Organizing the coactivity structure of the hippocampus from robust to flexible memory

New memories are integrated into prior knowledge of the world. But what if consecutive memories exert opposing demands on the host brain network? We report that acquiring a robust (food-context) memory constrains the hippocampus within a population activity space of highly correlated spike trains that prevents subsequent computation of a flexible (object-location) memory. This densely correlated firing structure developed over repeated mnemonic experience, gradually coupling neurons of the superficial CA1 pyramidale sublayer to whole population activity. Applying hippocampal theta-driven closed-loop optogenetic suppression to mitigate this neuronal recruitment during (food-context) memory formation relaxed the topological constraint on hippocampal coactivity and restored subsequent flexible (object-location) memory. These findings uncover an organizational principle for the peer-to-peer coactivity structure of the hippocampal cell population to meet memory demands.

Every day, we use our existing knowledge to guide the actions we make in our environment, integrating new information to gain further knowledge about the world.Therefore, building new memories does not take place in a state of tabula rasa, but against a background of prior experiences that have been accumulated across the lifespan and have shaped their host brain networks (1,2).
The hippocampus network uses the collective activity of the population of its neurons to support everyday memory (3,4).In principle, the level and structure of the activity coupling between individual neurons could reflect a critical tradeoff between the robustness versus the flexibility of the whole population in processing information.That is, strong peer-to-peer coupling could yield highly correlated spike trains, increasing the consistency of activity patterns within the population for robust memory expression.In contrast, weaker population coupling could release network activity space for new patterns, allowing more diverse mnemonic representations for dynamically adaptable behavior.Owing to convergent innervation on post-synaptic targets (e.g., subiculum, entorhinal cortex), adjusting population coupling to ongoing demand would influence information transmission of hippocampal inputs to downstream reader neurons (3,5,6).However, the hippocampus may have to switch between robust versus flexible computations depending on current demands.What are the consequences of placing the hippocampal population into a robust computational mode for subsequent memories that instead require flexible information processing?
To address this question, we first trained six mice (always fed ad libitum) to acquire a strong contextual memory.On 16 consecutive days, mice explored two arenas (Fig. 1A,  B).During the first 10 days ('Food-context conditioning'), we paired one arena (context X) with two regular chow pellets (fig.S1A).The other arena (context Y) contained one chow pellet and one high-fat-diet (Hfd) pellet (fig.S1A), which mice encountered for the first time and did not eat much on day 1 (Fig. 1C).By repeating these foraging sessions on each subsequent conditioning day, mice showed escalated food intake in context Y (Fig. 1C; from day 1 to day 10, a fold-change of 21.62 ± 6.70 versus 0.63 ± 0.24 in context Y versus X; mean ± s.e.m.).Food locations were randomized every day in each context to promote mnemonic association of food items not to fixed places but whole contexts (fig.S1B).To probe discriminative food-context association, we then measured the propensity of these mice to express context-biased feeding.By providing both arenas with new food items in post-conditioning days ('Novel food test'; days 11 and 12; Fig. 1B and fig.S1C), we observed higher novel food intake in context Y compared to context X (Fig. 1D; a fold-change of 3.47 ± 1.40 in context Y versus X; mean ± s.e.m.).Thus, mice in the Hfdconditioned context readily overcame the rodent natural tendency to express food neophobia (7,8).Mice also exhibited lower Hfd intake when provided in a third arena (context Z) never paired with any food (fig.S1D).Body weight remained stable across days (fig.S1E).
We next switched task demand to assess novelty detection in these contexts paired with food.For this, we used a hippocampus-dependent continuous Novel Object Recognition task ('cNOR test'; from day 13 to day 16; Fig. 1A, B and fig.S2A-C).On each cNOR day, we re-exposed mice to either context X or Y ('Re-exposure'; without food) before they encountered four novel objects ('Sampling'; Fig. 1A, B and fig.S2A, B).Across four more exploration sessions that day, we iteratively replaced one of the initially sampled objects with a new one ('Testing').This procedure yielded a set of three familiar (already-seen) objects and one novel (first time-seen) object in each cNOR test (Fig. 1B and fig.S2A).We measured novelty detection in each cNOR test n using the time spent exploring the novel object over the total time spent on all four objects, thereby probing memory for objects explored in session n − 1. Mice showed novel object preference in context X but not in context Y (Fig. 1E).Exploratory behavior measured by locomotor speed, distance travelled, and time spent with objects did not differ across contexts (fig.S2D-H).Altogether, the first demand in this 2-memory paradigm was for animals to repeatedly associate food resources to selective contexts across many days, yielding a robust memory able to shape contextual feeding.This later prevented mice from coping with a second demand: to dynamically update another memory for continually detecting novel items across many sessions each day.Thus, the robust (food) memory acquired in context Y interfered with the subsequent flexible (object-location) memory in that context.

Robust memory increases neuronal coactivity and population coupling in the hippocampus
We aimed to identify the neuronal correlates of this cross-memory interference.During active behavior, groups of principal cells recruited from the population of hippocampal neurons cooperate within the timeframe of theta-band (5-12Hz) oscillations to support codes and computations for memory (3).We recorded cell ensembles and local field potentials in the CA1 stratum pyramidale of these mice.Using the action potentials discharged by principal cells in theta cycles during exploration of each object in cNOR days (Fig. 1F and fig.S2I), a generalized linear model (GLM) trained on session n − 1 and applied in test n identified each object-location compound with up to 93.5% accuracy (range 9.4 -93.5 %; mean 55.0 % compared to a chance level of 25.0 %; mean ± s.e.m. number of principal cells = 51.1 ± 3.7 per GLM), consistent with work showing population-level object representation in the hippocampus (9,10).In context X, the mean accuracy of this population-level decoding started at 38.6 ± 3.2 % for the novel object-location compound (Fig. 1G) and improved by the following trial (fig.S2J), indicating rapid mnemonic integration of each object.This across-test gain in object-location representation did not occur in context Y (Fig. 1G and fig.S2J).In fact, the mean decoding accuracy there started at higher levels for the novel object-location (54.9 ± 3.4 %; P<0.001, two-sided paired permutation test, compared to context X), without significant changes in the following tests (Fig. 1G and fig.S2J).In both context X and Y, object-location memory was not affected by provision of Hfd (in neutral context Z) beforehand that day (fig.S3).Compared to context X, the contribution of individual cells to each novel object-location decoding during test n in context Y resembled its previous one expressed in session n − 1 at that location while having another (familiar) object.This was reported by the higher similarity between the population decoding vector that contained the set of neuron-wise GLM coefficients representing the novel object during test n in context Y versus that representing the familiar object at the same location during session n − 1 (Fig. 1H, I).In line with this observation, the across-test modulation in single-neuron contributions to population decoding vectors when encountering a novel object (i.e., the changes in the magnitude of individual GLM coefficients) was weaker in context Y compared to X (Fig. 1J).
We next computed the CA1 place maps expressed during the cNOR task (Fig. 2A and fig.S4A).Context Y did not exhibit an over-representation of the randomized Hfd location (fig.S4B).By quantifying the cross-session similarity of place maps from re-exposure to sampling to individual object recognition tests on each day, we observed that CA1 principal cells exhibited higher place map stability (i.e., lower spatial remapping) across contiguous sessions in context Y (Fig. 2B and fig.S4C).With the GLM object-location decoding, this result on the hippocampal place code supported the notion of a more rigid memory in the Hfd-paired context Y, but a flexible (cross-session updated) memory in context X.
We hypothesized that this representational rigidity reflects the organization of the population activity into a non-permissive structure.An operational principle serving many brain functions, including memory, is to leverage the collective activity of neural populations as an emergent property beyond that of individual cells (11)(12)(13)(14).We reasoned that Hfd context-conditioning yielded highly correlated firing patterns that created a dense network activity space for strong contextual (food) memory.But this later conflicted with the switch to a different demand where continually processing familiar versus novel stimuli would instead require sparser, weakly correlated patterns for disentangling discrete (objectlocation) representations.The correlational structure of the population activity was markedly different in context Y compared to X (Fig. 2C-J).We quantified the coactivity association of each cell pair (i, j) by predicting the theta-nested spike discharge of neuron j from the activity of neuron i while regressing out the activity of the remaining population (Fig. 2C).This procedure returned a matrix of β regression weights (Fig. 2D) that represented the neurons pairwise coactivity structure of the population in each context.For both context X and Y we constructed weighted neuronal graphs (with no self-connections) where each node is a cell and the edge linking any two nodes represents the coactivity of that cell pair (Fig. 2D-G).Neuronal graphs contained stronger triads of coactive nodes in context Y than X, as reported by higher clustering coefficients (Fig. 2H and fig.S5A; mean increase (95% CI): 11.0% (7.3-14.1%)).The population coactivity strength level, calculated for each node as the average weight of all its edges, was higher in Y (fig.S5B), with no difference in the mean neurons' firing rate across contexts (fig.S5C).The hippocampal population exhibited this denser coactivity structure in context Y without a reduction in geodesic path length, calculated as the mean shortest path between any two nodes (Fig. 2I and fig.S5A) (15).This suggested that the hippocampus, which usually displays the features of a small-world network (fig.S6A-C) that could allow for flexible information updating through efficient synchronization, has in fact acquired in context Y the rigidity of a more coherent, lattice-like network (fig.S6D-F) (16)(17)(18).These neuronal graphs that are composed of both correlated and anti-correlated spike trains (i.e., positive and negative edges) indeed showed more stable population activity patterns in context Y, as suggested by higher structural balance (fig.S6G-I).These topological alterations developed across the 10 conditioning days (fig.S7A) to continue altering the level and structure of population coactivity in post-conditioning days (fig.S7B), affecting the baseline re-exposure to context Y prior to any testing.Hippocampal graphs yet shared some common correlation structure across contexts (fig.S8), suggesting a coactivity backbone for cross-context generalization.

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To explore the development of a dense population activity structure in context Y, we investigated the one-to-many relationship between individual neurons and the rest of the population.We measured the coupling of each principal cell instantaneous firing rate in theta cycles to the concomitant summed activity of all other recorded cell members of the population ('population rate;' Fig. 2C).Consistent with the higher topological clustering (Fig. 2H), the average population coupling of individual neurons was stronger in context Y (Fig. 2J and fig.S5A; mean increase (95% CI): 16.6% (12.6-20.8%)).This increased population coupling reflected a stronger cross-neuron spiking relationship: shuffling the spike times across neurons and theta cycles, while preserving each neuron's mean rate and the population rate distribution, cancelled the increased population coupling seen in context Y (fig.S9A).This heightened coupling developed across the 10 conditioning days to mark the re-exposure to context Y during post-conditioning days even before any test (fig.S9B).Restricting food-context conditioning to two days in five additional mice did not alter subsequent cNOR memory (fig.S10A-D), allowing successful object-location decoding in both contexts with similar CA1 population activity level and structure (fig.S10E-J).Contextual food conditioning thus seemed to increase the recruitment of principal cells as "choristers of a larger hippocampal orchestra" (19) when daily repeated for well over a week.

Mitigating neuronal recruitment during robust memory formation relaxes hippocampal coactivity and restores flexible memory
We sought to relate hippocampal population activity to memory expression more directly by first manipulating an underlying neural pathway.The CA3 region features extensive excitatory recurrent connections and Hebbian synaptic plasticity (20)(21)(22)(23).It could therefore promote population coupling in the downstream CA1, which has little to no recurrent excitation (6,24).To test this, we transduced CA3 principal cells with the neural silencer Archaerhodopsin-T in four Grik4-Cre mice versus the GFP-only control in two Grik4-Cre mice (Fig. 3A, B).Bilateral implantation of tetrodes combined with optic fibers allowed monitoring of CA1 ensembles while actuating a theta phase-informed controller for realtime suppression of CA3 principal cells (Fig. 3A and fig.S11A-C).In CA3 Grik4 ::ArchT mice, but not CA3 Grik4 ::GFP mice, applying this closed-loop intervention during each Hfd conditioning session subsequently restored in post-conditioning test days object-location memory with CA1 place map similarity (cross cNOR-session remapping) and population coupling in context Y to levels seen in context X (Fig. 3C-E and fig.S11D-H).
We then examined the CA1 population after contextual food conditioning.Using the activity-dependent immediate-early-gene cFos, we quantified neuron recruitment in the CA1 pyramidale layer of six mice exposed to context Y with Hfd for 10 days (Fig. 4A and fig.S12).In parallel, six control mice explored context Y without food while six others ate Hfd in their homecage.Mice undergoing Hfd-context Y conditioning showed higher density of cFos + neurons compared to both controls (Fig. 4B).Contrasting cFos expression in the superficial versus the deep pyramidale sublayers using the marker Calbindin 1 (Fig. 4A) (25)(26)(27) suggested that Hfd-context conditioning preferentially recruited CA1 superficial cells (Fig. 4B).This is consistent with recent studies highlighting that CA1 pyramidal cells Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts segregate along the anatomical axes of the hippocampus (e.g., with respect to molecular markers, neural connectivity, and electrophysiological properties), indicating a functional specialization based on somatic location (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).
We identified neurons recorded in the CA1 superficial pyramidale sublayer, using the electrophysiological profile of each tetrode (fig.S13) (15).The representational rigidity affecting object-location memory update in context Y (Fig. 1J) corresponded to reduced modulation in the individual contribution of superficial cells to the population decoding from cNOR session n − 1 to test n (Fig. 4C and fig.S14A).This suggested that superficial cell population did not distinguish the novel object from the familiar previously encountered at the same location in context Y, only representing the location itself.In line with this, the spatial map rigidity (i.e., higher place field stability) observed across cNOR sessions in the Hfd-conditioned context Y (Fig. 2B) was explained by superficial cell maps (fig.S14B), with no representational bias to the Hfd location (fig.S14C).Superficial cells formed stronger coactivity triads in the network (fig.S15A-E), increasing their population coupling in context Y (Fig. 4D and fig.S15F-H).Deep pyramidale sublayer cells did not show such enhanced activity profile (fig.S15).The CA1 representational rigidity that prevented flexible object-location memory in context Y following Hfd conditioning was thus primarily explained by enhanced recruitment of superficial pyramidale sublayer cells.
We hypothesized that harnessing the rise in population coupling during robust contextual conditioning would restore subsequent flexible memory.We used an intersectional optogenetic strategy to target cells recruited in the CA1 superficial pyramidale sublayer during Hfd-context Y conditioning.CA1 superficial pyramidale sublayer cells are genetically defined by the molecular marker Calbindin-1 (25,27,34).They express cFos during contextual Hfd conditioning (Fig. 4A, B).We thus bred double-transgenic Calb1-Cre;cFos-tTA mice and generated a viral construct for the two-term Boolean logic (36) expression of the yellow light-driven neural silencer ArchT-EYFP (or its EYFP-only control) dependent on the two recombinases Cre and FlpO (Fig. 5A).

Discussion
Our results show that a robust (food-context) memory raises the population coupling of CA1 superficial pyramidale sublayer neurons, creating a dense network coactivity structure.CA1 pyramidal cells, the primary output of the hippocampus, segregate along the anatomical axes with different activity behaviors, indicating that cells arranged along the radial axis contribute differentially to information processing (25)(26)(27)(28)(29)(30)(31)35).Compared to the deep, the superficial sublayer is enriched in context-modulated cells with slower response dynamics to environmental changes (28,29), being more active in cue-poor environments and preferentially using a rate code driven by intra-hippocampal inputs; while deep cells are more active in cue-rich environments and use a phase code driven by entorhinal inputs (30).Our findings show that the hippocampus network preferentially engages superficial cells in strong peer-to-peer coactivity for robust contextual memory, at the risk of subsequent mnemonic rigidity.This finding is consistent with recent studies showing that CA1 superficial cells are more recruited into replay events and show stronger synaptic potentiation after novel experiences compared to deep cells (37,38).The dense coactivity structure emerged over many conditioning days and could prevent the network to switch between alternative coding schemes or cell assemblies, and override representations by the deep sublayer counterparts (28,30).The development of this coactivity structure required upstream CA3 activity, consistent with the observation that Schaffer collateral excitation is stronger in calbindin-expressing CA1 principal cells (25).With respect to subsequent behavior, this acquired hippocampal topology of heightened coactivity related to strong contextual (feeding) response and impaired novel information processing.Neural networks have been proposed to fall into the broad class of 'small-world' networks, a middle ground between regular and random networks where the combination of high clustering of elements (a property of regular networks) with short path lengths between elements (a property of random networks) would allow important properties of complex networks such as increased computational power and effective synchronizability (16,17,(39)(40)(41).Our observation that repeated food-context conditioning affects the coactivity structure of the network by increasing neuronal clustering without shortening node-to-node paths suggests that the hippocampal topology can deviate from a small-world toward a more coherent, regular lattice.This way, the joint activity of an increased number of neurons operating as a cohesive population would permit robust information flow to downstream receiver neurons, possibly at the expense of other (e.g., novel) input channels.
The hippocampus could broadcast this heightened coactivity to several recipient circuits and reader neurons.For instance, recent work points to a contribution of the nucleus accumbens in translating hippocampal dynamics of appetitive memory into a behavioral readout (42) or the hypothalamus in driving non-homeostatic contextual feeding (43).The clustered spiking activity that developed in the hippocampus across food-context conditioning days is Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts also likely to influence neocortical circuits for memory storage via systems consolidation (44,45).With hippocampal support neuronal ensembles in prefrontal cortex can be rapidly formed to then undergo a process of functional maturation over weeks (46).This maturation could allow prefrontal cortex ensembles to converge onto a lower-dimensional activity space to extract latent rules and common relational features across multiple experiences, gradually developing a knowledge structure of the world (47,48).Importantly, the instantiation of this highly clustered topology can be prevented: applying cell type-selective, network pattern-informed neuronal suppression during contextual learning rebalances population activity and restores flexible memory.Together, these findings suggest that the plastic organization of hippocampal coactivity supports a network tradeoff between robust and flexible computations, shaping continual integration of new memories and their adaptability to cognitive demands.

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One-Sentence Summary
The density of peer-to-peer neuronal coactivity determines robust versus flexible memory expression in the hippocampus.

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Fig. 1 .
Fig. 1.Robust contextual (food) memory prevents subsequent flexible (object) memory.(A, B) Behavioral tasks with open-field contexts (A) and multiday layout (B) for a twomemory paradigm.(C) Animals' food intake during contextual conditioning (each data point represents one mouse).(D) Estimation plot showing the effect size for the difference in novel food intake between context X and Y after conditioning.(E) Percentage of time exploring novel versus familiar objects during cNOR tests in context X or Y. (F) Schematic of the GLM predicting the identity of each object-location compound from population vectors of theta-nested principal cell spiking.A sample of CA1 ensemble spike data for one

Fig. 2 .
Fig. 2. Robust memory increases neuronal coactivity and population coupling.(A) Example firing maps across the consecutive cNOR sessions for one mouse day in context X (top) versus context Y (bottom).Each row shows one principal cell (numbers indicate peak rate for each map).(B) Estimation plot showing the place field similarity for the pairs of place maps expressed by individual cells across two contiguous cNOR sessions (e.g., Sampling and Test 1) in context X versus context Y (each data point represents one cell).(C) Schematic of the population-level analyses (see methods).Coactivity between any two (i, j) neurons measured as the β regression weight from the GLM assessing their firing