Subjects and Behavioral and Electrophysiological Procedures

Subjects were four adult male Long-Evans rats, housed individually, kept on food and water restriction, and monitored closely to maintain good health and a minimum of 85% free feeding weight. Animals were given free access to water at the end of the day and on weekends. All animal procedures were approved by the Boston University Institutional Animal Care and Use Committee.

On the first day of training, rats explored a figure-eight maze consisting of a rectangular track (122 cm × 91 cm; 48” × 36”) bisected lengthwise by a 122-cm (48”)-long central stem ( Figure 1 A; Movie S1 ). A 41-cm (16”) segment of the center stem was replaced with a commercially available treadmill (Columbus Instruments) adapted to the maze. Two ports for delivering water reward were located in the corners of the maze closest to the start of the central stem, and a third water port was located at the end of the treadmill. The water ports produced an audible click when they were activated.

For clarity, the term “session” is used to refer to an entire training or testing period within a day (typically 40–60 min), the term “trial” is used to refer to one full lap on the maze (starting and ending at either the left or right water port), and the term “run” is used to refer to the period during which the treadmill was moving within a trial (from the moment the treadmill starts to the moment the stop command is sent to the treadmill). The brief period the treadmill took to come to a complete stop was ignored in subsequent analyses. Starting on the second day of training, rats began each session at the start of the central stem. Throughout training, the rats were prevented from turning around. Once a rat progressed forward so their hind legs were on the treadmill, they were given a small water reward at the end of the treadmill and allowed 2 s to drink. The treadmill was then activated at a low speed (5–10 cm/s), and the rat was blocked from running forward off the treadmill while it was moving. The treadmill run was manually aborted, and the treadmill stopped immediately, if the rat either turned around or if its hind legs reached the back edge of the treadmill. The treadmill run was restarted (using the same settings but restarting the elapsed time) once the rat returned to the treadmill facing forward. Aborted runs that occurred during recording sessions were ignored in subsequent analyses. The rat was given another small water reward for running continuously until the treadmill stopped automatically. This reward typically caused the animal to spend the majority of the treadmill run with its mouth positioned close to the water port. The rat was then allowed to either remain on the treadmill or exit the treadmill and finish the lap. If the rat remained on the treadmill, the treadmill was started again, with the same rules as before. When the rat exited the treadmill, it was forced to turn either left or right and was rewarded for reaching the water port in the corner of the maze. Another trial was started when the rat reached the center stem.

During the first few trials, each run lasted only 5–10 s. As the rat grew accustomed to the treadmill, both the treadmill speed and the time required to receive a reward were gradually increased until the rat was consistently running 49 cm/s (maximum speed) for greater than 16 s. At this point, the protocol was changed to either a “distance-fixed” or a “time-fixed” protocol, and the rat was required to complete one trial for each run on the treadmill. In both protocols, the speed on each lap was randomly selected from within a predetermined range. The treadmill speed was held constant throughout each full treadmill run, and a new speed was randomly selected at the start of each treadmill run. In the “distance-fixed” protocol, the duration of each run was adjusted so that the distance traveled was constant (700 cm), regardless of the treadmill speed. In the “time-fixed” protocol, the duration of each run was constant (16 s), regardless of the speed. The minimum speed was chosen based on the lowest speed in which the individual rat ran smoothly on the treadmill. If the treadmill runs too slowly, the rat stops running smoothly and, instead, repeatedly runs forward then rides the treadmill back. The maximum speed was limited by the endurance of the rat and the need to run enough laps to fully sample the range of available speeds. The range of speeds used for recordings was 30 cm/s to 49 cm/s.

Once the rat was comfortable with the randomly varying speeds, it was trained to alternate left and right reward arms until it met a criterion of steady running on the treadmill through the range of speeds used, for at least 40 trials per session, with at least 75% accurate alternation. The total period of training prior to the first recording was between 5 and 25 weeks.

Following training, rats were implanted with microdrives containing 16 independently drivable tetrodes angled ∼25° in the posterior direction, which entered the skull through a craniotomy just anterior to the fissure between parietal and postparietal skull bone (approximately anterior-posterior [AP] = −8.0 mm; medial-lateral [ML] = −4.6 mm). Each tetrode consisted of four strands of 0.0005” (12.7-μm) Stablohm 800 wire (California Fine Wire Company), gold plated to reduce impedance to between 180 and 220 kΩ at 1 kHz. At the end of surgery, each tetrode was lowered ∼2–3 mm below the dorsal surface. Rats were allowed at least 1 week recovery before training resumed. Tetrodes were slowly advanced toward the MEC.

Electrical recordings were made using two different commercially available systems. The first system was the 96-channel Plexon Multichannel Acquisition Processor (MAP). On this system, each channel was amplified (1,000×–10,000×) and band-pass filtered for high-frequency spiking activity (154 Hz–8.8 kHz). Spike channels were referenced to another electrode to remove movement-related artifacts. Action potentials were detected by threshold crossing and digitized at 40 kHz.

The second recording system was a 64-channel Neuralynx Digital Lynx. Each of the 64 channels (4 channels each, for 16 tetrodes) first passed through a unity-gain VLSI headstage and then into pre-amplifiers. The signals were then amplified (5,000×–20,000×) and band-pass filtered in the 0.3 Hz–6 kHz range and digitized at 32 kHz. Signals were digitally processed online to detect and capture action potentials indicated by threshold crossing on one of the four channels within a tetrode. Custom-built adapters were used to allow the Plexon headstages to interface with the Neuralynx electrode interface board.

To analyze the spatial firing properties of neurons, rats were trained to forage for cut pieces of Kellogg’s Froot Loops cereal pieces, distributed randomly throughout an open field environment. Rats were allowed to forage for enough time to visit every region of the environment, typically 10–20 min.

One of two environmental arenas was used for open field foraging. The first environment was located near the Neuralynx recording system and consisted of a box 162 cm × 115 cm (64” × 45”) with walls 30 cm tall. A white cue card was present on one wall of the chamber to provide a stable visual landmark. This environment is hereinafter called the “Neuralynx open field.” This environment was used for prescreening and locating grid cells in rats 1, 3, and 4. In addition, for 19 out of 43 sessions for Rat 1 and for 34 out of 43 sessions for rat 3, open field foraging recorded on the Neuralynx open field was paired with treadmill recordings from the Plexon system to classify neurons firing on the treadmill as either grid cell or non-grid cell (see the following section, “ Analysis Methods ”).

The second environment was placed on top of the figure-eight maze (near the Plexon MAP system) and consisted of a black platform (122 cm × 152 cm; 48” × 60”) with no walls. This platform is hereinafter called the “Plexon open field.” This environment was used for prescreening and locating grid cells in rats 2 and 4 and to conduct open field foraging recordings immediately following treadmill recording sessions for 24 out of 43 sessions for rat 1, for 9 out of 43 sessions for rat 3, and for all sessions for both rats 2 and 4.

Once theta rhythmic neural activity (including grid cells) was detected, treadmill recording sessions were initiated. At this point, tetrodes were turned a maximum of ∼32 μm/day, and electrodes were allowed to settle overnight before each recording session. Rats were regularly tested on either the Neuralynx open field (rats 1, 3, and 4) or the Plexon open field (rats 2 and 4) to look for signs of new neural activity. Recordings were made on the treadmill on days that appeared to have neural activity that had not been previously recorded. All treadmill recordings were made using the Plexon MAP system.

For rat 1, all 42 daily recording sessions were distance fixed. For rat 2, all 23 recording sessions were time fixed. For rat 3, the first 34 recording sessions used the time-fixed protocol, and the remaining nine recording sessions were distance fixed. For rat 4, all 27 recording sessions were time fixed. For rat 3, the first few training sessions using the distance-fixed protocol were not used for data analysis.