Hippocampal ensembles represent sequential relationships among an extended sequence of nonspatial events

Recording device and surgery Each chronically implanted custom microdrive contained 20 independently drivable tetrodes. Each tetrode consisted of four twisted nichrome wire (13 µm in diameter; California Fine Wire) and gold-plated to achieve final tip impedance of ~250 kΩ (measured at 1 kHz). Voltage signals recorded from the tetrode tips were referenced to a ground screw positioned over the cerebellum, and filtered for single-unit activity (154 Hz to 8.8 kHz). The neural signals were then amplified (10,000–32,000X), digitized (40 kHz) and recorded to disk with the data acquisition system (MAP, Plexon). The microdrive was implanted over the left hippocampus, centered on coordinates: -4.0 mm AP, 3.5 mm ML. Unit preprocessing and spike sorting Action potentials from individual neurons were manually isolated offline using a combination of standard waveform features across the four channels of each tetrode (Offline Sorter, Plexon). Proper isolation was verified using interspike interval distributions for each isolated unit (assuming a minimum refractory period of 1 ms) and cross-correlograms for each pair of simultaneously recorded units on the same tetrode. Odor sequence task Five naïve rats were initially trained to nosepoke and reliably hold their nose for 1.2 s in the odor port for a water reward. Odor sequences of increasing length were then introduced in successive stages (A, AB, ABC, ABCD, and ABCDE) upon reaching behavioral criterion of 80% correct over three sessions per training stage. In each stage, rats were trained to correctly identify each presented item as either InSeq (by holding their nosepoke response for at least 1.2 s to receive a water reward) or OutSeq (by withdrawing their nose before 1.2 s to receive a reward). Note that OutSeq items could be presented in any sequence position except the first (i.e., sequences always began with odor A, though odor A could also be presented later in the sequence as an OutSeq item). All procedures were conducted in accordance with the Institutional Animal Care and Use Committee (Boston University and University of California, Irvine). For more information on data collection and procedures, please see the included references or feel free to contact us!