Survival in complex environments necessitates a flexible navigation system that incorporates memory of recent behavior and associations. Yet, how the hippocampal spatial circuit represents latent information independent of sensory inputs and future goals has not been determined. To address this, we imaged the activity of large ensembles in subregion CA1 via wide-field fluorescent microscopy during a novel behavioral paradigm. Our results demonstrated that latent information is represented through reliable firing rate changes during unconstrained navigation. We then hypothesized that the representation of latent information in CA1 is mediated by pattern separation/completion processes instantiated upstream within the dentate gyrus (DG) and CA3 subregions. Indeed, CA3 ensemble recordings revealed an analogous code for latent information. Moreover, selective chemogenetic inactivation of DG-CA3 circuitry completely and reversibly abolished the CA1 representation of latent information. These results reveal a causal and specific role of DG-CA3 circuitry in the maintenance of latent information within the hippocampus.

In vivo calcium videos were recorded with a miniscope (v3; miniscope.org) containing a monochrome CMOS imaging sensor (MT9V032C12STM, ON Semiconductor) connected to a custom data acquisition (DAQ) box (miniscope.org) with a lightweight, flexible coaxial cable ¹⁴. The DAQ was connected to a PC with a USB 3.0 SuperSpeed cable and controlled with Miniscope custom acquisition software (miniscope.org). The outgoing excitation LED was set to between 3-6%, depending on the mouse to maximize signal quality with the minimum possible excitation light to mitigate the risk of photobleaching. Gain was adjusted to match the dynamic range of the recorded video to the fluctuations of the calcium signal for each recording to avoid saturation. Behavioral video data were recorded by a webcam mounted above the environment. Behavioral video recording parameters were adjusted such that only the red LED on the CMOS of the miniscope was visible. The DAQ simultaneously acquired behavioral and cellular imaging streams at 30 Hz as uncompressed avi files and all recorded frames were timestamped for post-hoc alignment.

            All recording environments were constructed of a grey Lego base and black Lego bricks (Lego, Inc) according to the dimensions specified in the main text and supplemental figures. All external walls had a height of 22 cm; all internal walls had a height of 15 cm. All hallways were 5 cm wide; due to the width mice typically ran the length of the hallway rather than turning around in the hallway. During recording, the environment was dimly lit by a nearby computer screen, which served as directional cue. A white-noise generator was placed above the environment to mask uncontrolled ambient sounds. Each recording session lasted 20 minutes, and only one session was recorded per day to avoid photobleaching. The mouse was always placed in the corner of the hallway at the start of the session and was allowed to explore the environment for 15 to 30 s prior to data acquisition. Following each recording the environment was cleaned with disinfectant (Prevail).

            For CA3 inactivation experiments, 5 mg/kg of clozapine-N-oxide (CNO + 0.7% DMSO) was injected Intraperitoneally 1 hr and 15 min prior to recording. This timepoint was chosen based on a separate experiment in which we monitored the time course of hippocampal theta power reduction during hm4di/CNO-mediated inhibition of the medial septum (data not shown). Mice were returned to their home cage between the injection and the start of the recording session. We conducted two separate control experiments to rule out the possibilities that our results could be explained by injection procedure, expression of hm4di-mcherry, or to non-specific effects of CNO itself. As our first within-animal control, we injected sterile saline instead of CNO and repeated the recording and analysis procedures (Fig. 4b-d). The order of Saline and CNO recordings was interleaved within mouse, and whether the first recording session for a mouse followed a Saline or CNO injection was randomized. As a second control, to ensure that any differences between Saline and CNO sessions were attributable to the interaction between hm4di and CNO, and not an effect of CNO or its metabolites alone, CNO injection experiments were repeated in a second across-mouse control group which did not express hm4di (Fig. 4c-d).

Calcium imaging data were preprocessed prior to analyses via a pipeline of open source MATLAB (MathWorks) functions to correct for motion artifacts¹⁶, segment cells and extract transients^17,18, and infer the likelihood of spiking events via deconvolution of the transient trace through a second-order autoregressive model¹⁹. The motion-corrected calcium imaging data were manually inspected to ensure that motion correction was effective and did not introduce additional artifacts. Following this preprocessing pipeline, the spatial footprints of all cells were manually verified to remove lens artifacts. Position data were inferred from this LED offline following recording using a custom written MATLAB (MathWorks) script and were manually corrected if needed. The experimenter manually segmented data recorded in the compartment and hallway, as well as the most recent entryway based on the recorded position data prior to all further analyses.

Each session is included as a separate .mat file containing the structures 'processed' and 'properties'. Most relevant fields are:

processed.p = [x; y] position data 

processed.trace = firing rates estimated via autoregressive deconvolution 

processed.FiltTraces = dF/F traces prior to deconvolution

processed.splithalf = various cell selection criteria

processed.roi = coordinates for segmenting each entryway, the compartment, and the hallway