Sequential-context-dependent hippocampal activity is not necessary to learn sequences with repeated elements

Abstract

Learning sequences of events (e.g., a-b-c) is conceptually a simple problem that can be solved using asymmetrically linked cell assemblies [e.g., "phase sequences" (Hebb, 1949)], provided that the elements of the sequence are unique. When elements repeat within the sequence, however (e.g., a-b-c-d-b-e), the same element belongs to two separate "contexts," and a more complex sequence encoding mechanism is required to differentiate between the two contexts. Some neural structure must form sequential-context-dependent, or "differential," representations of the two contexts (i.e., b as an element of "a-b-c" as opposed to "d-b-e") to allow the correct choice to be made after the repeated element. To investigate the possible role of hippocampus in complex sequence encoding, rats were trained to remember repeated-location sequences under three conditions: (1) reward was given at each location; (2) during training, moveable barriers were placed at the entry and exit of the repeated segment to direct the rat and were removed once the sequence was learned; and (3) reward was withheld at the entry and exit of the repeated segment. In the first condition, hippocampal ensemble activity did not differentiate the sequential context of the repeated segment, indicating that complex sequences with repeated segments can be learned without differential encoding within the hippocampus. Differential hippocampal encoding was observed, however, under the latter two conditions, suggesting that long-term memory for discriminative cues present only during training, working memory of the most recently visited reinforcement sites, or anticipation of the subsequent reinforcement site can separate hippocampal activity patterns at the same location.

Figure 1.

The arena and schematics of routes for each sequence task. A photograph of the arena with clothespin-mounted light-emitting diodes (which served as cues) mounted around the perimeter is shown to the left. Neural data were recorded from the rat through a lightweight tether. The three figures on the right show top-down, schematic views of the routes for each sequence. The legs of each sequence are shown as lines ending in arrowheads, in which arrowheads note the location where reward was given. Dotted lines represent the first segment of a context. In CCW contexts (blue), a CCW turn at the choice point is the most efficient for reaching the next goal, whereas in CW contexts (red), the most efficient turn direction at the choice point is a CW turn. Parallel red and blue lines denote the repeated segment(s). All other segments are shown in black (e.g., in the skipped-reward task). In the barrier-trained task, the black rectangles represent wooden blocks that were used during training to guide the rat through the sequence. Once the rat had learned the sequence, these blocks were removed from the arena. In the skipped-reward task, the probability of reward at the ends of the repeated segment (denoted by an X) was reduced across sessions until no reward was given, allowing rats to make continuous trajectories into, through, and out of the repeated segment. To make the two tasks and corresponding spatial locations less similar to the rat, the direction of the repeated segment was rotated by 90° between the barrier-trained and skipped-reward tasks (as shown in the schematic). To enable easier comparisons of unit responses, the orientation of sequences has been rotated in subsequent figures such that the repeated segments are vertical.

Figure 2.

Behavioral analysis for the three sequence tasks. For each sequence, the schematic of the sequence (see Fig. 1) is shown at the top left, the behavioral accuracy scores (i.e., the percentage of traversals that met the behavioral criterion) for each rat are shown in the bar graphs at the bottom left, and the actual paths followed by each rat are shown in the right portion. The paths labeled “Selected,” a subset of “All,” are those that met the performance criteria described in Materials and Methods and were used for subsequent analysis. Behavioral performance is shown for the complex-sequence task (A), barrier-trained task (B), and skipped-reward task (C). Only traversals that occurred during delayed-cue blocks are considered (see Materials and Methods).

Figure 3.

Rats solved the sequence tasks using a spatial rather than a motor strategy. In both graphs, the horizontal axis shows the number of delayed-cue blocks (sets of 3 complete sequences, during which the illumination of the cue light was delayed by 5 s) after exposure to a novel or previously learned but rotated sequence. The change or rotation occurred while the rat was performing the previously learned sequence and was initiated at the start of a cued block of trials. The vertical axis shows the percentage of zones reached before cue onset. The “Novel” sequence shows the disruption in performance during delayed-cue segment traversals when rats were exposed to a sequence with which they had no previous experience (for details, see Materials and Methods). The “Rotated” sequence shows the disruption in performance during delayed-cue segment traversals when rats were exposed to a rotated version of a previously learned sequence. As denoted by an asterisk, performance during the first block after the change in both conditions is significantly worse than the average performance on the preceding 10 blocks (t test; p < 0.05). In addition, the relearning curves are similar.

Figure 4.

The complex-sequence task was solved without differential encoding in the hippocampus. Each row depicts data from the same rat during a single session; each subpanel depicts a different cell and the corresponding cell identification number. The actual paths taken by the rat during clockwise (light gray paths; blue dots and bars) and counterclockwise (dark gray paths; red dots and bars) contexts are overlaid, showing the similarity between the paths that were taken. The location of the rat when each spike occurred is overlaid on the paths. The mean binned firing rates and SEs are shown by bar graphs adjacent to the paths, with calibration bars shown in the bottom right of each subpanel. The cells displayed were chosen because they showed the greatest difference between the two sequential contexts. None of the cells analyzed exhibited statistically significant differences in firing rate attributable to sequential context in any bin (ANOVA; p < 0.05).

Figure 5.

Training with barriers (the barrier-trained task) produced persistent differential activity. Each row depicts data from three different cells during the same recording session from the same rat, performing the task without the aid of barriers. The asterisk denotes significant sequential-context-specific activity as determined by either a significant main effect of context or bin-by-context interaction (ANOVA; p < 0.05). All other details are the same as those given in Figure 4.

Figure 6.

Training without rewards at the entry or exit of the repeated segment (the skipped-reward task) induced differential activity. In the schematic at the top, X symbols denote zones in which reward was withheld. Each row depicts data from three different cells during the same recording session from the same rat. All other details are the same as those given in Figure 4.

Figure 7.

Ensemble neural activity discriminated between the two contexts for barrier-trained and skipped-reward tasks but not for the complex-sequence task. Black bars show the behavioral proficiency, i.e., the percentage of segment traversals in which the rat met behavioral criterion, as described in Materials and Methods. Light gray bars show the mean percentage of correct classifications of context based on raw firing rate data using a Fisher linear discriminant analysis of the neural firing patterns, i.e., how different the ensemble firing patterns on the repeated segment were between the two contexts. Dark gray bars show the mean percentage of correct classifications of context based on firing rate data adjusted for path deviations and velocity. CS, The complex-sequence task; BTi, the initial day of the barrier-trained task; BTc, the criterion day of the barrier-trained task; SRi, the initial day of the skipped-reward task; SRc, the criterion day of the skipped-reward task. The asterisk denotes prediction that was significantly greater than chance, as defined in Materials and Methods. The lack of an error bar for “SRi” was attributable to having only one rat for this condition.

Figure 8.

Separability of neural activity increases with repeated exposures. The horizontal axis shows the number of the training day on the skipped-reward task; “C” represents the criterion day for each rat (day 6 for rat 2 and day 10 for rat 3). The vertical axis shows the separability score as determined by discriminant analysis. Data are shown for two rats (squares for rat 2; dots for rat 3). Data for day 1 for rat 3 were not used because of poor proficiency on the task; data for days 4 and 5 for rat 2 were not used because too few units were recorded on those days to allow analysis. Guide rails, which limited lateral position variability on the repeated segment, were present on each day.

Figure 9.

Tasks in which differential hippocampal responses occurred activated a larger percentage of pyramidal cells. The vertical axis shows the percentage of pyramidal cells in one recording task that showed a significant main effect of bin along the repeated segment (i.e., showed a place-selective response regardless of context). Sequence tasks were grouped according to whether the discriminant analysis (as summarized in Fig. 7) could predict the context at better than chance levels. “Non-separable” denotes tasks in which prediction was no greater than chance; “Separable” denotes tasks in which prediction was greater than chance. The separable tasks showed a significantly larger percentage of units with place-specific responses on the repeated segment (ANOVA; F_(1,9) = 5.3491; p < 0.05). CS, The complex-sequence task; BTi, the initial day of the barrier-trained task; BTc, the criterion day of the barrier-trained task; SRi, the initial day of the skipped-reward task; SRc, the criterion day of the skipped-reward task.