AbstractPrincipal neurons in rodent medial entorhinal cortex (MEC) generate high-frequency bursts during natural behavior. While in vitro studies point to potential mechanisms that could support such burst sequences, it remains unclear whether these mechanisms are effective under in-vivo conditions. In this study, we focused on the membrane-potential dynamics immediately following action potentials, as measured in whole-cell recordings from male mice running in virtual corridors (Domnisoru et al., 2013). These afterpotentials consisted either of a hyperpolarization, an extended ramp-like shoulder, or a depolarization reminiscent of depolarizing afterpotentials (DAPs) recorded in vitro in MEC stellate and pyramidal neurons. Next, we correlated the afterpotentials with the cells’ propensity to fire bursts. All DAP cells with known location resided in Layer II, generated bursts, and their inter-spike intervals (ISIs) were typically between five and fifteen milliseconds. The ISI distributions of Layer-II cells without DAPs peaked sharply at around four milliseconds and varied only minimally across that group. This dichotomy in burst behavior is explained by cell-group-specific DAP dynamics. The same two groups of bursting neurons also emerged when we clustered extracellular spike-train autocorrelations measured in real two-dimensional arenas (Latuske et al., 2015). No difference in the spatial coding properties of the grid cells across all three groups was discernible. Layer III neurons were only sparsely bursting and had no DAPs. As various mechanisms for modulating the ion-channels underlying DAPs exist, our results suggest that the temporal features of MEC activity can be altered while maintaining the cells’ spatial tuning characteristics.Significance StatementDepolarizing afterpotentials (DAPs) are frequently observed in principal neurons from slice preparations of rodent medial entorhinal cortex (MEC), but their functional role in vivo is unknown. Analyzing whole-cell data from mice running on virtual tracks, we show that DAPs do occur during behavior. Cells with prominent DAPs are found in Layer II; their inter-spike intervals reflect DAP time-scales. In contrast, neither the rarely bursting cells in Layer III, nor the high-frequency bursters in Layer II, have a DAP. Extracellular recordings from mice exploring real two-dimensional arenas demonstrate that grid cells within these three groups have rather similar spatial coding properties. We conclude that DAPs shape the temporal but not the spatial response characteristics of principal neurons in MEC.Author contributionsAll authors designed research. DÉC, CF, and JN performed research and analyzed data (equal contribution). AVMH wrote and edited the paper with support from MS and the other authors.