Perirhinal and postrhinal cortices are necessary for retrieving latently-acquired stimulus associations

14 The perirhinal and postrhinal cortices reside in the parahippocampal region of the medial 15 temporal lobe. They receive and process sensory information from cortical and subcortical 16 sources and support hippocampal functions via direct connectivity and indirectly via the 17 entorhinal cortex. Previous studies using the sensory preconditioning paradigm have shown that 18 the perirhinal and postrhinal cortices are necessary for associating cues during preconditioning 19 and retrieving the associations during conditioning. However, the question of whether these 20 regions are also required for stimulus association retrieval during re-exposure to the 21 preconditioning cue had not been addressed. Using a chemogenetic approach, we temporarily 22 suppressed the perirhinal or postrhinal cortex in adult male rats during the preconditioning cue 23 test phase. Both suppression groups showed impaired sensory preconditioning compared to sham 24 surgery controls, as indicated by significantly reduced preferential responding. Implications of 25 our findings are discussed in relation to the importance of the PER and POR in context 26 processing and episodic-like memory in animal models. 27 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 3 Significance statement 28 Although Pavlovian conditioning between two stimuli is widely used in neuroscience, 29 associative learning in the real-world is often too complex to be modeled by first-order 30 conditioning alone. Proper responding may require integrating multiple associations via common 31 elements, i.e. higher-order conditioning, the neural basis of which is not well understood. 32 Whereas existing research indicates the perirhinal and postrhinal cortices in the medial temporal 33 lobe contribute to certain forms of sensory preconditioning (SPC, a type of higher-order 34 conditioning), we suggest their contributions lie in the encoding and retrieving latently acquired 35 associations, processes which are central to all forms of SPC. We argue this interpretation also 36 captures perirhinal and postrhinal contributions to contextual and episodic memory, hence 37 offering a unifying explanation regarding the contributions of these cortices across multiple 38 paradigms. 39 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 4

40 The medial temporal lobe (MTL) plays pivotal roles in a range of memory functions 41 underpinning human and animal behavior. In the rodent MTL, the perirhinal cortex (PER) and 42 postrhinal cortex (POR, homologous to the primate parahippocampal cortex) are critically 43 involved in various forms of learning and memory (Burwell et al., 1995; Estela-Pro & Burwell, 44 2021; Peng & Burwell, 2021). Both regions receive polymodal and unimodal associational input 45 from other cortical and subcortical structures (Burwell & Amaral, 1998a; Furtak et al., 2007; 46 Tomas Pereira et al., 2016). Information from the PER and POR reaches hippocampal CA1 and 47 the subiculum through direct projections and indirect connections via the entorhinal cortex 48 (Agster & Burwell, 2013). The PER specializes in processing item features (i.e. “what”), 49 whereas the POR mainly processes spatial properties of items and the environment ( i.e. 50 “where”). Through robust, reciprocal connections, the PER and POR work together to represent 51 the local environment, including the spatial layout of the items, objects, and patterns contained in 52 the spatial context (Burwell & Amaral, 1998b; Estela, 2020; Furtak et al., 2012; Heimer-McGinn 53 et al., 2017). The information is used by the hippocampus to support episodic, recognition, and 54 other forms of memory (Diana et al., 2007; Eichenbaum et al., 2012; Eichenbaum et al., 2007; 55 Yonelinas et al., 2022). 56 57 Sensory preconditioning is a form of second-order conditioning involving the integration of 58 multiple associations formed through separate experiences (Brogden, 1939). In the 59 preconditioning phase, subjects receive paired presentations of two neutral stimuli. Next, in the 60 conditioning phase, subjects undergo first-order Pavlovian conditioning between one of the 61 neutral stimuli and an unconditioned stimulus (US); this neutral stimulus hence becomes a 62 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 5 conditioned stimulus (CS). Finally, in the test phase, the other neutral stimulus (the 63 preconditioning stimulus, PCS) is presented to the subject. The PCS is expected to elicit the 64 conditioned response, despite never having been directly paired with the US. 65 66 Sensory preconditioning may be supported by two cognitive mechanisms. The PCS may become 67 directly associated with the US via “representation-mediated learning” (Holland, 1981). In this 68 case, the CS evokes a representation of the PCS during conditioning, and an association is 69 established between the PCS representation and the US. Alternatively, the PCS may elicit 70 conditioned responding indirectly via an “associative chain” composed of the PCS-CS 71 association formed during preconditioning and the CS-US association formed during 72 conditioning (Rizley & Rescorla, 1972). Multiple factors seem to influence which mechanism 73 supports a sensory preconditioning procedure (Holmes et al., 2022). However, we are not aware 74 of any study that has systematically manipulated these factors and evaluated their influence. 75 Moreover, it is unclear whether different neural substrates are engaged by these two 76 mechanisms. Both the PER (Holmes et al., 2013; Nicholson & Freeman Jr, 2000; Wong et al., 77 2019) and the POR (Taylor-Yeremeeva et al., 2021) are shown to be critically involved in 78 sensory preconditioning. In these studies, however, the experimental procedures likely favored 79 representation-mediated learning over associative chaining as the primary underlying 80 mechanism. Therefore, whether the involvement of the PER and POR in sensory preconditioning 81 extends to associative chaining is an open question. 82 83 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 6 In the current study, we utilized a sensory preconditioning procedure previously shown to rely on 84 associative chaining at the time of PCS testing (Jones et al., 2012) and investigated the 85 involvement of the PER and POR. Since we reasoned that PER and POR contributions to 86 sensory preconditioning are the basis for their broader roles in latent learning (i.e. learning in the 87 absence of explicit reward or punishment), we hypothesized that the two regions should be 88 required for retrieving neutral associations for chaining as well. We therefore predicted reduced 89 responding to the PCS when either the PER or the POR was temporarily unavailable during the 90 PCS test. We employed the Designer-Receptor-Exclusively-Activated-by-Designer-Drugs 91 (DREADDs) technique to create temporary suppression by systematic injection of clozapine-N-92 oxide prior to behavioral sessions (Smith et al., 2016; Urban & Roth, 2015). 93 94

95 Subjects 96 The subjects were 34 adult male Long-Evans rats (Rattus norvegicus) acquired from Charles 97 River Laboratories (Wilmington, MA). Upon arrival at our facility, they were pair-housed in 98 ventilated cages, placed on a reverse 12-hour light/dark cycle, and given ad libitum access to 99 food and water. They were food restricted to 85-90% free-feed body weight starting from one 100 week before surgeries until the end of the experiments. All subjects were about 3-4 months old at 101 the time of surgeries and weighed between 300g to 400g. Subjects were handled by multiple lab 102 personnel before surgery and during post-surgical recovery. Three subjects died during or shortly 103 after long surgical procedures, one reacted badly to CNO injection procedures and was removed, 104 and three subjects were removed due to equipment malfunction. This left 10 in the sham group, 105 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 7 11 in the PER group, and 9 in the POR group. All procedures were conducted according to NIH 106 guidelines and protocols approved by the Brown University Institutional Animal Care and Use 107 Committee. 108 Table 1. Stereotaxic coordinates for bilateral DREADD virus injections into PER or POR. Site AP (mm) ML (mm) DV (mm) Angle PER1 3.2 +/- 5.0 6.3 13° PER2 4.4 +/- 5.0 6.2 13° PER3 5.6 +/- 5.0 5.9 13° PER4 6.8 +/- 5.0 5.5 13° POR1 -0.5* +/- 5.0 4.5 16° POR2 0.2* +/- 5.0 2.75 16° In the anterior-posterior axis (AP), positive values indicate locations posterior to the reference point, and negative values indicate locations anterior to the reference. The bregma was used as reference for PER sites, whereas the lambda was the reference for POR sites (marked by *). Dorsal-ventral (DV) values indicate distances to the skull surface. Surgery 109 The timeline for the procedures is shown in Figure 1 (panel A). Subjects were randomly assigned 110 to the PER group, POR group, or Sham group before surgery. Pre-operative preparation included 111 injections of glycopyrrolate, diazepam, and buprenorphine, and the subjects’ heads were shaved 112 to expose the surgery site. Subjects were anesthetized with isoflurane and secured with ear bars 113 in a stereotaxis apparatus. The bregma and lambda were arranged in the same horizontal plane 114 (+/- 0.1 mm) by adjusting the incisor bar. Craniotomies over the injection coordinates were made 115 using a dental drill, and the dura was pierced prior to pipette insertion. For rats in the PER and 116 POR group, bilateral injections of the DREADD virus AAV8-CaMKIIa-hM4d(Gi)-mCherry 117 were made using a Hamilton syringe pump at 0.1 µl/min for 5 minutes per site (Table 1). The 118 pipette tip remained at the injection coordinate for an additional 10 minutes before slowly being 119 retracted. Rats in the Sham group received craniotomies only. Afterwards, gel foam soaked in 120 sterile saline was placed over the cortex, and the incision was sutured. All subjects were given at 121 least 2 weeks to recover from the surgery before behavioral procedures began. 122 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 8 123 Apparatus 124 Behavioral procedures were performed in conditioning chambers (Med Associates) placed inside 125 sound attenuating cabinets. The chambers were made with two aluminum side panels, three 126 Plexiglas panels as front, back, and the ceiling. A food port located on one of the side panels was 127 used to deliver pellets and detect the frequency and duration of nose pokes via infrared sensors. 128 A speaker and a mechanical relay were located on the same side panel near the roof to produce 129 the auditory stimuli used in the behavioral experiments. Four auditory stimuli (about 75 dB) 130 were used for sensory preconditioning. The white noise and click (0.5 Hz) were used as PCS+ 131 and PCS-, whereas a tone (2.5 kHz) and a siren (alternating between 1 kHz and 4 kHz) were used 132 as CS+ and CS-. The stimulus arrangement was counter-balanced across subjects. The floor was 133 made of stainless-steel rods connected to a shock generator and scrambler that were used to 134 deliver foot shocks for fear conditioning. Video cameras mounted behind the back Plexiglas 135 panel captured the fear conditioning sessions and saved the videos to a hard drive. Additional 136 cues were added to the chamber for the fear conditioning experiment to differentiate the new 137 contexts from the base context. These context cues included laminated sheets with distinctive 138 visual patterns and artificial odors, such as vanilla and almond. 139 140 Behavioral procedures 141 Sensory preconditioning. The general procedure was based upon prior studies from Schoenbaum 142 and colleagues (Hart et al., 2020; Jones et al., 2012; Sadacca et al., 2018). Subjects first 143 underwent a magazine training session, in which a total of 20 pellets were delivered via a 144 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 9 variable-interval schedule. For this and all following sessions, the inter-trial intervals varied 145 pseudo-randomly from three to six minutes. The procedure contained four phases (Figure 1B). 146 The first was the preconditioning phase. Subjects were placed individually into conditioning 147 chambers for two daily sessions of 12 preconditioning trials each. Each trial contained a serial 148 presentation of one of two pairs of auditory stimuli. No reward was delivered during this phase. 149 Next, in the conditioning phase, subjects received six sessions of first-order Pavlovian 150 conditioning, each with 12 trials. During the CS+ trials, three food pellets total were delivered 151 into the food port at 5, 7, 9 seconds after CS+ onset. No pellets were delivered during CS-. The 152 third phase was the PCS testing phase. About 30 minutes prior to testing, all subjects received an 153 intraperitoneal injection of clozapine N-oxide (CNO, 5mg per 1kg of body weight) and were 154 returned to the home cage. The first 6 trials were CS+ and CS- reminder trials identical to trials 155 in the conditioning phase. Next, subjects were presented with 12 PCS probe trials arranged in 156 four 3-trial blocks in which the PCS+ and PCS- were presented without rewards. Finally, 48 157 hours after PCS testing, a 12-trial CS probe session was conducted about 30 minutes after 158 intraperitoneal CNO injections. In each trial, subjects were presented with the CS+ or CS without 159 reward delivery. All trial types in each session were counter balanced. 160 Fear conditioning. At least 30 days after the CS test session, a fear conditioning procedure was 161 conducted to behaviorally verify the efficacy of DREADDs inhibition (Figure 1C). Though the 162 same conditioning chambers were used, each subject was assigned a chamber different from the 163 one where they underwent sensory preconditioning. Multiple visual, tactile, and olfactory cues 164 were also introduced into the chambers to distinguish them from previous sensory 165 preconditioning contexts. On day 1 (shock training with CNO), subjects received one session of 166 signaled fear conditioning 30 minutes after CNO injections. Subjects were placed into context A. 167 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 10 After three minutes, three pure tones (10s, 2 kHz, 75dB) were delivered, each co-terminating 168 with a foot shock (1s, 1.0 mA). The inter-stimulus-interval was one minute. Subjects remained in 169 the chamber for an additional minute after the last tone-shock pairing before being returned to 170 their home cages. On day 2 (context extinction without CNO), subjects were again placed into 171 context A for 9 minutes without tone or shock delivery. On day 3 (tone extinction without CNO), 172 subjects were put into context B. After 2 minutes, they received a continuous tone presentation 173 lasting for 7 minutes. The specific configurations of context A and B were counter-balanced 174 among subjects. All sessions were recorded with Ethovision 15 and saved to a computer hard 175 drive for analysis. 176 Figure 1. Schematics for experimental timeline and behavioral procedures. A) Overall timeline for the experiment, from the start of the surgeries until the end of behavioral testing. B) The Pavlovian sensory preconditioning procedure based on studies such as Jones et al. (2012) combined with CNO injections, indicated by the syringe symbol. Pellets were delivered only during the last 5 s of CS+ trials, indicated by upward arrows. C) A standard one-session fear conditioning procedure, followed with context extinction and tone extinction. CNO injections were only given on the first day. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 11 Behavioral and statistical analysis 177 Sensory preconditioning. Custom MED-PC scripts were written to control the stimuli and 178 generate output containing the timestamps of all nose pokes into the food port. The output file 179 was then analyzed by a custom-written Python script to compute the latency and total duration of 180 nose pokes during periods of interest. For preconditioning, the nose poke time percentage 181 differences were calculated between PCS+ and PCS-, and between CS+ and CS-. A positive 182 difference score indicated a higher nose poke time percentage during the PCS+ versus PCS-, or 183 the CS+ versus CS-. Separate one-way ANOVAs were conducted to detect whether any initial 184 preference existed before conditioning started. For conditioning trials in the conditioning phase 185 and CS reminder trials in PCS test, pellets were delivered during the second half (5 s) of the 186 CS+, therefore conditioned responding was assessed by comparing the nose poke time 187 percentages during the first half of the CS+ and CS-. For PCS and CS test trials, nose poke time 188 percentages during the second half of the stimuli were compared. The difference scores were 189 computed. First, a mixed-design ANOVA was conducted on difference scores from the 190 conditioning sessions, with GROUP (Sham, PER, POR) as the between-subject factor and 191 SESSION (1 to 6) as the within-subject factor. This test examined whether any group differences 192 occurred during first-order Pavlovian conditioning, even though no CNO injection was made 193 prior to the conditioning sessions. Second, a one-way ANOVA with GROUP as the between-194 subject factor was conducted on difference scores from PCS trials in the PCS test session under 195 inactivation. Further, one-sample t tests were conducted to compare each group’s PCS difference 196 score to 0. The Bonferroni-Holm procedure was applied to control for family-wise type I error 197 rate (Abdi, 2010). These tests provided additional evidence on whether sensory preconditioning 198 was successful in the Sham group, and whether suppression of the PER or POR led to deficits. 199 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 12 The same analysis was conducted for CS test trials, also under DREADD suppression. These 200 tests evaluated whether first-order conditioning was successful in the Sham group, and whether 201 suppression of the PER or POR led to deficits. Finally, response latencies during PCS+ and CS+ 202 trials were tested for group differences with a one-way ANOVA to determine whether attentional 203 deficits contributed to group differences in nose poke time percentages. 204 Fear conditioning. The sessions were broken into 1-minute blocks, then a motion analysis 205 software, custom-written in Python, was used to measure the total duration of freezing within 206 each minute. Briefly, adjacent video frames were contrasted and processed to detect changes in 207 pixel intensity that indicated movement from the subject. The subject was determined to be 208 freezing when the period without movement exceeded a preset threshold. This software was 209 validated with human observation in prior fear conditioning experiments (data not included). 210 Expression of contextual fear was evaluated by analyzing freezing behavior on day 2 (context 211 extinction). A mixed-design ANOVA was conducted with BLOCK as the within-subject factor 212 and GROUP as the between-subject factor. Post hoc two-sided Dunnett’s t tests were used to 213 compare each of the PER and POR group to the Sham group. Tone-elicited fear behavior on day 214 3 (tone extinction) was examined using the same tests. 215 216 Histology 217 After the last fear conditioning session, the subjects were deeply anesthetized with isoflurane and 218 received an overdose of Beuthanasia-D (100mg/kg, i.p.). They were then perfused with 219 phosphate-buffered saline, followed by a 10% formalin solution. The brains were post-fixed for 220 at least 48 hours in formalin and then were transferred to a 30% sucrose solution for at least 221 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 13 another 48 hours. The brains were sectioned on a microtome into three series of 40μm sections. 222 For each brain, one series was later stained with thionine, and another was stained with DAPI for 223 fluorescence microscopy. Fluorescent images were taken in blue and red channels showing 224 DAPI-stained neurons and neurons transfected by the DREADD virus, respectively. For 225 histological analysis, each brain was scored based on expression in the rostral and caudal halves 226 of the target area and on the degree of extra-target spread. 227 228

229 Histology 230 Of the 11 rats in the PER group, viral transduction was observed bilaterally, and along the 231 rostrocaudal extent of the target region in eight (Figure 2A). Thus, three were removed for 232 insufficient transduction. For two of the eight cases, there was also moderate involvement in the 233 ventral temporal association cortex and caudal hippocampus in both hemispheres, and these 234 cases were also removed, leaving six in the PER group. Of the 9 rats in the POR group, viral 235 transduction was observed bilaterally, and along the rostrocaudal extent of the region in seven 236 (Figure 2B). For two cases, there was insufficient transduction, and those cases were removed. 237 For three cases of the seven, there was slight involvement in the most caudal PER and/or caudal 238 entorhinal cortex, and these cases were retained. In some cases, there was slight unilateral 239 involvement of the temporal association cortex (TEv) dorsal to the POR, and these cases were 240 also retained in the study. 241 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 14 Figure 2. Schematics of PER and POR showing target locations along with representative labeling of viral transduction. A) Four levels of the PER with the target regions illustrated by dashed lines. Labeling was distributed across the rostrocaudal extent (see text for details). B) Four levels of the POR with the target regions illustrated by dashed line. Again, labeling was distributed across the rostrocaudal extent (see text for details). C) Example of labeling in PER. See inset in A for location. D). Example of labeling in POR. See inset in B for location. DAPI staining is captured in the blue channel, and the DREADD virus is captured in the red channel. Preconditioning and conditioning 242 The groups did not differ on nose poke time percentage differences during preconditioning 243 sessions in their initial preference for PCS+ versus PCS- or CS+ versus CS-. A mixed-design 244 ANOVA confirmed no significant DIFFERENCE*GROUP interaction (p = 0.792) and no main 245 effects for DIFFERENCE (p = 0.625) or GROUP (p = 0.99). As expected, no impairment was 246 found in any group for first-order conditioning (Figure 3). All groups significantly increased 247 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 15 nose poke time during the first 5s of CS+, compared to CS-, and the changes were not 248 significantly different among groups. A mixed-design ANOVA on nose poke time percentage 249 differences for the 6 conditioning sessions showed a significant main effect for SESSION, F(5, 250 100) = 4.61, p = 0.001, but not for the SESSION*GROUP interaction (p = 0.247) or for GROUP 251 (p = 0.849). 252 Figure 3. Nose poke time percentage difference during the conditioning phase. Data was only analyzed for the first half (5 s) of CS+ and CS- because pellets were delivered during the second half of CS+. Error bars indicate standard error. 253 PCS test 254 The PCS test was conducted 30 minutes after the subjects received a CNO injection. The test 255 started with 6 reminder trials that were identical to conditioning trials in that pellets were 256 delivered during the last 5s of the CS+. The main measure for the sensory preconditioning effect 257 was nose poke time the percentages during PCS+ and PCS- (Figure 4A). As predicted, the sham 258 group showed a robust response sensory preconditioning effect, but the PER and POR groups did 259 not (Figure 4). A one-way ANOVA was conducted to compare the nose poke time percentage 260 difference between the last 5s of PCS+ versus PCS-. There were significant differences among 261 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 16 groups, F(2, 20) = 3.82, p = 0.039 (Figure 4B). Planned comparisons revealed a marginally 262 significant difference between the Sham group and the PER group, t(20) = 2.02, p = 0.057, and a 263 significant difference between the Sham and the POR group, t(20) = 2.53, p = 0.020. Further, 264 one-sample t tests showed that only the sham group had a significantly positive nose poke time 265 percentage difference, t(9) = 3.71, p = 0.005. Neither the PER, t(5) = 0.97, p = 0.378, nor POR 266 group t(6) = 0.001, p = 0.999, was significantly different from 0. Thus, after CNO injections, the 267 sham group had significantly higher responding to PCS+ than PCS- during the last 5 s, whereas 268 the PER and POR groups did not. This result provided evidence for an impairment in retrieving 269 the PCS-CS association when either the PER or POR is under temporary DREADDs 270 suppression. 271 272 Figure 4. Nose poke time percentage during the last 5s of PCS trials in the PCS test phase with DREADD suppression. A) nose poke time percentages during PCS+ and PCS-. P-value symbols indicate significant differences between stimuli. B) nose poke time percentage difference between responding during PCS+ and PCS-. Positive values indicate higher responding during PCS+. P- value symbols indicate significant differences between groups. #: p < 0.10, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Error bars indicate standard error. 273 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 17 CS test 274 To assess first-order conditioning, a CS test session was conducted 48 hours after the PCS test, 275 also following CNO injections. All groups showed a longer nose poke time percentage during 276 the last 5s of the CS+, compared with the last 5s of the CS- (Figure 5A). The percentage time 277 differences between CS+ and CS- were analyzed with one-way ANOVA (Figure 5B). The 278 omnibus ANOVA was not significant, F(2, 20) = 2.39, p = 0.117. Planned comparisons revealed 279 no significant difference between the Sham and PER groups, t(20) = 1.66, p = 0.112), but a 280 marginally significant difference between the Sham and POR groups, t(20) = 2.01, p = 0.064. 281 One-sample t tests showed that all groups had significantly positive difference scores during the 282 CS test: sham, t(9) = 7.96, p < 0.001; PER, t(5) = 3.40, p = 0.019; POR, t(6) = 6.79, p < 0.001. 283 Overall, all groups demonstrated significantly longer nose poke time percentage during the CS+, 284 compared with the CS-, indicating successful first-order conditioning. 285 Figure 5. Nose poke time percentages during the last 5s of CS trials in the CS test with DREADD suppression. A) nose poke time percentages during CS+ and CS-. Asterisks indicate significant one-sample t-tests on the difference between CS+ and CS-. B) nose poke time percentage differences between CS+and CS-. Error bars indicate standard error. 286 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 18 PCS and CS response latency 287 To confirm that DREADD suppression did not lead to attentional deficits in the PER or the POR 288 group, nose poke latencies after stimulus onset during the PCS+ and CS+ test trials were 289 analyzed separately with one-way ANOVA (Figure 6). The omnibus ANOVA was not 290 significant for CS+ trials (p = 0.308) or PCS+ trials (p = 0. 379), and none of the planned 291 comparisons between the Sham and virus groups was significant. Therefore, deficits observed in 292 nose poke time percentages cannot be explained by delayed reaction in response to stimulus 293 onset. 294 Figure 6. Latencies of nose poking in the test sessions under suppression. A) nose poke latencies since the onset of PCS+ during test trials. B) nose poke latencies since the onset of CS+ during test trials. Error bars indicate standard error. 295 Fear conditioning 296 In a fear conditioning paradigm, subjects received an injection of CNO (i.p.) on day 1, followed 297 by tone-shock conditioning in context A. On day 2, they received extinction in context A without 298 the tone. And on day 3, they underwent tone extinction in context B. No CNO injections were 299 made on day 2 or 3. For freezing time on day 2 (Figure 7A), a mixed-design ANOVA did not 300 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 19 reveal a significant MINUTE*GROUP interaction, F(16, 160) = 0.968, p = 0.494. However, 301 there was a significant main effect of GROUP, F(2, 20) = 6.87, p = 0.005, as well as a main 302 effect of MINUTE, F(4.9, 160) = 2.47, p = 0.039. Further simple contrast tests revealed a 303 significant difference between the Sham and the PER group, p = 0.002, but not between the 304 Sham and the POR group, p = 0.79. Therefore, when re-exposed to the shock context, the PER 305 group displayed a significantly lower amount of freezing when compared to the sham group, 306 indicative of impaired contextual fear memory. No reduction in freezing was observed in the 307 POR group, indicating normal contextual fear memory. Since DREADDs suppression was 308 induced only during tone-shock training (day 1) but not on day 2, this suggests that the 309 acquisition of contextual fear was impaired in the PER group, but not in the POR group, when 310 compared to the Sham group. 311 Freezing to the tone on day 3 was also analyzed by a mixed-design ANOVA to detect the 312 presence of tone-elicited fear (Figure 7B). A significant main effect of MINUTE was revealed, 313 F(3.74, 74.81) = 11.76, p < 0.001, but no significant MINUTE *GROUP interaction, F(7.48, 314 74.81) = 1.34, p = 0.203, or main effect of GROUP, F(2, 20) = 1.77, p = 0.196. Simple contrast 315 tests revealed a marginally significant difference between the Sham and the PER group (p = 316 0.82). Further tests revealed a significant linear trend for MINUTE, F(1, 20) = 36.60, p < 0.001. 317 The results indicated that all three groups gradually displayed less freezing to the tone, with no 318 significant group difference. Therefore, fear conditioning to the tone was not impaired by PER or 319 POR suppression during conditioning. 320 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 20 Figure 7. Freezing time percentage in each minute during context extinction (day 2, panel A) and tone extinction (day 3, panel B). The tone was presented from the start of the third minute during tone extinction. Error bars indicate standard error. 321

322 Chemogenetically suppressing bilateral PER or POR during the PCS test impaired sensory 323 preconditioning by disrupting the retrieval of PCS-CS associations. First-order conditioning 324 remained intact in all groups under the influence of CNO. The overall results suggest that, 325 although the PER and POR are not necessary for conditioned responding to directly conditioned 326 auditory stimuli used in this experiment, both are required for retrieving neutral stimulus-327 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 21 stimulus (S-S) associations when re-exposed to the PCS. These findings appear partially 328 inconsistent with existing studies using temporary PER or POR suppression. However, we argue 329 that this is because the sensory preconditioning effect in our study relied on a mechanism 330 different from procedures in previous studies. Whereas existing studies collectively showed that 331 the PER and POR are necessary for sensory preconditioning when it primarily depends on 332 representation-mediated learning, our findings indicate they are crucial for associative chaining 333 as well. 334 335 Our sensory preconditioning procedure was slightly modified from a previous study in which 336 sensory preconditioning deficits occurred when temporarily suppressing the orbitofrontal cortex 337 during the PCS test (Jones et al., 2012). This pattern of deficit typically indicates that responding 338 to the preconditioning cue is likely supported by associative chaining (Holmes et al., 2022; Jones 339 et al., 2012). In contrast, previous studies of the PER or POR employed procedures that likely 340 relied on representation-mediated learning (Holland, 1981). A recent study found reduced 341 responding to the PCS if chemogenetic POR suppression took place during preconditioning or 342 conditioning, but not if it occurred during PCS testing (Taylor-Yeremeeva et al., 2021). In Wong 343 et al. (2019), reduced sensory preconditioning was observed when a sodium channel blocker was 344 injected into the PER before conditioning or before the test (albeit of a smaller degree). The 345 authors concluded that PER-dependent memory integration in their procedure occurred primarily 346 via mediated learning during conditioning, though it wasn’t ruled out that some associative 347 chaining also took place at the time of testing. Collectively, these studies and ours suggest the 348 PER and POR are necessary for retrieving neutral S-S associations, regardless of whether these 349 associations are used for mediated learning or associative chaining. Moreover, both mechanisms 350 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 22 were shown to be necessary for encoding associations between neutral stimuli during 351 preconditioning (Holmes et al., 2013; Taylor-Yeremeeva et al., 2021). Overall, the evidence 352 supports the notion that the critical contribution of the PER and POR to sensory preconditioning 353 is the encoding and retrieving of the associations formed when the stimuli involved are neutral. 354 355 The ability to associate neutral stimuli in the absence of explicit reward or punishment fits well 356 with the roles of the PER and POR in automatically representing the spatial context and using 357 contextual cues to guide spontaneous or learned behavior (Peng & Burwell, 2021). At the most 358 basic level, the context exists as a loose collection of environmental elements that contain multi-359 sensory information without meaningful connections. But with the contribution from MTL 360 structures such as the PER and POR, associations formed among the elements become the basis 361 for a cohesive representation of the context. For example, neurons in the POR increase firing 362 rates when the rat encounters a particular object at a particular location, but not when 363 encountering the same object at other locations or other objects at the same location (Furtak et 364 al., 2007). This “object-location” conjunctive coding likely emerges as object information from 365 the PER is combined with spatial information from POR via robust direct interconnections 366 (Agster & Burwell, 2013; Heimer-McGinn et al., 2017). The PER and POR are necessary in a 367 variation of sensory preconditioning in which the preconditioning phase requires establishing an 368 association between a neutral cue and the context (Iordanova et al., 2009; Taylor-Yeremeeva et 369 al., 2021). 370 371 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 23 In typical context learning paradigms, the context is usually composed of static stimuli that 372 remain constant throughout a session. In contrast, associations formed and retrieved in sensory 373 preconditioning are among transient stimuli with clear onsets and offsets. Therefore, the critical 374 involvement of the PER and POR in sensory preconditioning suggests their contributions extend 375 beyond the well-known ability for context learning; they may also be capable of representing 376 transient cues inside the context. In other words, they are not only able to represent stimuli 377 existing in the three-dimensional space, but also stimuli in the dimension of time. This is 378 consistent with findings about events and sequences representations in other parts of the MTL, 379 such as the entorhinal cortex (Robinson et al., 2017) and the hippocampus (Allen et al., 2016; 380 MacDonald et al., 2013; Pastalkova et al., 2008). The PER is necessary for bridging the temporal 381 gap between segments of a discontinuous auditory stimulus (Bang & Brown, 2009; Kholodar-382 Smith, Allen, & Brown, 2008) and between a CS and US in trace fear conditioning (Kholodar-383 Smith, Boguszewski, & Brown, 2008). It is likely supported by persistent firing PER neurons 384 that can maintain activation for seconds to several minutes (Navaroli et al., 2012). The PER is 385 also important for incidental learning of odor sequences (Allen et al., 2020; Jayachandran et al., 386 2019). The POR also plays a role in processing localized temporal events with a significant 387 spatial component, in which case it may cooperate with the PER cortex via their functional 388 connectivity (Heimer-McGinn et al., 2017). 389 390 Combining the evidence that both areas are necessary for incidental learning of event 391 associations together with their well-established roles in representing context, we propose that 392 the rodent PER and POR may collectively construct and maintain neural representations of 393 stimulus sequences in contexts that serve as rudimentary forms of event representation. These 394 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 24 representations contain salient features of an event, such as the occurrence of transient cues, their 395 associations and temporal relationships, and the context. In other words, these “event elements” 396 in the PER and POR constitutes the “gist”, but not the full perceptual aspects of an event 397 (Moscovitch et al., 2016; Robin & Moscovitch, 2017). These representations may be available to 398 the hippocampus as the “backbone” for episodic memory (Agster & Burwell, 2013; Furtak et al., 399 2007). Alternatively, they may also be used by areas outside the MTL for hippocampal-400 independent behaviors, such as expressing remote fear memory (Burwell et al., 2004), or when 401 hippocampal functioning is disrupted (Fanselow, 2010; Wiltgen et al., 2006). This does not 402 detract from the key role of the hippocampus in episodic memory (Eichenbaum, 2017; 403 Eichenbaum et al., 2012; Moscovitch et al., 2016; Robin & Moscovitch, 2017). Instead, it fits 404 into the idea that two types of episode memory representations dynamically co-exist in the brain, 405 with one being “schematic” or “gist-like” that mostly resides in the neocortex, and the other one 406 being detail-rich and hippocampal-dependent (Moscovitch et al., 2016; Robin & Moscovitch, 407 2017). The PER and POR have reciprocal connections with the hippocampus and various 408 prefrontal areas (Agster & Burwell, 2013; Furtak et al., 2007; Hwang et al., 2018), therefore they 409 potentially serve as a region where the two types of episodic representations overlap or interact. 410 Other cortical areas such as the orbitofrontal cortex and the retrosplenial cortex may be involved 411 as well (Hart et al., 2020; Jones et al., 2012; Robinson et al., 2014; Todd et al., 2019). 412 413

from the contextual fear conditioning with DREADD inactivation during training were 414 partially inconsistent with existing literature. Prior studies found that normal contextual fear 415 learning required both the PER and POR (Bucci et al., 2000; Bucci et al., 2002). Although in the 416 current study the PER group displayed less freezing during the context extinction test as 417 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 25 expected, the POR group had similar levels of context-elicited freezing compared to the Sham 418 group. We argue this discrepancy regarding effects of POR inactivation was because the fear 419 conditioning contexts were not sufficiently distinctive from the sensory preconditioning context, 420 even though we did introduce various visual and olfactory cues to the chambers for fear 421 conditioning. Consequently, unlike typical fear conditioning in which the context is novel to the 422 subjects, our subjects were already partially familiar with context where shocks occurred. This 423 may suggest that although contextual representations initially depend on both the PER and POR, 424 as the context becomes increasingly familiar, the PER alone gradually becomes more efficient at 425 representing the context thanks to visuospatial input from the POR and other areas, until the PER 426 alone may be sufficient for new learning, such as contextual fear conditioning. 427 428 In conclusion, we utilized the DREADD technique to show that both the PER and POR are 429 necessary for retrieving neutral S-S associations in a sensory preconditioning procedure that 430 likely depends on the chaining of associations at PCS testing. Combined with their well-431 established roles in context processing, these findings suggest the PER and POR collectively 432 support a rudimentary, gist-like representation of event elements that could form the basis for 433 episodic-like memory in rodents. Although latent learning paradigms such as sensory 434 preconditioning do not capture the full spectrum of episodic-like memory in animals (Clayton et 435 al., 2003), they nevertheless provide us with a powerful tool to investigate the neural 436 mechanisms underlying different aspects of episodic-like memory in animal models. 437 438 439 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 26, 2024. ; https://doi.org/10.1101/2024.05.26.595958doi: bioRxiv preprint 26

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