Neuronal activity – driven oligodendrogenesis in selected brain regions is required for episodic 1 memories 2 3

The formation of long-term episodic memories requires the activation of molecular mechanisms in several regions of the medial temporal lobe, including the hippocampus and anterior cingulate cortex (ACC). The extent to which these regions engage distinct mechanisms and cell types to support memory formation is not well understood. Recent studies reported that oligodendrogenesis is essential for learning and long-term memory; however, whether these mechanisms are required only in selected brain regions is still unclear. Also still unknown are the temporal kinetics of engagement of learning-induced oligodendrogenesis and whether this oligodendrogenesis occurs in response to neuronal activity. Here we show that in rats and mice, episodic learning rapidly increases the oligodendrogenesis and myelin biogenesis transcripts olig2, myrf, mbp, and plp1, as well as oligodendrogenesis, in the ACC but not in the dorsal hippocampus (dHC). Region-specific knockdown and knockout of Myrf, a master regulator of oligodendrocyte maturation, revealed that oligodendrogenesis is required for memory formation in the ACC but not the dHC. Chemogenetic neuronal silencing in the ACC showed that neuronal activity is critical for learning-induced oligodendrogenesis. Hence, an activity-dependent increase in oligodendrogenesis in selected brain regions, specifically in the ACC but not dHC, is critical for the formation of episodic memories.Oligodendrogenesis is required in the anterior cingulate cortex but not in the hippocampus for long-term memory consolidation.


Introduction 34
Long-term memories are initially fragile but become resilient to disruption through consolidation, 35 a temporally graded process that engages cascades of molecular mechanisms in select brain regions. 36 Episodic memories become consolidated by rapidly recruiting molecular changes in several brain regions, 37 including the hippocampus, medial prefrontal cortex (mPFC), and anterior cingulate cortex (ACC) 38  (Hughes et al., 2018). These studies examined 53 the role of oligodendrocytes by assessing brain-wide oligodendrogenesis, however, several questions 54 remain to be addressed. First, are distinct brain regions differentially engaging oligodendrocytes 55 mechanisms in learning and memory? Second, what is the fine temporal engagement of learning-induced 56 oligodendrogenesis? And, finally, does this oligodendrogenesis require neuronal activation? 57 Steadman et al. (2020) reported that the acquisition of spatial memory in mice is accompanied by 58 an increase in oligodendrocyte precursor cells (OPCs) proliferation and/or differentiation mechanisms in 59 induced increase in OLIG2 protein levels (Fig. 1E, G). We measured the number of proliferating OPCs by 138 counting cells co-stained with fluorescently labeled EdU and antibodies to platelet-derived growth factor 139 receptor alpha (PDGFR), a marker of OPCs (Rivers et al., 2008). We quantified newly differentiated To confirm the effect of myrf deletion in OPCs on oligodendrocyte differentiation in the brain, 162 TAM-treated P-Myrf floxed/floxed and P-Myrf +/+ mice received an injection of EdU one hour before IA 163 training to label proliferating cells and were perfused one day later. Oligodendrogenesis was significantly 164 inhibited in the ACC in P-Myrf floxed/floxed mice, as demonstrated by the significant reduction in the number 165 of cells that were positive for EdU, OLIG2, and CC1 in P-Myrf floxed/floxed mice compared to P-Myrf +/+ 166 littermate controls (Fig. 3A). 167 To test the effect of Myrf knockout on memory formation, TAM was administered to 168 Myrf floxed/floxed and P-Myrf +/+ mice seven days before IA training, and the mice were tested at 1, 7, and 28 169 days after training. P-Myrf floxed/floxed mice exhibited significant memory reduction at all time points 170 compared to P-Myrf +/+ controls (Fig. 3B). To exclude the potential effects of multiple testing, a second 171 experiment was conducted in which P-Myrf floxed/floxed and P-Myrf +/+ littermates were tested only at 28 days 172 after training, and we again observed significant impairment in memory retention (Fig. 3C). We 173 concluded that brain-wide oligodendrogenesis is required for long-term memory formation and that 174 inhibiting oligodendrogenesis before training impairs memory retention at both recent and remote time 175 points post-training. 176 To determine whether oligodendrogenesis contributes to the persistence or storage of memory, 177 we administered TAM to P-Myrf floxed/floxed and P-Myrf +/+ mice 14 days after training, when the 178 consolidation process has significantly advanced (Bambah-Mukku et al., 2014; Squire et al., 2015). 179 Memory retention was tested 14 days after knockout, corresponding to 28 days after training, as well as at 180 36 days and 56 days after training. No difference was detected between groups (Fig. 3D), indicating that 181 oligodendrogenesis is not required for the persistence, retrieval, or storage of long-term memory. 182 Finally, to determine whether mechanisms involving oligodendrogenesis play a role in the formation of 183 non-aversive episodic memories, P-Myrf floxed/floxed and P-Myrf +/+ littermates were injected with TAM 184 seven days before being trained in novel object location (nOL), a hippocampus-dependent learning Thus, Myrf-dependent oligodendrogenesis is also required for the formation of non-aversive 189 hippocampus-dependent memories. 190 To exclude that the memory impairments we observed were due to other behavioral responses 191 such as heightened anxiety-like responses or locomotor impairments, we tested P-Myrf floxed/floxed and P-192 In order to investigate whether oligodendrogenesis is differentially implicated in distinct brain 202 regions and memory processes, we employed a Myrf knockdown strategy. Because the P-Myrf floxed/floxed 203 global knockout approach used previously affects other tissues and organs where Myrf is expressed in 204 addition to the central nervous system, such as the gastrointestinal tract and kidney, employing a region-205 targeted approach also addresses possible off-target effects of Pdgfr-driven global Myrf deletion. We 206 achieved region-specific and temporally restricted Myrf knockdown by using stereotactic injections to 207 deliver an antisense oligodeoxynucleotide (ASO-ODN) specific against Myrf (Myrf-ASO), and, as a 208 control, a related scrambled sequence (Myrf-SCR). We injected the ODNs bilaterally into the brain region 209 of interest at various times before and after training. is required in the ACC for memory acquisition or consolidation. 215 To verify knockdown of myrf, Myrf-ASO and Myrf-SCR were injected bilaterally 15 minutes 216 before training, and myrf mRNA levels were measured in the ACC one hour after training, when there is a 217 significant learning-dependent increase in myrf expression (Fig. 1C). Rats treated with Myrf-ASO had 218 significantly lower myrf mRNA levels compared to those treated with Myrf-SCR (Fig. 4A). Rats injected 219 with Myrf-ASO exhibited no significant differences in MBP protein expression in the ACC one day after 220 training, suggesting that Myrf-ASO treatment does not lead to demyelination (Fig. 4B). 221 In order to test whether MYRF is required for learning, we bilaterally injected Myrf-ASO or 222 Myrf-SCR into the ACC 15 minutes before training and tested the effect 1 hour after training. We 223 detected no differences in memory between the two groups ( Fig. 4C), indicating that MYRF is 224 dispensable in the ACC for learning and short-term IA memory. To test whether MYRF is required for 225 memory consolidation, bilateral injections of Myrf-ASO or Myrf-SCR were administered in the ACC 15 226 minutes before and six hours after training, then memory was tested one day after training. Rats injected 227 with Myrf-ASO exhibited significant memory impairment one day after training compared to rats that had 228 received Myrf-SCR injections (Fig. 4D), and the impairment persisted at 28 days after training (Fig. 4D). 229 A reminder shock given one day after the remote memory test was unable to reinstate memory, indicating 230 that the memory impairment was not due to a suppressed memory response but likely resulted from 231 disrupted memory consolidation. Furthermore, retraining one day later of rats who had been injected with 232 Myrf-ASO resulted in a long-lasting memory, thereby excluding the possibility that they had experienced 233 memory loss due to damage to the ACC caused by surgery or injections. 234 By contrast, when Myrf-ASO was injected bilaterally into the dHC 15 minutes before and six 235 hours after IA training, we observed no effect on memory retention; memories of the two treatment 236 groups were similar at one day and 28 days after training (Fig. 4E). The lower level of retention in the 237 dHC relative to the ACC with stereotactic injections is typically observed. Thus, we concluded that Myrf-238 dependent oligodendrogenesis in the ACC is critical for the consolidation but not the acquisition of IA 239 and is not required in the dHC. To determine the role of oligodendrogenesis in the ACC on behavioral responses, Myrf +\+ and 256 Myrf flox\flox littermates were treated with the same viral and TAM injection protocol as above but tested 257 for memory retention at one day and seven days after training. Compared to Myrf +\+ littermates, 258 Myrf flox\flox mice showed a significant memory impairment at both time points after training. 259 To test whether ACC-specific oligodendrogenesis is required for learning and short-term 260 memory, another cohort of Myrf +\+ mice and Myrf flox\flox littermates were treated with the same viral 261 injection and TAM protocol but tested at one hour after IA training. No differences between groups were 262 observed (Fig. 5D, E), leading us to conclude that oligodendrogenesis in the ACC is necessary for 263 memory consolidation but dispensable for memory acquisition and short-term memory in mice, just as in 264

rats. 265
To test whether oligodendrogenesis is required for memory formation in the hippocampus, AAV-266 Mbp-CreER T2 was bilaterally injected into the dHC of Myrf +\+ and Myrf flox\flox littermates using the 267 protocol described above. No differences in memory retention were observed at one day or seven days 268 post-training compared to control groups (Fig. 5F), leading us to conclude that oligodendrogenesis is 269 required in the ACC for memory consolidation but not for learning or short-term memory. By contrast, 270 oligodendrogenesis is dispensable in the dHC for the formation of hippocampus-dependent memories. was assessed by confocal microscopy two weeks after viral infection and found to be mostly confined to 284 the ACC (Fig. 6A). The mice were tested one day after training. Treatment with C21 significantly 285 impaired memory retention compared to vehicle injection (Fig. 6B), suggesting that neuronal activity in 286 the ACC is required for memory formation. 287 To determine whether blocking neuronal activity in the ACC affected learning-dependent 288 oligodendrogenesis, AAV-hSyn-hM4D(Gi)-mCherry was bilaterally injected into the ACC and fourteen 289 days later the mice were injected with either C21 or vehicle in combination with EdU two hours before 290 receiving IA training. The mice were perfused one day after training and oligodendrogenesis was assessed 291 by performing immunohistochemistry with an antibody to OLIG2 then quantifying cells that were 292 positive for both EdU and OLIG2. Trained mice injected with C21 had significantly fewer cells with both 293 EdU and OLIG2 staining compared to mice injected with vehicle control, implying that 294 oligodendrogenesis was greatly impaired (Fig. 6C). Thus, we concluded that neuronal activity in the ACC 295 is required for learning-induced oligodendrogenesis. 296 297

Discussion 298
This study showed that episodic learning, modeled by an IA paradigm in rats and mice, induces a 299 rapid expression of the oligodendrocyte-specific mRNAs olig2, myrf, mbp, and plp1 in the ACC but not 300 in the dHC, though we did detect an increase in enpp6 in the dHC. The reason for this increase in enpp6 is 301 unclear; ENPP6 is a choline phosphodiesterase involved in lipid metabolism and myelin biogenesis 302 Our western blot analyses confirmed that levels of OLIG2 significantly increased in the ACC 307 following learning, supporting the idea that oligodendrogenesis is rapidly upregulated in this brain region 308 in response to experience. Interestingly, MBP protein levels did not change, despite a significant increase 309 in mbp mRNA levels. This dichotomy might be due to the fact that there is a large pool of MBP in the 310 brain, so relatively small changes of MBP induced by a learning event may be difficult to be detected. 311 Another oligodendrocyte-specific protein, CASPR, which is an axonal membrane protein involved in 312 myelin sheet growth, significantly increased after learning in the ACC, confirming the idea that learning 313 rapidly activates oligodendrocyte-specific mechanisms and myelination in that region. The upregulation 314 of both mRNAs and proteins accompanied associative learning but were not found in unpaired behavioral 315 paradigms, which served as a control for the separate experiences of context and footshock, indicating 316 that oligodendrocyte-mediated mechanisms are involved in associative memory processes. 317 Our results also extended previous findings on motor, spatial, and contextual fear memories by 318 showing that global disruption of oligodendrogenesis impairs novel object location memories, 319 strengthening the conclusion that oligodendrogenesis is a fundamental mechanism required for long-term 320 memory formation. 321 Furthermore, by using multiple genetic and molecular approaches in rats and mice targeting 322 specific brain regions of interest we provided evidence that Myrf-dependent oligodendrogenesis is 323 required in the ACC but not the dHC, confirming the data across species. Thus, only certain brain regions 324 in a given memory system recruit oligodendrogenesis for memory consolidation. To our knowledge, this 325 is the first demonstration of a differential requirement for oligodendrogenesis in selected brain regions for 326 memory formation, and specifically for hippocampus-dependent memories. (mPFC), but not the hippocampus. They suggested that myelin remodeling following training might be 332 restricted to brain regions associated with long-term consolidation of hippocampus-dependent memories. 333 However, because these studies utilized a global knockout approach, they could not determine whether 334 oligodendrogenesis in specific brain regions is required for memory formation. Identification of region-335 and circuitry-specific requirements for oligodendrogenesis and/or myelination in different types of 336 learning and behavioral stimuli is important because it will offer critical knowledge for better 337 understanding the role of myelin in healthy brain functions as well as in diseases. Such a knowledge will 338 also expand our understanding of the circuitry that supports responses to learning. 339 Why oligodendrogenesis is required in the ACC but not the hippocampus is an open question, and 340 one possible explanation is that oligodendrogenesis may subserve long-term changes required for memory 341 storage. It is known that in cortical regions including the ACC, but not in the hippocampus, episodic and 342 spatial memories are stored for the very long term via a process that requires time and is known as system 343 consolidation (Dudai et al., 2015). During system consolidation the memories that initially recruit Chan, 2020) may be a mechanism supporting the long-lasting memory storage, which in cortical regions 359 persists for weeks, months, or even years. Whether the hippocampus is instructive for the cortical 360 oligodendrogenesis changes induced by learning is possible and is in agreement with the findings that 361 global oligodendrogenesis knockout impairs activity coupling between hippocampus and ACC. Indeed, 362 Steadman (2019) and Pan (2020) both speculated that experience-dependent myelination might promote 363 the coupling of ensembles across regions to support the generation of a coordinated memory network 364 because when they blocked myelin formation throughout the brain, the activity and coordination in neural 365 ensembles across the hippocampus and PFC networks was altered. 366 In the present study, we also dissected the requirement for oligodendrogenesis in various phases 367 of memory. We found that oligodendrogenesis in the ACC is necessary for the consolidation process but 368 not for the initial acquisition of memory (learning) or remote storage. In fact, inhibiting 369 oligodendrogenesis before training did not affect short-term memory or acquisition, nor was there an 370 effect on memory when oligodendrogenesis was inhibited at a remote time point. However, disruption of 371 oligodendrogenesis after training impaired long-term memory tested one day later, and the impairment 372 (2020) reported that myrf knockout mice trained in contextual fear conditioning (CFC) had intact recent 382 memory recall at 1 day after training but impaired remote memories at 28 days after training. We found 383 that global and ACC-targeted knockout of myrf in mice as well as ACC-specific ODN-mediated 384 knockdown of MYRF in rats impaired recent memories, tested at one day after IA training. The 385 impairments persisted in both rats and mice tested up to 28 days after training, leading us to conclude that 386 MYRF-dependent oligodendrogenesis is rapidly upregulated and engaged following learning to 387 selectively support a rapid phase of memory consolidation. It is possible that task-related differences in 388 the kinetics of MYRF requirements exist, and that CFC has a slower cortical recruitment of 389 oligodendrogenesis relative to water maze and IA tasks. Knowing the role of oligodendrogenesis in 390 specific memory processes and temporal phases of memory provides valuable information for future 391 development of temporally targeted treatments for cognitive symptoms of demyelinating diseases. 392 In sum, our data support the view that activity-regulated oligodendrogenesis in selected brain 402 regions underlies hippocampus-dependent memory consolidation. We suggest that this induced 403 oligodendrogenesis provides the myelination necessary to support the stabilization process required to 404 store information long-term.   door closed when the animal entered the dark compartment with all four limbs, and a foot shock (2 s, 446 0.9 mA in rats and 0.7mA in mice) was administered. The animal was removed from the dark 447 compartment (10 s after the shock for rats and immediately after for mice) and returned to its home cage. 448 Memory tests were performed at designated time points by placing the animal back in the lit compartment 449 and measuring their latency to enter the dark compartment. Foot shocks were not administered during 450 memory testing, and testing was terminated at 900s. Reminder foot shocks (R.S.), with identical duration 451 and intensity to those used in training (i.e., 2 s, 0.9 mA), were administered in a novel, neutral chamber 452 with transparent walls in a different experimental room. The animal was placed into the neutral chamber 453 for 10s before receiving a single R.S. The animal was removed from the chamber immediately after the 454 R.S. and returned to its home cage. 455 Control groups consisted of 1) untrained (U.T.) animals which were handled like the 456 experimental but, instead of undergoing training, remained in their home cage, and 2) unpaired (UP) 457 animals, which underwent the I.A. box exposure procedure without receiving a shock and, one hour later, 458 given a foot shock immediately after being placed on the grid of the dark chamber and then immediately 459 returned to the home cage. 460 461

Rat cannula implants and injections 540
Rats were anesthetized with ketamine (75 mg/kg) mixed with xylazine (10 mg/kg), and stainless-541 steel guide cannulas (C313G-SPC; 26-gauge P1 Technologies, Roanoke, VA) were implanted bilaterally 542 using a stereotaxic apparatus (Kopf Instruments, Tujunga, CA) through holes drilled in the overlying 543 skull to target the ACC (0.2 mm anterior, 0.5 mm lateral, -1.3 mm ventral from bregma). The 544 guide cannulas were fixed to the skull with dental cement. Rats were administered meloxicam (3 mg/kg, 545 subcutaneous) and let recover for at least 14 days before undergoing behavioral experiments. The ODN, which served as control, contained the same relative AS-ODN base composition but in random 553 order and showed no homology to any mammalian sequence in the GenBank database, as confirmed 554 using a basic local alignment search tool (BLAST). All ODNs were phosphorothioated on the three-555 terminal bases at each end to protect against nuclease degradation. ODNs were synthesized, reverse-phase 556 cartridges purified, and purchased from Gene Link (Hawthorne, NY). Rats were euthanized at the end of 557 the behavioral experiments to confirm cannula and injection placement. Toward this end, 40 µm coronal 558 sections were sliced following fixation of the brains in 10% formalin; then, the sections were examined 559 under a light microscope to verify cannula placement. Rats with incorrect placement were excluded from 560 the study. 561 562

Object location memory 563
Mice were habituated, trained, and tested in a square, open field (29 × 29 × 18 cm) with white 564 Plexiglas walls and floor measured at 12.5 (±2.5) lux in the center of a dim room. Visual cues were 565 provided within the box and on the walls of the room. Behavior was recorded with a video camera 566 positioned above the arena. Mice were first habituated to the arena for 10 minutes for 3 consecutive days 567 before the training. Twenty-four hours after the last habituation session, each animal was returned to the 568 arena for its training session. Training consisted of exposing the mice to two identical objects constructed 569 from Mega Bloks (Montreal, Canada) secured to the floor of the arena. Object sizes were no taller than 570 twice the size of the mice. Mice were initially placed facing a corner, away from the objects, and were 571 allowed to explore the arena and objects for 10 min. 4 hours after training; each animal was tested in the 572 arena. During testing, one object remained in the same location as during training, whereas the second 573 object had been moved to a novel location. Animals were placed in the arena facing the same direction as 574 during training and were allowed to explore for 10 min. The placement of the object in the novel location 575 was counterbalanced between subjects. The arena and objects were cleaned between sessions. Video files 576 were coded and scrambled. The experimenter was blind to treatment and scored the total time the mice 577 spent actively exploring each object in each session. Active exploration was defined as the mice pawing 578 at, sniffing, or whisking with their snout directed at the object from a distance of less than ∼1 cm. Sitting 579 on or next to an object was not counted as active exploration. Mice with less than 10s total exploration 580 time were excluded. If mice explored more than 15s, the exploration percentage was taken at 15s of total

Mouse viral injections and C21 administration 592
Mice were anesthetized with isoflurane. The skull was exposed, and holes were drilled in the 593 skull bilaterally above the ACC or dHC. A Hamilton (Reno, NV) syringe with a 33 gauge needle, 594

Statistical analyses 611
Data were statistically analyzed using Prism software. The student's t-test was used to compare 612 statistical differences between two experimental groups. When more than two groups were compared, 613 data were analyzed with one-or two-way repeated-measure ANOVA followed by Bonferroni post hoc 614

Competing interests 637
The authors declare that no competing interests exist.  Ma a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a ag g g g g g g g g g g g g g g g g g g g g g g g g