DIRECTION AND FREQUENCY-DEPENDENT PATTERNS OF NEURONAL ENTRAINMENT IN MOUSE CORTICAL SLICES

Cortical neurons show cell-class dependent entrainment to electrical stimulation that varies with frequency and field orientation. Previous studies have suggested that pyramidal neurons preferentially entrain to low-frequency fields aligned with their apical dendrites, whereas interneurons respond more strongly at higher frequencies with weaker orientation dependence. Acute cortical slices of primary motor cortex were prepared from wild-type mice. We examined how neurons responded to stimulation of different frequencies and directions, and whether distinct coherence patterns correlated with specific neuronal subtypes.
Low-frequency AC stimulation (band-limited 5–50 Hz white noise) was applied either across (Direction A) or along (Direction B) cortical layers. Spikes from 110 well-isolated neurons (27 slices) were obtained following automated sorting (Kilosort4) and manual curation (Phy2). We analysed entrainment by calculating spike-stimulus coherence spectra for stimulation delivered in Direction A or Direction B.
Coherence spectra were decomposed using nonnegative matrix factorization (NNMF) to extract recurring entrainment patterns, and normalised feature weights were classified with K-means clustering. Three dominant factors accounted for the variability across neurons: one captured low-frequency entrainment specific to Direction A, while the other two reflected broadband entrainment associated with each direction. Clustering identified two principal neuron groups: Cluster 1 (46 neurons), showing low-frequency Direction A entrainment, and Cluster 2 (64 neurons), showing broadband responses to one or both directions.
These findings reveal distinct frequency- and direction-dependent response profiles, suggesting functional subpopulations within cortical circuits. Future work will combine waveform and inter-spike interval analysis with optogenetic tagging to relate response-based clusters to defined neuronal cell types.