Extracellular stimulation in neocortex lacks specificity.

By activating axons, extracellular stimulation elicits synaptic responses in patch-clamped cells. However, because axonal signaling is both orthodromic and antidromic, extracellular stimulation may lack specificity. For example, stimulating neocortical layer (L) 4 to recruit ascending inputs to L2/3 pyramidal cells (PCs) may also antidromically activate presynaptic L2/3 PCs, as their descending axons traverse L4. This contaminates L4→L2/3 activation with L2/3→L2/3 responses, which is problematic since these pathways have different properties. Using 2-photon calcium imaging, we found L2/3 PCs that responded to L4 stimulation even after synaptic blockade, demonstrating antidromic activation. Our findings highlight limitations of extracellular stimulation specificity in neocortex.