Extended Data Fig. 4: Additional data on combined imaging and hippocampal electrophysiology; reactivation events are not a result of epileptiform events, transient brain motion, or eye motion.

a. Schematic for two-photon calcium imaging in visual association cortex while recording from contralateral hippocampal area CA1 using a silicon multi-electrode probe. b. Same example aversive-cue reactivation event as in Fig. 2g, but with additional data shown. Bottom left: mean activity time courses (fractional change in fluorescence, ΔF/F₀) in response to food cues, neutral cues, and aversive cues (columns) for all simultaneously recorded neurons (rows) from the same session, organized by preferred response. Bottom right: deconvolved activity traces of all recorded cortical cells in the period surrounding the aversive-cue reactivation event (red arrow). Top: purple traces: CA1 local field potential trace and zoom-in. When band-pass filtered in the ripple band (150-300 Hz; gray trace), the LFP shows a transient increase just before the detected reactivation event. c-e. The Emx1-Cre;Ai93;CaMK2a-tTA mouse line (Madisen et al., 2015) used in our work has been reported to exhibit epileptiform events in some cases (Steinmetz et al., 2017). Early experiments in our lab using mice obtained directly from the Allen Brain Institute did appear to exhibit visible epileptiform events as detected in the neuropil signal in cortical imaging. While none of these events overlapped with any cue reactivation events, we excluded this mouse from the study. We then acquired mice from Jackson labs (line 024108 rather than 024103 used in (Steinmetz et al., 2017)) which may have been further back-crossed. To further address the possibilities that epileptiform events occurred in the 8 mice used in our study and that these might overlap with the cue reactivations described in our study, we characterized the amplitude and width of all transient events in the neuropil from spontaneous activity recordings in all of our mice (as in (Steinmetz et al., 2017)). As described below, we found that 7/8 mice did not show any evidence of epileptiform events, while 1/8 mice only demonstrated a small number of epileptiform events. Panel c: scatter plot of peak width vs. event peak prominence (height above local background), combined across 7/8 mice included in this study. Typical low-amplitude or high-width peaks (Steinmetz et al., 2017) are shown as blue dots, whereas brief, high-amplitude peaks that may reflect epileptiform events are shown as black dots (defined using a conservative manual threshold as in (Steinmetz et al., 2017)). In these 7 mice, we found almost no epileptiform events total across over a dozen sessions per mouse (0,0,0,0,1,2,3 events total per mouse; events rates ranged between 0 and 0.00003 epileptiform events/second). Panel d: same as c, but for the eighth mouse included in the current study. In this mouse, we found 87 epileptiform events total across all sessions, amounting to 0.0014 epileptiform events/second, or approximately one such ~150 ms event every 12 minutes. Across all sessions, a total of two epileptiform events were observed within 1 second of any reactivation event, and both of these were neutral-cue reactivation events. Panel e: same as c, but for the mouse excluded from this dataset. Detectable epileptiform events were very rare in this mouse (0.008 events/s). Critically, we found that across all 8 included mice, 0/1444 food cue reactivations, 0/1258 aversive cue reactivations, and 2/1040 neutral cue reactivations occurred within 1 second of an epileptiform event (and the two overlapping neutral cue reactivations were from a mouse with 2 epileptiform events detected in total across all sessions). Thus, epileptiform events are exceptionally rare in the mice used in this study, and those that occurred did not occur near cue reactivation events. To further confirm these observations, we re-analyzed the 7 imaging sessions from two mice in which we imaged from the identical mouse line while recording cortical and hippocampal local field potentials using a 16-channel silicon probe acutely implanted contralateral to the imaging window (see Fig. 2g). The epileptiform events previously described using cortical electrophysiology (Steinmetz et al., 2017) appear as large local field potential (LFP) spikes, characterized by high amplitude (>1000 μV; normal LFP amplitudes are 5–15x smaller) and long duration (>10 ms). Briefly, we used the exact methods as in Steinmetz et al., 2017, and analyzed 20-s LFP traces surrounding 250 cue reactivation events identified using contralateral imaging (5000 seconds of data). We found a total of 5 epileptiform events from our LFP data (average rate of 1 events / 1000 s). Critically, all 5 of these events occurred >2 seconds away from any cue reactivation event. f. Pupil area (normalized to mean area estimated during locomotion in darkness, a state involving dilated pupils) was constricted throughout reactivation events. g. There was no transient increase in brain motion (root-mean-squared motion in the imaging plane) in the moments surrounding a reactivation event (green: food-cue reactivations; blue: neutral-cue reactivations; red: aversive-cue reactivations). h. There was no transient increase in eye motion (root-mean-squared motion) in the moments surrounding a reactivation event. Error bars are ± SEM across sessions (f) and across events (g-h).

Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).

Steinmetz, N. A. et al. Aberrant Cortical Activity in Multiple GCaMP6-Expressing Transgenic Mouse Lines. eNeuro 4, ENEURO.0207–17.2017–33 (2017).