Distinct cell-type contributions and network topography of theta-nested gamma oscillations in the medial entorhinal cortex

Theta-nested gamma oscillations in the medial entorhinal cortex (mEC) are essential for spatial coding and memory, but the underlying cellular mechanisms remain unclear. We combined optogenetics, whole-cell electrophysiology, intracellular voltage imaging, and local field potential (LFP) recordings in acute slices from CaMKIIα-ChR2 mice to investigate how excitation and inhibition shape theta-gamma coupling in layer II/III mEC. During theta-frequency stimulation, fast-spiking interneurons received strong gamma-frequency excitation and fired rhythmic bursts, whereas stellate and pyramidal neurons fired more sparsely and were dominated by gamma-frequency inhibition. This sparse firing could support the selective firing of grid cells. Excitatory post-synaptic currents in interneurons preceded inhibitory currents and LFP gamma by ∼3 ms, supporting a pyramidal-interneuron network gamma (PING) mechanism. Pyramidal neurons fired on the descending phase of the gamma cycle, whereas stellate cells and fast-spiking interneurons fired before and after the trough, respectively. Intracellular voltage imaging revealed network gamma synchronization among excitatory neurons at a population level, with topographic clustering of subthreshold membrane potentials, but not spike timing, while individual neurons often skipped gamma cycles. These findings identify the dominant role of reciprocal E-I interactions in generating theta-nested gamma oscillations and highlight distinct cell-type contributions to the temporal dynamics of the mEC. Further, a biophysically realistic computational model predicted gamma cycle skipping in stellate cells and burst firing in fast-spiking interneurons during PING. Our experimental and computational results provide mechanistic insight into how the intrinsic properties of mEC cell types generate oscillatory activity in a manner that could support grid cell function and spatial computation. Significance Statement: Theta-nested gamma oscillations in the medial entorhinal cortex (mEC) are essential for spatial navigation and memory, yet the underlying circuit mechanisms remain incompletely understood. Using optogenetics, voltage imaging, electrophysiology, and biophysically realistic computational modeling, we show that reciprocal interactions between excitatory neurons and fast-spiking interneurons generate robust gamma oscillations through a pyramidal-interneuron network gamma (PING) mechanism. We reveal cell-type specific differences in gamma phase-locking and demonstrate that principal neurons synchronize at the population-level across large laminar distances (up to 800 µm). Further, the voltage activity, but not spike timing, of principal neurons exhibited topographic clustering. These findings clarify how local circuits in mEC generate temporal dynamics critical for grid cell coding and further constrain computational models of spatial representation.