Lateral Entorhinal Cortex Suppresses Drift in Cortical Memory Representations

Abstract

Memory retrieval is thought to depend on the reinstatement of cortical memory representations guided by pattern completion processes in the hippocampus. The lateral entorhinal cortex (LEC) is one of the intermediary regions supporting hippocampal-cortical interactions and houses neurons that prospectively signal past events in a familiar environment. To investigate the functional relevance of the activity of the LEC for cortical reinstatement, we pharmacologically inhibited the LEC and examined its impact on the stability of ensemble firing patterns in one of the efferent targets of the LEC, the medial prefrontal cortex (mPFC). When male rats underwent multiple epochs of identical stimulus sequences in the same environment, the mPFC maintained a stable ensemble firing pattern across repetitions, particularly when the sequence included pairings of neutral and aversive stimuli. With LEC inhibition, the mPFC still formed an ensemble pattern that accurately captured stimuli and their associations within each epoch. However, LEC inhibition markedly disrupted its consistency across the epochs by decreasing the proportion of mPFC neurons that stably maintained firing selectivity for stimulus associations. Thus, the LEC stabilizes cortical representations of learned stimulus associations, thereby facilitating the recovery of the original memory trace without generating a new, redundant trace for familiar experiences. Failure of this process might underlie retrieval deficits in conditions associated with degeneration of the LEC, such as normal aging and Alzheimer's disease.SIGNIFICANCE STATEMENT To recall past events, the brain needs to reactivate the activity patterns that occurred during those events. However, such reinstatement of memory traces is not trivial because it goes against the natural tendency of the brain to restructure the activity patterns continuously. We found that dysfunction of a brain region called the LEC worsened the drift of the brain activity when rats repeatedly underwent the same events in the same room and made them behave as if they had never experienced these events before. Thus, the LEC stabilizes the brain activity to facilitate the recovery of the original memory trace. Failure of this process might underlie memory problems in elderly and Alzheimer's disease patients with the degenerated LEC.

Keywords: ensemble decoding; hippocampus; medial prefrontal cortex; memory retrieval; rats; representational drift.

Figure 1.

Task schedule and the effect of LEC inhibition on behavior. A, The sequence of stimulus presentations in each epoch. B, The experimental schedule. C, The schematic representations of cannula location and the site of single-unit recording. D, The time line of each test session including four identical conditioning epochs (epochs 1–4) and the pharmacological manipulation. E, The proportion of trials in which rats expressed CRs (%; n = 4 rats, mean ± SEM); *p < 0.05, **p < 0.01 in two-way repeated-measures ANOVA followed by post hoc Tukey's HSD.

Figure 2.

Stabilization of PrL ensemble firings by high task demands. A, Greyscale plots showing the normalized firing rate (maximum = 1 in each neuron) during two epochs before the drug infusion (Aa, CS-US block; Ab, CS alone block). Neurons were sorted based on the CS-induced firing rate during the first epoch, from the largest increase (neuron 1) to the largest decrease (neuron 1005). Top, Black bars indicate the CS, whereas black rectangles mask US artifacts. B, Pearson correlation coefficient of ensemble firing rates between two epochs (n = 20 runs with 300 subsampled neurons, mean ± SEM). C, Trajectories of ensemble firing patterns projected onto the top three principal components (PC). D, Accuracy of decoding of the task events from ensemble firing rates in trials in the same epoch (Within) or different epochs (Cross). Each dot represents decoding accuracy in each run with 300 subsampled neurons. Horizontal bars indicate the median; ***p < 0.001 in Wilcoxon signed-rank tests.

Figure 3.

Reconfiguration of PrL ensemble representations of task events following LEC inhibition. A, Greyscale plots showing the normalized firing rate (maximum = 1 in each neuron) during the epochs before and after aCSF (Aa, Ab) or muscimol infusions (Ac, Ad). Neurons were sorted based on the CS-induced firing rates during the epoch before the infusion. Top, Black bars indicate the CS, whereas black rectangles mask US artifacts. B, The correlation coefficient of ensemble firing rates between two epochs before and after the infusion (n = 20 runs with 300 subsampled cells, mean ± SEM); ***p < 0.001 in two-way mixed ANOVA followed by t tests. C, Trajectories of ensemble firing patterns projected onto the top three principal components (PC) in sessions with aCSF (Ca) and muscimol infusions (Cb).

Figure 4.

Impaired reinstatement of ensemble firing patterns by LEC inhibition. A, Decoding accuracy of the task events within each epoch (Within) or between two epochs (Cross) in the CS-US block (Aa) and the CS-alone block (Ab). Each dot represents decoding accuracy in each of 20 runs with 300 subsampled neurons. Horizontal bars indicate the median; **p < 0.01, ***p < 0.001 in Wilcoxon rank-sum tests. B, Confusion matrices indicating proportions of task events that were identified by the classifier correctly (warm colors along the diagonal) or misidentified as a different event (lighter colors off diagonal) in the CS-US block (Ba) and the CS-alone block (Bb). The expected task phase (y-axis) shows the task phase from which a firing rate pattern was extracted. The predicted task phase (x-axis) shows which task phase the decoder classified the firing rate pattern as. The color indicates the probability of firing patterns in each task phase being categorized in one of the four task phases. Although the US was not delivered during the CS-alone block, firing rates were calculated during time windows corresponding to the Interval and Post-US period in the CS-US block. C, Decoding accuracy in cross-epoch decoding analyses calculated separately for each of the four task phases in the CS-US block (Ca) and the CS-alone block (Cb), as in A.

Figure 5.

Diverse effects of LEC inhibition on firing patterns of individual PrL neurons. Examples of firing patterns of individual neurons during the epochs before (black) and after (blue) muscimol infusions. The light and dark lines show the firing pattern in the CS-alone and CS-US block, respectively. Light gray bars indicate the CS, and black bars mask US artifact.

Figure 6.

Effects of LEC inhibition on spontaneous firing rates and across-trial firing stability of PrL individual neurons. A, Spontaneous FR of each neuron before (x-axis) and after the infusion (y-axis; 1 dot per neuron, color indicates the result of random permutation tests, p < 0.05) in aCSF and muscimol sessions. B, The cumulative distributions of spontaneous FRs. Inset, The difference in spontaneous FRs between, before, and after the infusion (1 dot per neuron). C, The difference in a measure quantifying the stability of firing rates across trials before and after the infusion (1 dot per neuron). Firing rates were calculated during each of the four task phases. The horizontal bars indicate the median; *p < 0.05, **p < 0.01, ***p < 0.001 in Wilcoxon rank-sum test.

Figure 7.

Effects of LEC inhibition on the sparsity and consistency of task event representations in the PrL. A, In aCSF and muscimol sessions (Aa), neurons were categorized into those with a significant FR increase (UP), decrease (DN), or no change (–) in response to the CS (p < 0.05 in random permutation tests). The width of arrows indicates the fraction of neurons that changed their category after the infusion, whereas their color indicates the type of change (gray, no change; green, loss of significance; orange, gain of significance). For neurons with significant CS-evoked FR responses before the infusion (Ab), the fraction of neurons maintained the same type of responses after the infusion (Retention). For neurons with significant CS-evoked FR responses after the infusion, the fraction of neurons that gained the responses after the infusion (Recruitment). Error bars indicate 95% confidence intervals. The cumulative distribution of the magnitude of CS-evoked firing responses in the CS-US block before and after the infusion (Ac). The magnitude was quantified by the RI that becomes zero for no change and −1/1 for the most robust decreased/increased firing rates. Inset, The change in the response magnitude after the infusion (1 dot per cell). The horizontal bars indicate the median. B, The same for US-evoked FR responses. C, In aCSF and muscimol sessions (Ca), neurons were categorized into those with significantly higher CS-evoked FR rates in the CS-US (U) than CS-alone block (U > A), significantly lower rates (U < A), or no difference (−); p < 0.05 in random permutation tests. A: CS Alone. U: CS-US. The definition of the arrow width and color is the same as in A. For neurons with significant FR differentiation before the infusion (Cb), the fraction of neurons that maintained the same FR differentiation after the infusion (Retention). For neurons with significant FR differentiation after the infusion, the fraction of neurons that gained the differentiation after the infusion (Recruitment). Error bars indicate 95% confidence intervals. The cumulative distribution of the degree of differentiation of CS-evoked firing responses between the CS-US block and CS-alone block (Cc). The differentiation was quantified by the DI that becomes 0 for no differentiation and −1/1 for lower/higher firing rates in the CS-US block than the CS-alone block. Inset, The change in the firing differentiation from before to after the infusion (1 dot per cell). The horizontal bars indicate the median.

Figure 8.

Loss of beneficial effects of high task-demand on PrL stability after LEC inhibition. A, Greyscale plots show the normalized firing rate during the two epochs after aCSF (Aa, Ab) or muscimol infusions (Ac, Ad). Neurons were sorted based on the CS-evoked firing rate during the preceding epoch. Black bars on the top indicate the CS, whereas black rectangles mask US artifacts. B, The correlation coefficient of ensemble firing rates between two epochs after the infusion (n = 20 runs with 300 subsampled cells, mean ± SEM); ***p < 0.001 in two-way mixed ANOVA followed by t test. C, Trajectories of ensemble firing patterns projected onto the top three principal components (PC).

Figure 9.

Exaggerated drift of PrL ensemble firing patterns by LEC inhibition. A, Decoding accuracy of the task events from ensemble firing rates in trials in the same epoch (Within) or different epochs (Cross) in the CS-US block (Aa) and the CS-alone block (Ab). Each dot represents decoding accuracy in each of 20 runs with 300 subsampled cells. Horizontal bars indicate the median; *p < 0.05, **p < 0.01, ***p < 0.001 in Wilcoxon rank-sum tests. B, Confusion matrices indicating proportions of task events that were identified by the classifier correctly (warm colors along the diagonal) or misidentified as a different event (lighter colors off diagonal) in the CS-US block (Ba) and the CS-alone block (Bb). The definition of axes and color are the same as in Figure 4B. C, Decoding accuracy in cross-epoch decoding analyses calculated separately for each of the four task phases in the CS-US block (Ca) and the CS-alone block (Cb), as in A.

Figure 10.

Correlations between the across-trial firing stability and memory expression. The correlation coefficient of ensemble firing rates between two sets of trials with or without CR expression. The analysis was conducted in each session separately and applied only to sessions in which the rats expressed CRs in at least 30% of CS-US trials (aCSF, n = 7 sessions in epochs 3 and 4; muscimol, n = 8 sessions in epoch 3 and five sessions in epoch 4; mean ± SEM); **p < 0.01 indicates a significant main effect of Response type, #p < 0.05 indicates a significant Drug × Response type interaction in two-way mixed ANOVA.