==== Front Proc Natl Acad Sci U S AProc. Natl. Acad. Sci. U.S.ApnaspnasPNASProceedings of the National Academy of Sciences of the United States of America0027-84241091-6490National Academy of Sciences 3001262020180322410.1073/pnas.1803224115Biological SciencesNeuroscienceHippocampus-dependent emergence of spatial sequence coding in retrosplenial cortex http://orcid.org/0000-0001-5523-657XMao Dun a12Neumann Adam R. aSun Jianjun aBonin Vincent bcd3Mohajerani Majid H. aMcNaughton Bruce L. ae13aCanadian Centre for Behavioural Neuroscience, Department of Neuroscience, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada;bNeuro-Electronics Research Flanders, 3001 Leuven, Belgium;cVlaams Instituut voor Biotechnologie, 3000 Leuven, Belgium;dDepartment of Biology, KU Leuven, 3000 Leuven, Belgium;eDepartment of Neurobiology and Behavior, University of California, Irvine, CA 926971To whom correspondence may be addressed. Email: zjumao@gmail.com or bruce.mcnaughton@uleth.ca.Edited by Gyorgy Buzsáki, New York University Neuroscience Institute, New York, NY, and approved June 20, 2018 (received for review February 21, 2018) Author contributions: D.M., V.B., M.H.M., and B.L.M. designed research; D.M., A.R.N., and J.S. performed research; D.M. analyzed data; and D.M., V.B., and B.L.M. wrote the paper. 2Present address: Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030. 3V.B. and B.L.M. contributed equally to this work. 31 7 2018 16 7 2018 16 7 2018 115 31 8015 8018 Copyright © 2018 the Author(s). Published by PNAS.2018This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).Significance Retrosplenial cortex (RSC) is a major relay of hippocampal formation output to other neocortical areas and is critical for spatial and some other forms of learning. We show here that the sparse, orthogonal, “place cell” sequence activity in RSC develops gradually over several days and is severely attenuated by hippocampal damage. These data support the theory that hippocampus endows RSC (and possibly other cortical areas) with an index-like, continuous representation of the context in which events occur, that could support coordinated retrieval of recent memory. Retrosplenial cortex (RSC) is involved in visuospatial integration and spatial learning, and RSC neurons exhibit discrete, place cell-like sequential activity that resembles the population code of space in hippocampus. To investigate the origins and population dynamics of this activity, we combined longitudinal cellular calcium imaging of dysgranular RSC neurons in mice with excitotoxic hippocampal lesions. We tracked the emergence and stability of RSC spatial activity over consecutive imaging sessions. Overall, spatial activity in RSC was experience-dependent, emerging gradually over time, but, as seen in the hippocampus, the spatial code changed dynamically across days. Bilateral but not unilateral hippocampal lesions impeded the development of spatial activity in RSC. Thus, the emergence of spatial activity in RSC, a major recipient of hippocampal information, depends critically on an intact hippocampus; the indirect connections between the dysgranular RSC and the hippocampus further indicate that hippocampus may exert such influences polysynaptically within neocortex. retrosplenial cortexhippocampusspatial sequence codingspatial learninghippocampal indexing theoryAlberta Innovates - Health Solutions (AIHS)501100000145Polaris awardDun MaoBruce L McNaughtonAlberta Innovates - Health Solutions (AIHS)501100000145Graduate studentshipDun MaoBruce L McNaughtonGouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (NSERC)501100000038#40352Majid MohajeraniBruce L McNaughtonGouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (NSERC)501100000038#RGPIN-2017-03857Majid MohajeraniBruce L McNaughtonFonds Wetenschappelijk Onderzoek (FWO)501100003130G0D0516NVincent BoninOnderzoeksraad, KU Leuven (Research Council, KU Leuven)501100004497C14/16/048Vincent BoninNational Science Foundation (NSF)100000001#1631465Bruce L McNaughtonCanada Foundation for Innovation (CFI)501100000196#33598Bruce L McNaughtonDOD | Defense Advanced Research Projects Agency (DARPA)100000185# HR0011-18-2-0021Bruce L McNaughton ==== Body The retrosplenial cortex (RSC) is a midline association region that integrates thalamic, (para)hippocampal, and neocortical information (1–5). Similar to the hippocampus (6), RSC is also essential for spatial learning and memory (7–9). Consistent with its proposed role in translating between world-centered and body-centered views (10), RSC neurons carry various navigation-related signals such as head direction, positional, and conjunctive allocentric and egocentric information (11–17). RSC neurons also show spatial activity resembling the activity of hippocampal CA1 place cells (15). The sources of spatial signals in RSC are unknown; however, hippocampus is an obvious possibility, and hippocampal lesions or inactivation impair immediate early gene expression in RSC (18, 19). On the other hand, both rodent and human studies have suggested the opposite direction of information flow, that RSC may send sensory and contextual information to the hippocampus (20, 21), possibly through RSC projections to the medial entorhinal cortex (22). Here we studied the emergence of spatial activity in RSC upon repeated exploration of the same environment and tested the impact of the hippocampus on this activity. Results We investigated RSC neuronal activity in mice in a head-fixed, treadmill assay (15, 23) (Fig. 1A). Fifteen adult transgenic mice specifically expressing calcium indicator GCaMP6 in excitatory neurons (24, 25) were divided into three experimental groups: control, unilateral hippocampal lesion, and bilateral hippocampal lesions (n = 5 in each group; NMDA lesion) (Fig. 1B). Mice in the lesion groups sustained extensive neuron/tissue loss in the dorsal hippocampal formation (Fig. 1C and SI Appendix, Fig. S1). Movement trajectories were similar across groups (average speed between 30- and 120-cm position: control, 15.4 ± 1.7; unilesion, 17.7 ± 1.8; bilesion, 18.9 ± 3.5 cm/s; all mean ± SEM, n = 5 in each group; P = 0.61, one-way ANOVA) (Fig. 1D). We measured cellular activity in the superficial layers (100 μm to 200 µm deep) of dysgranular RSC using two-photon calcium imaging (26) (SI Appendix, Fig. S2). We inferred activity from raw calcium fluorescence signals using deconvolution (SI Appendix, Fig. S2) (27). We studied the degree to which RSC neurons encode the animal’s position on the treadmill. Consistent with our previous work (15), a substantial fraction of RSC neurons showed repeated activation at specific positions on the treadmill (Fig. 1E and SI Appendix, Fig. S1D), similar to the activity of hippocampal place cells. Fig. 1. Experimental design and retrosplenial place cell activity. (A) Treadmill locomotion assay. Two-photon calcium imaging was performed in head-fixed mice running on a treadmill belt endowed with tactile cues. (B) Cellular imaging of neuronal activity through a glass window in RSC in both hemispheres in mice with intact hippocampus (control), unilateral hippocampal lesion (unilesion), and bilateral hippocampal lesions (bilesion). (C) Scatter plot of the remaining volume (normalized) of the hippocampus in the left and right hemispheres. Colors correspond to animal groups shown in B. (D) Mean movement speed profiles as a function of position for the three experimental groups. Error bars are SEM over animals. (E) Calcium time courses (raw and deconvolved) of an example RSC place cell. Position and speed traces are shown below. deconv, deconvolved; norm, normalized. Hippocampal projections to RSC are mostly ipsilateral (2); we therefore tested whether a unilateral hippocampal lesion disrupts “place” cell activity in ipsilateral RSC. We compared activity in the lesioned and intact hemispheres measured from the same animals. Unilateral hippocampal lesion had no discernible impact on RSC spatial activity (Fig. 2). RSC neuronal ensembles showed sequential activation that was locked to position during movement in both the intact and lesioned hemispheres (Fig. 2 A and B). To quantify the encoding of spatial information by RSC neuronal population, we built a Bayesian decoding model (SI Appendix, Methods and Fig. S3A) to predict the animal’s position from all imaged neurons using separate sets of trials for training and testing (Fig. 2 A and B) (28). We observed no significant difference in the place cell fraction (P = 0.30, paired t test) or the position decoding error (P = 0.98, paired t test) between the lesioned and intact hemispheres (Fig. 2C). Fig. 2. Unilateral hippocampal lesion does not impair ipsilateral RSC place cell activity. (A) (Top) Raw calcium time courses of 229 simultaneously imaged RSC neurons in the intact hemisphere of an example unilesion mouse. Neurons were sorted by the positions that elicited their maximum responses. (Bottom) Real (blue) and Bayesian decoded (red) position traces. (B) The same as A but for the lesioned hemisphere RSC of the same mouse. (C) Mean place cell fractions and position decoding errors in unilesion mice. Note that two mouse lines were used here (Thy1 and Ai93). Error bars are SEM over sessions. For unknown reasons, there were intrinsic differences between these lines in the observed place cell fractions. While unilateral hippocampal lesions did not disrupt ipsilateral RSC spatial activity, RSC could also receive spatial information from the intact, contralateral hemisphere (2). We compared RSC place cell activity between intact animals and animals with bilateral hippocampal lesions. Indeed, bilateral hippocampal lesions significantly impaired place cell activity in RSC (Fig. 3). RSC spatial sequence activity was severely disrupted by bilateral hippocampal lesions (Fig. 3 A and B). The fraction of RSC neurons showing stable place fields dropped dramatically in animals with bilateral lesions (place cell fraction: control, 0.50 ± 0.02; bilesion, 0.29 ± 0.04; mean ± SEM, n = 5 each group; P = 0.0005, one-tailed t test) (Fig. 3C). An alternative, spatial information-based criterion of place cell selection yielded similar results (SI Appendix, Fig. S3 B–G). Position estimates inferred from Bayesian decoding of population activity (using all cells) were also severely disrupted in bilaterally lesioned animals (decoding error: control, 14.60 ± 1.98 cm; bilesion, 27.68 ± 2.20 cm; mean ± SEM; P = 0.0011, one-tailed t test) (Fig. 3D). These effects were not explained by other variables such as neuron or trial counts (SI Appendix, Fig. S3 H and I). Notably, while the proportion of place cells was reduced in animals with bilateral hippocampal lesions, the properties of neurons with place cell activity were similar (place field width: P = 0.28; spatial information: P = 0.21, two-tailed t test) (Fig. 3 E and F and SI Appendix, Figs. S3J and S4). The extent of hippocampal lesions may explain the residual place cell activity observed in bilaterally lesioned animals, since the magnitude of the effect was proportional to the proportion of hippocampal damage (SI Appendix, Fig. S3J). These results indicate that the hippocampus is necessary for the expression of place cell activity in RSC. Fig. 3. Bilateral hippocampal lesions severely impair place cell activity in RSC. (A) (Top) Raw calcium time courses of 255 simultaneously imaged RSC neurons from an example control mouse. Neurons were sorted by the positions that elicited their maximum responses. (Bottom) Real (blue) and Bayesian decoded (red) position traces. (B) The same as A but from an example bilateral lesion mouse. (C) (Left) Mean place cell fractions of individual mice in the control and bilesion groups (all Thy1 mice). Error bars are SEM over sessions. (Right) Bar plots of the average place cell fractions for the two groups. Error bars are SEM over animals. (D) The same as C but for decoding errors. (E) Bar plot of the mean place field width. Colored dots correspond to the mean place field width of individual mice in the control and bilesion groups. (F) The same as E but for the mean spatial information of identified place cells in each animal. We next investigated how RSC place cell activity emerges over repeated exploration of the same environment and what the role of the hippocampus is in this process. We monitored the activity of the same RSC neuronal population over a period of 2 wk to 3 wk in daily imaging sessions (control, n = 4; bilesion, n = 4). Activity from the same neuronal cell bodies could be imaged across days (Fig. 4A and SI Appendix, Fig. S5 A and B). Place cell activity increased gradually with experience in the control group but not in mice with bilateral hippocampal lesions (place cell fraction change: control, r = 0.96, P = 0.0006; bilesion, r = 0.61, P = 0.17) (Fig. 4D). Similar to what is observed in the hippocampus (29), RSC population representations of position on the track changed dynamically across days (Fig. 4B), and more adjacent days had more highly correlated population activity as experience increased (Fig. 4 B and C). The control group showed more repeatable population representations across days than the bilateral lesion group (population vector correlations: control, 0.19 ± 0.02; bilesion, 0.09 ± 0.02, P = 0.02, two-tailed t test) (Fig. 4E and SI Appendix, Fig. S5C). Position tuning curves (occupancy-normalized neuronal responses as a function of position) of individual neurons were also more correlated between days in control mice than in bilateral lesion mice (P = 0.02, two-tailed t test) (Fig. 4E). These effects were not explained by the number of place cells included in the calculation (SI Appendix, Fig. S5F). We did not observe a difference in spatial activity correlations (population vector correlations or position tuning correlations of individual neurons) between the two hemispheres in mice with unilateral hippocampal lesion (SI Appendix, Fig. S5 D and E). These results indicate that RSC population representation of spatial context improves progressively over time and that the hippocampus is necessary for the emergence of spatial sequence coding in RSC. Fig. 4. Hippocampal destruction disrupts experience-dependent emergence of spatial coding in RSC. (A) Average fluorescence images (centered on the target neuron) and position activity maps of the target neuron imaged on days 1, 3, 5, and 7. Position tuning curves (white traces) are overlaid on the position activity maps. (B) Sorted, trial-averaged position activity maps for all RSC place cells from an example control mouse on days 1, 3, 5, and 7. Same neuronal population was imaged across days. Neurons were selected and sorted by corresponding days. (C) Population vector correlation matrices between days for data shown in B. (D) Mean place cell fractions and decoding errors as a function of imaging session for the control and bilateral lesion animals. Error bars are SEM over animals. (E) Mean population vector correlations of the same position (distance < 15 cm, dashed area in SI Appendix, Fig. S5C) and mean position tuning (white traces in A) correlations for all place cells as a function of different imaging intervals. Error bars are SEM over animals. norm, normalized; Pop Vec Corr, population vector correlation. Discussion Our data indicate that the spatial context coding in RSC improves with experience, and this process relies on instructive signals from the hippocampus. This may reflect a direct impact of the hippocampus or an indirect effect through intermediate regions after lesioning the hippocampus. Indeed, the specific RSC subregion we studied, the dysgranular RSC, receives weak direct hippocampal input (1, 3, 4). Our results demonstrate the importance of the hippocampus in shaping neocortical activity. The pronounced experience-dependent spatial activity observed may reflect a general principle of the influences of hippocampal outflow on the association neocortex in terms of spatiotemporal contextual processing. Sequential activation of large groups of neurons has been observed in several cortical regions, including the posterior parietal and prefrontal cortices (30, 31). These sequences reflect information processing along the spatial and/or temporal dimensions, with concurrent sensory experience and events superimposed. Being uniquely situated at the intermediate layer within the default mode network (DMN) (32), RSC may be critical for episodic memory processes by mediating functional interactions between the cortical and subcortical DMN subsystems (33, 34). The RSC may play a critical role in the transfer of hippocampal place/memory sequence codes to other regions of the neocortex, to associate information across different cortical modalities (35–37) and to guide complex behaviors (38). Methods All animal procedures were performed in compliance with protocols approved by the ethical research committee of the University of Lethbridge. Fifteen adult male and female transgenic GCaMP6 mice [20 g to 25 g, 2 mo to 4 mo old at the time of surgery, including 13 Thy1 GCaMP6s GP4.3 mice and 2 Ai93 (TITL-GCaMP6f) || CaMK2a-tTA || Rasgrf2-2A-dCre mice] were used in this study. Mice were divided into three experimental groups: sham lesion (control, n = 5 Thy1 mice), unilateral hippocampal lesion (n = 3 Thy1 mice and n = 2 Ai93 mice), and bilateral hippocampal lesions (n = 5 Thy1 mice). Mice were habituated and trained to run on a linear treadmill track with head fixed. Full methods can be found in SI Appendix, Methods. Supplementary Material Supplementary File We thank V. Lapointe and A. Demchuk for help with histology, and F. Battaglia for comments on the manuscript. This research was supported through the Alberta Innovates–Health Solutions Polaris award (to B.L.M.) and a graduate studentship (to D.M.), Natural Sciences and Engineering Research Council of Canada Discovery Grant 40352 (to M.H.M.) and RGPIN-2017-03857 (to B.L.M.), Research Foundation–Flanders (Fonds voor Wetenschappelijk Onderzoek - Vlaanderen) Grant G0D0516N (to V.B.), KU Leuven Research Council Grant C14/16/048 (to V.B.), National Science Foundation Grant 1631465 (to B.L.M.), Canada Foundation for Innovation Grant 33598 (to M.H.M. and B.L.M.), and Defense Advanced Research Projects Agency Grant HR0011-18-2-0021 (to B.L.M.). The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The data related to this work has been deposited on Gin and is available at https://web.gin.g-node.org/dunmao/RSC_HPC. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1803224115/-/DCSupplemental. ==== Refs 1 van Groen T Wyss JM 1992 Connections of the retrosplenial dysgranular cortex in the rat J Comp Neurol 315 200 216 1545009 2 Wyss JM Van Groen T 1992 Connections between the retrosplenial cortex and the hippocampal formation in the rat: A review Hippocampus 2 1 11 1308170 3 Cenquizca LA Swanson LW 2007 Spatial organization of direct hippocampal field CA1 axonal projections to the rest of the cerebral cortex Brain Res Brain Res Rev 56 1 26 4 Sugar J Witter MP van Strien NM Cappaert NL 2011 The retrosplenial cortex: Intrinsic connectivity and connections with the (para)hippocampal region in the rat. An interactive connectome Front Neuroinform 5 7 21847380 5 Van Groen T Wyss JM 2003 Connections of the retrosplenial granular b cortex in the rat J Comp Neurol 463 249 263 12820159 6 Morris RG Garrud P Rawlins JN O’Keefe J 1982 Place navigation impaired in rats with hippocampal lesions Nature 297 681 683 7088155 7 Sutherland RJ Whishaw IQ Kolb B 1988 Contributions of cingulate cortex to two forms of spatial learning and memory J Neurosci 8 1863 1872 3385478 8 Czajkowski R 2014 Encoding and storage of spatial information in the retrosplenial cortex Proc Natl Acad Sci USA 111 8661 8666 24912150 9 Vann SD Aggleton JP 2005 Selective dysgranular retrosplenial cortex lesions in rats disrupt allocentric performance of the radial-arm maze task Behav Neurosci 119 1682 1686 16420172 10 Byrne P Becker S Burgess N 2007 Remembering the past and imagining the future: A neural model of spatial memory and imagery Psychol Rev 114 340 375 17500630 11 Chen LL Lin L-H Green EJ Barnes CA McNaughton BL 1994 Head-direction cells in the rat posterior cortex. I. Anatomical distribution and behavioral modulation Exp Brain Res 101 8 23 7843305 12 Cho J Sharp PE 2001 Head direction, place, and movement correlates for cells in the rat retrosplenial cortex Behav Neurosci 115 3 25 11256450 13 Alexander AS Nitz DA 2015 Retrosplenial cortex maps the conjunction of internal and external spaces Nat Neurosci 18 1143 1151 26147532 14 Smith DM Barredo J Mizumori SJ 2012 Complimentary roles of the hippocampus and retrosplenial cortex in behavioral context discrimination Hippocampus 22 1121 1133 21630374 15 Mao D Kandler S McNaughton BL Bonin V 2017 Sparse orthogonal population representation of spatial context in the retrosplenial cortex Nat Commun 8 243 28811461 16 Alexander AS Nitz DA 2017 Spatially periodic activation patterns of retrosplenial cortex encode route sub-spaces and distance traveled Curr Biol 27 1551 1560.e4 28528904 17 Vedder LC Miller AMP Harrison MB Smith DM 2017 Retrosplenial cortical neurons encode navigational cues, trajectories and reward locations during goal directed navigation Cereb Cortex 27 3713 3723 27473323 18 Albasser MM Poirier GL Warburton EC Aggleton JP 2007 Hippocampal lesions halve immediate-early gene protein counts in retrosplenial cortex: Distal dysfunctions in a spatial memory system Eur J Neurosci 26 1254 1266 17767503 19 Kubik S Miyashita T Kubik-Zahorodna A Guzowski JF 2012 Loss of activity-dependent Arc gene expression in the retrosplenial cortex after hippocampal inactivation: Interaction in a higher-order memory circuit Neurobiol Learn Mem 97 124 131 22100445 20 Cooper BG Mizumori SJ 2001 Temporary inactivation of the retrosplenial cortex causes a transient reorganization of spatial coding in the hippocampus J Neurosci 21 3986 4001 11356886 21 Auger SD Zeidman P Maguire EA 2015 A central role for the retrosplenial cortex in de novo environmental learning eLife 4 e09031 22 Czajkowski R 2013 Superficially projecting principal neurons in layer V of medial entorhinal cortex in the rat receive excitatory retrosplenial input J Neurosci 33 15779 15792 24089485 23 Royer S 2012 Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition Nat Neurosci 15 769 775 22446878 24 Dana H 2014 Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo PLoS One 9 e108697 25250714 25 Madisen L 2015 Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance Neuron 85 942 958 25741722 26 Goldey GJ 2014 Removable cranial windows for long-term imaging in awake mice Nat Protoc 9 2515 2538 25275789 27 Pnevmatikakis EA 2016 Simultaneous denoising, deconvolution, and demixing of calcium imaging data Neuron 89 285 299 26774160 28 Davidson TJ Kloosterman F Wilson MA 2009 Hippocampal replay of extended experience Neuron 63 497 507 19709631 29 Ziv Y 2013 Long-term dynamics of CA1 hippocampal place codes Nat Neurosci 16 264 266 23396101 30 Harvey CD Coen P Tank DW 2012 Choice-specific sequences in parietal cortex during a virtual-navigation decision task Nature 484 62 68 22419153 31 Fujisawa S Amarasingham A Harrison MT Buzsáki G 2008 Behavior-dependent short-term assembly dynamics in the medial prefrontal cortex Nat Neurosci 11 823 833 18516033 32 Buckner RL Andrews-Hanna JR Schacter DL 2008 The brain’s default network: Anatomy, function, and relevance to disease Ann N Y Acad Sci 1124 1 38 18400922 33 Kaboodvand N Bäckman L Nyberg L Salami A 2018 The retrosplenial cortex: A memory gateway between the cortical default mode network and the medial temporal lobe Hum Brain Mapp 39 2020 2034 29363256 34 Vann SD Aggleton JP Maguire EA 2009 What does the retrosplenial cortex do? Nat Rev Neurosci 10 792 802 19812579 35 Teyler TJ DiScenna P 1986 The hippocampal memory indexing theory Behav Neurosci 100 147 154 3008780 36 McNaughton BL 2010 Cortical hierarchies, sleep, and the extraction of knowledge from memory Artif Intell 174 205 214 37 Treves A Rolls ET 1994 Computational analysis of the role of the hippocampus in memory Hippocampus 4 374 391 7842058 38 Yamawaki N Radulovic J Shepherd GM 2016 A corticocortical circuit directly links retrosplenial cortex to M2 in the mouse J Neurosci 36 9365 9374 27605612