Fifteen male Sprague-Dawley rats were obtained from Envigo (Indianapolis, IN; 114 days old and weighed 328 g at the beginning of the experiment). Rats were housed individually and maintained on a 12:12 light/dark cycle, with light onset at 7 a.m. and offset at 7 p.m. Water was available ad libitum, except during testing sessions. The rats received 45-mg chocolate pellets and chow pellets (F0229 and F0164, respectively; Bio-Serv, Frenchtown, NJ) during experimental sessions. Daily rations were 15 g consisting of pellets consumed during experimental sessions and 5102-Rat-Diet (PMI Nutritional International, St. Louis, MO). All procedures followed the Guide for Care and Use of Laboratory Animals and were approved by the Bloomington Institutional Animal Care and Use Committee at Indiana University. One rat was euthanized for health reasons before completing Experiment 1. One rat tracked rewards in Experiment 1 and was removed from all data analyses. From the remaining 13 animals, all 13 rats completed Experiment 1 and 2. Experimenter scheduling conflicts, long interruptions in behavioral testing, variable numbers of sessions required to meet criteria for experiments and deterioration in baseline performance and in health led to: 2 rats were not assessed in Experiment 3, 2 rats were not assessed in Experiment 4A, 4 rats were not assessed in Experiment 4B, 5 rats were not assessed in Experiment 5.

Method Details

Apparatus Four open-field arenas constructed from acrylic plexiglass served as the distinctive contexts for encoding and memory assessments. All arenas contained “food holes” that were used for placement of scented lids. Each food hole was circular (5 cm diameter, 2.5 cm depth), which allowed a cup to be firmly snapped in place so that the cup lay flush with the floor and could be covered with a lid placed loosely on top. The arena used for list encoding was square (61-cm length, 61-cm width, and 30-cm height) and had 12 equidistant food holes, arranged along the perimeter of the walls. Two open-field arenas that differed in size, shape, and color were used for the second to last item and fourth to last item memory assessments (the assignment of these two arenas to second to last and fourth to last contexts was counterbalanced across rats). One arena was circular, white, with a 94-cm diameter floor, and enclosed within a 30-cm high wall. The inside of the arena consisted of 18 food holes arranged in two concentric circles (6 food holes were positioned in the inner ring, and 12 food holes were positioned in the outer ring). The other arena was circular, with a 46-cm diameter floor, and a transparent 30-cm high wall. The floor pattern in this arena consisted of 3 concentric circles, with the inner, middle, and outer circles that were black, white, and black, respectively. The inside of the arena consisted of 8 equidistant food holes positioned along the walls. The arena used for new-old odor recognition was gray and triangular (walls measured 51-cm length and 30-cm height). In this arena, nine equidistant food holes were arranged along the perimeter of the three walls. Arenas were cleaned with 2% chlorhexidine solution after each animal completed its daily session.

Stimuli Odors were presented with opaque plastic lids that were odorized by storing them in sealed plastic containers. Plastic containers were filled with 90 mL of an oil odorant or approximately 150 mL of a dry spice powder odorant, and lids were odorized for at least 2 weeks before being presented to the rats. A metal grating was used in each container to separate the lids and the odorants in order to prevent direct contact. Odorants were refreshed approximately every 2 months in order to maintain scent potency and consistency. Odorants included: (i) 60 replay odorants: allspice, amaretto, anise seed, apple, apricot, asparagus, banana, bay leaf, black walnut, blackberry, blueberry, brandy, bubble gum, butterscotch, caraway seed, carob powder, celery seed, champagne, cheddar cheese, cherry, chicory root, cilantro, cinnamon, clove, coconut, coffee, coriander, cotton candy, cumin, dill weed, nutmeg, onion powder, orange oil, peach, pecan, pineapple, pistachio, pumpkin, raspberry, root beer, rosemary leaf, sage leaf, sassafras, sesame, spearmint, spinach powder, strawberry, sumac, summer savory, sweet basil, tarragon, thyme, tomato, turmeric, Mexican vanilla, wasabi, watermelon, white willow, and Worcestershire. (ii) 21 new-old odor recognition task odorants: annatto, beet powder, black pepper, blue cheese, cardamom, crème de menthe, eggnog, fennel seed, galangal, ginger, grape flavor oil, hot chili, juniper berry, lemon grass, lime oil, malt vinegar, marshmallow, mushroom, paprika, soy sauce, and tangerine. (iii) 14 odor discrimination task odorants: butter rum oil, camphor, caramel, castor oil, English toffee, horehound, licorice oil, peppermint, pina colada, praline oil, salt water taffy, tropical punch, tutti-frutti, and winter green. All stimuli used as odors were purchased from The Great American Spice Company (Rockford, MI).

General Methods One session was conducted each day, approximately 5 days per week. During each session, the rat was removed from its home cage and placed in a holding cage, where it also resided during inter-trial intervals. Holding cages were the same as cages used in vivarium housing, except bedding, food, and water were not present. The behavioral testing room contained four open-field arenas and a platform for the holding cage. All arenas and platforms were elevated approximately 76-cm above the floor and were configured in an equidistant arc. During sessions, the default position for the experimenter was located in the middle of the arc, approximately 1-m from the center of the arenas. For each trial, the experimenter removed the rat from the holding cage and placed the rat in the designated arena facing away from the experimenter (i.e., positioned with head pointed away from experimenter). Next, the experimenter returned to the default position where he/she remained with hands at his/her sides until the rat displaced the lid of the designated odor. The experimenter then removed the rat from the arena and returned it to the holding cage. When odors were presented as stimuli multiple times during each session, new lids were used to prevent the rat from relying on scent marking. The following variables were randomized each session: order of second to last and fourth to last memory assessments in each trial, the odors used, and location of the odor lids.

Pre-training Before odors were introduced, rats were trained to search the arenas and displace unscented lids from food holes to obtain food rewards.

Memory replay training Because learning the rules in our approach may be difficult for the rats, we developed a training approach to optimize learning. To this end, we implemented training strategies that provided the rat with immediate feedback, allowed the rat to continue each trial until they displaced the lid to the correct item, and used a large reward for an initial correct choice. Since timely feedback promotes learning, we utilized a strategy that provided the rat with immediate feedback to facilitate acquisition of the second and fourth to last rules. To minimize delay between the response and the delivery of the food reward, the cup under the correct choice was baited with a single food pellet, thereby providing immediate access to the food after lid displacement. To further optimize learning, we provided the rat with feedback in every trial by allowing the rat to continue each trial if the initial response was incorrect. To this end, if the rat’s initial lid displacement was to an incorrect item, the trial continued until lid displacement of the correct item occurred (the second choice was not included in calculations of accuracy). Because our approach baited the cups of correct items and allowed the rat to continue in each trial following an initial incorrect choice, it is possible for the rat to choose randomly and still obtain many food rewards. Thus, in order to incentivize learning while implementing features described above, we provided the rat with a large reward following a correct first choice. Specifically, a correct first response was rewarded with five additional food pellets, whereas a correct second choice was not rewarded with additional pellets. To this end, immediately following the rat’s initial correct lid displacement, the experimenter delivered additional food pellets to the cup; the experimenter did not initiate delivery of the additional food reward until after the rat’s initial correct response had occurred. In training, sessions consisted of approximately 7 trial-unique lists and corresponding memory assessments. At this stage, each list consisted of 5-8 trial-unique odors. Lists were presented to the rat in the distinct encoding context, one item at a time; the number of items in the list was randomly selected for each trial. Each list item presentation consisted of a single cup and odorized lid placed at a randomly determined location and baited with a single chow pellet. The rat was removed from the holding cage and placed in the list encoding context facing away from the experimenter. A response was defined as the vertical or horizontal displacement of the lid from the cup. The rat remained in the encoding context (i.e., arena) until it displaced the lid and consumed the food reward. Immediately after the rat displaced the lid it was removed from the arena and returned to the holding cage. This procedure continued until all items in the list were presented. Once the list presentation was complete, the rat received a memory assessment that involved the memory for the order of items presented in the list. Rats were trained to identify items that were encountered in distinct ordinal positions from the preceding list. To this end, in each memory assessment, the rat was rewarded for selecting the second to last or fourth to last items encountered in the list depending on the context in which the assessment occurred. Immediately after completing each list presentation, the rat was moved to a distinctive context for the memory assessment. Memory assessments consisted of a choice between two odors presented in the list in one context; one odor was from the second to last ordinal position in the list (the correct choice) and was baited with a single chocolate pellet, while the other odor was unbaited and from another ordinal position in the list (the incorrect choice, foil odor). In this context, a correct choice was defined as the first lid displacement for the second to last list item, whereas an incorrect response was defined as the first lid displacement for an odor from the different ordinal position. The same memory assessment procedure was conducted in the other memory assessment context, except the rat was rewarded for selecting the fourth to last item in the list. To promote learning, we minimized the delay between the rat’s response and delivery of the food reward by baiting the cup beneath the lid of the correct choice with a single chocolate pellet. Again, assessments consisted of two odors from the list, one odor from the fourth to last ordinal position (the correct choice; baited with a single chocolate pellet), whereas the unbaited foil odor was from elsewhere in the list (the incorrect choice). To provide feedback about the memory assessment rules, if the first choice was an incorrect response, the rat remained in the arena until a response to the correct choice occurred, but this second choice was not included in calculations of accuracy. When the rat displaced the incorrect lid first and the correct lid second, it was allowed to eat the pellet from the baited correct choice, but no additional pellets were provided at that point. To incentivize learning, we provided the rat with a large reward following a correct first choice. Specifically, we rewarded the rat with five additional chocolate pellets following a correct first response, whereas an initial incorrect choice was not rewarded with additional pellets. Assignment of second and fourth to last memory assessment contexts were counterbalanced across rats prior to the start of the experiment. Approximately 10 sessions were conducted in replay training.

Experiment 1: Rats identify the order of items encountered in the list Here, we asked if rats remember the order in which multiple events occurred. Experiment 1 was designed to assess whether rats could identify items that occupied distinct ordinal positions from a previously encountered list. The rat was presented with a trial-unique list of odors in an encoding context, followed by memory assessments about the order of items in a different context. After the list presentation, the experimenter moved the rat to one of two distinct contexts for the first memory assessment, followed by the second memory assessment in the other context. In one memory assessment context, selection of the second to last item encountered in the list was rewarded. In the other memory assessment context, selection of the fourth to last item encountered in the list was rewarded. Optimal performance is to select the second and fourth to last items in the memory assessments when in the corresponding context and reject items encountered from other ordinal positions elsewhere in the list. Thus, solving the memory assessments requires memory of the order of events that occurred in the preceding trial-unique list. In Experiment 1, the rats were presented with a variable length list that consisted of trial-unique odors in a distinctive encoding context, immediately followed by memory assessments that required memory of the order in which the preceding items were presented. Experiment 1 was the same as replay training, except that the lists ranged from 5-12 odors. Testing continued for each rat until performance in the memory assessments met the following criteria: (i) Mean accuracy observed in the second to last and the fourth to last memory assessment was at least 75% for six consecutive sessions. (ii) Mean accuracy observed in each memory assessment context during the two most recent sessions was at least 75%. Approximately 35 sessions were conducted in Experiment 1.

Experiment 2: Rats remember the order of items using episodic memory In Experiment 1, identifying the ordinal position of list items in the memory assessment may be accomplished using episodic memory replay. However, because items in the list were presented sequentially, high accuracy could be attained in the memory assessments by selecting the item with the typical memory trace strength of a second or fourth to last item, without relying on episodic memory replay. Here, the rat was presented with a list using atypical inter-trial intervals. Consequently, in the subsequent memory assessment, the second to last and fourth to last items were presented to the rat with atypical memory trace strengths for their corresponding ordinal positions. In Experiment 2, we asked whether rats relied on non-episodic memory trace strength cues or episodic memory replay when selecting items in the memory assessment. Experiment 2 was the same as Experiment 1 except that: (i) We doubled the duration of typical inter-item intervals between the last four items in the list and the memory assessment. (ii) In the memory assessment, foil odors consisted of items that exhibited the typical decay profile, or memory trace strength, of the second and fourth to last items. (iii) Only one memory assessment occurred after each list. Next, in the memory assessment we presented the rat with a choice between the second to last item (correct choice) and the last item (foil odor). In this pair, the foil odor had a memory trace strength that was typical for a second to last item, whereas the second to last item did not. In the memory assessment for the fourth to last item, we presented the rat with a choice between the fourth to last item (correct choice) and the second to last item (foil odor). In this pair, the foil odor had a memory trace strength that was typical for a fourth to last item, whereas the fourth to last item did not. Approximately 6 sessions were conducted in Experiment 2.

Experiment 3: Replay survives a 60-min retention interval Experiment 3 was designed to test the hypothesis that replay memory could survive a long retention interval, consistent with long-term memory. In Experiment 3, sessions were the same as those in Experiment 1 except that: (i) Sessions consisted of 2 trials, with no delay occurring during the first trial. (ii) In the second trial, a 60-min retention interval occurred between the list presentation and the memory assessments. The rat was returned to its home cage (located outside of the testing room) during the retention interval. After the 60-min delay had elapsed, the rat was returned to the testing room to complete the memory assessment. Thus, high accuracy in the memory assessment requires knowledge about the order of events that occurred in the list prior to the long retention interval. Prior to Experiment 3, rats completed approximately 7 sessions of training with 15-min delays, and 6 sessions of training with 30-min delays. Approximately 8 sessions were conducted in Experiment 3.

Experiment 4: Replay is resistant to interference from other memories Overview. If replay relies on episodic memory, then replay memory for the order of events should be resistant to interference from other memories. Experiment 4 was designed to assess whether replay memory could survive interference from new memories. To this end, we asked if rats could remember the order of events encountered in the list when both encoding and memory assessment were interleaved with a new-old odor recognition task that occurred in another context (i.e., List encoding→ New-old recognition→ Memory assessment). In the new-old recognition task, rats were trained to pick the “new” odor and avoid the “old” odor (i.e., the old odor was presented in the new-old recognition task earlier in the session). In each new-old recognition trial, selection of the new odor was rewarded with food, while selection of an old odor was not. Thus, high accuracy for identifying new odors in the new-old recognition task requires memory for odors presented earlier in the session. New-old recognition task. In new-old recognition training, the first trial consisted of one baited cup covered with an odorized lid. The rat was then placed in the arena and allowed to displace the lid and consume the chow pellet, or until 2 min elapsed. Next, the rat was removed from the arena and returned to the holding cage. The second trial consisted of two odors, a new odor and an old odor (i.e., re-presentation of the odor previously encountered in the new-old task earlier in the session). A correct choice was defined as the first lid displacement of the new odor, whereas an incorrect response occurred when the rat displaced the lid of an old odor prior to displacing the lid of a new odor. If the first lid displacement was to an incorrect choice, the rat remained in the arena until a response to the correct choice was made; the second choice was not included in the calculation of accuracy. If the rat made two consecutive correct responses, the subsequent third trial continued to increment in this fashion such that it consisted of one new odor placed in the arena along with the two previously presented old odors (incorrect choices); trials incremented until an incorrect response was made or until 4 lids occupied the arena (one new odor, three old odors). If the rat made an incorrect response, the number of odors in the arena was reset to one (only the new odor) in the subsequent trial. Incrementing resumed with each subsequent correct response until another incorrect response occurred, a maximum of 4 odors were simultaneously presented in the arena, or until 20 odors had been presented as new. Rats completed approximately 9 new-old recognition training sessions as described above. After new-old recognition training, the new-old recognition task was included in daily sessions along with memory replay testing. The procedure for the new-old recognition was the same as in the training described above except that: (i) A maximum of 16 odors were presented in the new-old recognition task. (ii) Following an incorrect response, the number of odors in the arena continued to increment (up to 4), rather than reset to one in the subsequent trial. (iii) The rat received the list, the memory assessments, and then the new-old recognition task (i.e., List encoding→Memory assessment→New-old recognition task); approximately 6 of these sessions were conducted. Experiment 4A In Experiment 4A, list presentations and subsequent memory assessment were interleaved with the new-old odor recognition task. Experiment 4A was the same as in the new-old recognition training described above, except that the new-old recognition memory task was interleaved in between list encoding and the memory assessment, rather than immediately following the memory assessment (i.e., List encoding→ New-old recognition task→Memory assessment). Approximately 6 sessions were conducted in Experiment 4A. Experiment 4B Experiment 4B was the same as Experiment 4A except that in the memory assessment, foil odors consisted of second and fourth to last rewarded odors in the new-old recognition task. For instance, in the memory assessment for the second to last item, following the interference task, we presented rats with a choice between the second to last odor encountered in the list (correct choice) and the second to last rewarded odor from the new-old recognition task (foil odor). In this pair, the foil odor was an attractive choice because it was a second to last odor in the recognition task, but it was not the second to last item encountered in the list. Likewise, the memory assessment for the fourth to last item consisted of the fourth last odor encountered in the list (correct choice) and the fourth to last rewarded odor from the new-old recognition task (foil odor). Approximately 5 sessions were conducted in Experiment 4B.

Experiment 5: Episodic memory replay is hippocampal dependent Experiment 5 was designed to assess whether episodic memory replay is hippocampal dependent. To test whether the hippocampus is critical for episodic memory replay, we temporarily suppressed hippocampal activity in the rats. In Experiment 5, rats received bilateral injections of the inhibitory chemogenetic actuator AAV8-hSyn-hM4Di-mCherry in the dorsal hippocampus (surgical procedures for AAV injections are described below). Experiment 5 sessions consisted of both replay (as described in Experiment 4B) and independent memory assessments to evaluate the non-specific impact on memory. The hippocampal-independent memory procedures consisted of the new-old recognition task (described above) and the odor discrimination task (described below). Thus, if the hippocampus is critical for episodic memory replay, then hippocampal suppression should impair the rat’s performance in the replay memory assessment and spare hippocampal independent new-old recognition and odor discrimination performance. Daily sessions consisted of memory assessments for replay, new-old recognition, and odor discrimination. Memory assessments in Experiment 5 were the same as in Experiment 4B with the addition of assessment in the odor discrimination task at the end of each daily session. Before receiving surgery, testing continued for each rat until performance in the Experiment 5 memory assessments met the following criteria: (i) Mean accuracy observed in the second to last and the fourth last memory assessments was at least 75% for six consecutive sessions. (ii) Mean accuracy observed in each memory assessment context during the two most recent sessions was at least 75%. (iii) Mean accuracy observed in the new-old recognition task during the two most recent sessions was significantly above chance. (iv) Mean accuracy observed in the odor discrimination task was at least 75% for six consecutive sessions. Following surgery, AAV intracranial injections, and recovery, rats were required to re-established baseline performance using the same criteria described above before receiving vehicle or the chemogenetic actuating drug clozapine N-oxide (CNO). Thus, high accuracy in the memory assessments was established before and after surgery prior to testing the impact of hippocampal suppression with CNO. Approximately 10 sessions were conducted in Experiment 5 after surgery to re-establish baseline. In the drug phase of Experiment 5, 5 sessions of vehicle and 5 sessions of CNO were interleaved in a randomized block design with five blocks of two sessions each. Vehicle or CNO (10 mg/kg i.p.) injections were administered ∼30-45 mins prior to testing in the daily session. The experimenters who collected the behavioral data were blind to drug conditions (identity of CNO versus vehicle injections). Odor discrimination task. Prior to AAV injections, rats were trained on an odor discrimination task. In the odor discrimination procedure the rat was presented with a fixed pair of odors. Odor discrimination pairs consisted of one odor that was always rewarded and the other odor that was never rewarded. In order to attain high accuracy in the odor discrimination trial, rats were required to learn the rule about which odor was rewarded. Ten odor discrimination trials were conducted at the end of the rat’s daily replay session. Odor assignments for each animal’s discrimination pair were randomly selected from a pool of odors prior to the start of Experiment 5. AAV Injections. AAV-hM4Di-mCherry was a gift from Bryan Roth (University of North Carolina, Chapel Hill) and obtained from Addgene (pAAV8-hSyn-hM4D(Gi)-mCherry; Addgene viral prep #50475-AAV8). AAV-hM4Di-mCherry was bilaterally injected into the hippocampus of each rat using a stereotaxic apparatus (Stoelting Co, Wood Dale, IL) under isoflurane anesthesia. Rats were approximately 16 months of age prior to surgery. Based on the Paxinos and Watson rat brain atlas, the following coordinates were used to target the dorsal hippocampus bilaterally at five sites in each hemisphere of every rat; AP −3.3, ML ± 1.5, DV −3.0; AP −3.6, ML ± 1.8, DV −2.8; AP −4.1, ML ± 2.2, DV −2.8; AP −4.5, ML ± 2.5, DV −2.6; AP −5.2, ML ± 3.2, DV −2.8 mm from bregma, the midline suture and the skull surface, respectively. Viral vector (1 μl) was infused at each of the 10 co-ordinates at a flow rate of 4 nl/sec using a 30G gastight syringe (Hamilton, Reno, NV) attached to an automated infusion pump (UMP3 UltraMicroPump; World Precision Instruments, Sarasota, FL). Rats were tested three weeks after surgery to allow for optimal viral expression as verified in our previous pilot studies. Immunohistochemistry. Rats were deeply anaesthetized with 25% urethane, then transcardially perfused with 0.1% heparinized 0.1 M phosphate buffered saline (PBS), followed by ice-cold 4% paraformaldehyde. Brains were dissected and kept in the same fixative for 24 hours, then cryoprotected in 30% sucrose for at least 3 days. Coronal sections (30 μm) of the hippocampus (spanning from approximately −2.3 mm to −6.3 mm from bregma) were cryostat cut and kept in an antifreeze solution (50% sucrose in ethylene glycol and 0.1 M PBS) prior to immunostaining. Tissue sections were collected in separate series so every tenth section within a series would be processed for immunohistochemistry. Free floating sections were washed in 0.1 M PBS, then incubated in blocking solution consisting of 10% goat serum and 0.1% Triton X-100 in 0.1 M PBS (blocking buffer was used as primary and secondary antibody diluent). Sections were then incubated for 72 hours at 4°C with rabbit polyclonal anti-mCherry primary antibody (1:3,000, ab167453, Abcam, Cambridge MA). Following primary antibody incubation, tissue sections were washed in 0.1 M PBS then incubated with goat anti-rabbit Alexa Fluor 488 (1:500, ThermoFisher, Waltham, MA) for 2 hours at room temperature. Following secondary antibody incubation, sections were washed in 0.1 M PBS then incubated in DAPI (0.1 μg/ml, ThermoFisher, Waltham, MA) for 10 minutes, washed in ddH20 and mounted with ProLong Diamond (ThermoFisher, Waltham, MA). Both the correspondence between anti-mCherry labeling and native mCherry staining and the rostrocaudal distribution and extent of Designer Receptors Exclusively Activated by a Designer Drug (DREADD) transduction was assessed using a Leica DMLB microscope. Site maps documenting this distribution were mapped by a single experimenter (LMC) for each animal, using the atlas of Paxinos and Watson as a guide and a composite that was representative of all subjects was constructed (see Figure S1 ). Sections were also photographed on a Retiga 1300 digital camera (Qimaging, Burnaby, BC, Canada) using QCapture Image Acquisition Software (Qimaging, Burnaby, BC, Canada) on the same Leica DMLB microscope used for qualitative mapping of mCherry expression. Minimal adjustments to color histograms were performed using the levels function in Photoshop Elements 15 (Adobe Systems, San Jose, CA) and all manipulations were performed uniformly to the entire image.

Assessment of non-episodic memory resources 27 Baddeley A. Working memory. 28 Baddeley A.D.

Hitch G. Working memory. Three lines of evidence suggest that data shown in Figure 2 were not produced by rats using non-episodic memory resources. First, retaining the last four items in memory would require rapid updating when each new item is presented, which would require working memory resources. Because a defining feature of working memory is that such memory is short lived [], performance following a long retention-interval challenge would occur at chance ( = 0.5). When the rats were challenged with 60-min delay, performance was above chance ( Figure 2 A; Expt. 3), which is inconsistent with the use of working memory resources. Second, one approach to rescue a working memory account is to propose that after updating, the last four items are stored in long-term memory in response to an expected long retention-interval challenge. Through training, if the rats learned to transfer the last four items to long-term memory, then accuracy would be at chance levels when presented with the first long retention-interval challenge. By contrast, episodic memory is an aspect of long-term memory; thus, above chance performance based on episodic memory replay is expected on the initial long-delay memory assessment. When challenged with the first long retention interval (15 min delay between encoding and assessment), accuracy in the memory assessment for the second to last and fourth to last items was high, and initial performance was above chance ( Table S5 ), suggesting that rats relied on long-term episodic memory. 29 Braver T.S.

Cohen J.D.

Nystrom L.E.

Jonides J.

Smith E.E.

Noll D.C. A parametric study of prefrontal cortex involvement in human working memory. 30 Owen A.M.

McMillan K.M.

Laird A.R.

Bullmore E. N-back working memory paradigm: a meta-analysis of normative functional neuroimaging studies. 31 Ragland J.D.

Turetsky B.I.

Gur R.C.

Gunning-Dixon F.

Turner T.

Schroeder L.

Chan R.

Gur R.E. Working memory for complex figures: an fMRI comparison of letter and fractal n-back tasks. Third, another dominant feature of working memory is that performance is susceptible to manipulations of memory load []. If rats relied on working memory resources, performance in the second to last memory assessment should be higher than in the fourth to last memory assessment. To test this hypothesis, we examined three measures of performance. Three lines of evidence suggest that performance in the second and fourth to last memory assessment are equivalent. First, accuracy increased with similar trajectories as a function of sessions in second and fourth to last assessments ( Figure S2 ). Second, rats achieved a high level of performance in both assessments after an equivalent number of sessions ( Figure S2 ). And, third, equivalent performance in the second to last and fourth to last assessments was observed across all experiments ( Figure 2 A; 2nd last versus 4th last, F(1, 7) = 2.89, p = 0.13); Experiments 1-4, F(4, 28) = 1.77, p = 0.16), and interaction F(4, 28) = 0.84, p = 0.51). Overall, learning and performance in the second and fourth to last memory assessments were equivalent, which does not support the use of working memory resources.

Unbaited memory assessments The rat can attain high accuracy in the second and fourth to last memory assessments by relying on episodic memory replay to identify the order of items. However, an alternative is that the rat is detecting the odor of the food pellet in the baited cups without relying on episodic memory replay. Because the correct items were baited with a food pellet in memory assessments, we asked whether rats relied on food pellet odor cues or item order by conducting unbaited trials immediately after Experiment 1 (i.e., after each rat met the learning criteria). In this session, four unbaited memory assessments were given to each rat in random order, counterbalanced so that at least one unbaited memory assessment occurred in each context every other trial. In the unbaited memory assessments, the designated second or fourth to last item was placed in the arena without a food reward (i.e., unbaited), along with the foil odor. Following a correct first response, the experimenter delivered five chocolate pellets to the cup for that item. Following an incorrect first response, the rat remained in the arena until a correct response occurred. If a correct choice followed an initial incorrect choice, the experimenter manually delivered one chocolate pellet to the cup of the correct item after a response to the correct item was observed. Our measure of accuracy was the proportion of correct first choices. Thus, if rats were using the odor cues of the food reward to guide their selection in the baited assessments, accuracy in the unbaited memory assessment, wherein the odor cues are removed, would occur at chance ( = 0.5). Alternatively, if rats were relying on memory, accuracy in this unbaited memory assessment would be above chance and equivalent to data from Experiment 1. The proportion correct for the baited second to last and fourth to last items in Experiment 1 immediately prior to the unbaited memory assessment is shown in Table S6 . When the rats were presented with unbaited memory assessments, above chance accuracy was observed ( Table S6 ); and performance in unbaited and baited memory assessments were not significantly different ( Table S6 ).

Choice between second to last versus fourth to last items In our experiments, identifying the ordinal position of list items in the memory assessment may be accomplished using episodic memory replay, but it is important to rule out the use of reward history. Because memory assessments presented rats with a choice between a second to last or fourth to last item and a foil odor, but not a choice between a second to last item and a fourth to last item, high accuracy could be attained by selecting the item with the greatest reward history, without relying on episodic memory replay to distinguish the second and fourth to last events. Here, we presented the rat with a memory assessment consisting of a choice between a second to last and a fourth to last item. Notably, each item in the memory assessment under these conditions possessed equated reward histories. Tests for equated reward history occurred after Experiment 1. Approximately 2 sessions were conducted ( Table S6 ).