test for the encountered stimuli (Kirchhoff et al., 2000), and that MTL activity associated with making recognition judgements is correlated with performance on a subsequent second test for items encountered as novel lures in the first test (Stark and Okado, 2003). We and others have argued that an encoding and a retrieval interpretation of novelty effects may, in fact, not be mutually exclusive: The same signals in the MTL that provide the neural basis for making recognition decisions about what is novel and what is familiar in the environment may also trigger processes related to the formation of memory representations for the novel information (Köhler et al., 2002; Henson et al., 2003). Further research is necessary, however, to provide more direct evidence in support of this idea. Implications for the Functional Organization of the MTL On the basis of several studies failing to find a differential involvement of MTL structures in relational and nonrelational memory tasks, Squire and colleagues (2004) have argued in a recent review that the data currently available in the literature do not support a functional model of MTL organization that is based on the dichotomy between relational and nonrelational memory processes. To the extent that not all hippocampal and parahippocampal regions activated in the present study showed a differential novelty response, our results provide further support for such a cautionary approach in interpretation. This is not to say, however, as Squire et al. (2004) also note, that it should be concluded from such data that there is no functional specialization within the MTL. Clearly, the fmri data reported here, together with evidence from past lesion and neuroimaging research (Vargha-Khadem et al., 1997; Davachi and Wagner, 2002; Giovanello et al., 2003, 2004; Sperling et al., 2003; Mayes et al., 2004), indicate that the distinction between relational and non-relational processing is valuable in revealing differences in functions between different MTL components. Yet, our data also suggest that there is no one-to-one mapping between this distinction at the functional level and the one between the hippocampus and the parahippocampal region at the neuronantomical level (see also Davachi and Wagner, 2002). Even functional accounts of MTL organization that distinguish between the perirhinal and parahippocampal cortices would not capture the complexity of the data reported here and in other fmri studies, given that there also appears to be differences in the response profile of different portions of the hippocampus along its longitudinal axis (e.g., Small et al., 2001; Pihlajamäki et al., 2004). Further progress in this field of study will quite likely require a more detailed consideration of the intrinsic and extrinsic neuroanatomical connectivity of different MTL structures (Lavenex and Amaral, 2000; Lavenex et al., 2004), and even of different subdivisions within distinct structures, such as the hippocampus (Amaral, 1993); recent advances in increasing the spatial resolution of fmri data in the MTL will be beneficial toward this end (e.g. Zeineh et al., 2003). 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