Two-photon imaging and the activation of cortical neurons

•Two-photon imaging and sparse activation of cortical neurons by microstimulation.•Experiments for the study of neuronal excitability with microstimulation.•Implications for neural prosthetics.

Based on two-photon calcium imaging, Histed et al. (2009) concluded that electrical microstimulation of cortical tissue in mammals activates a sparse and distributed population of neurons. This work has been cited by many as proof that electrical microstimulation is nonfocal, which means it may lack the precision needed for applications in neuroprosthetics. We affirm that the generation of stimulation-evoked behaviour is based primarily on the orthodromic conduction of signals originating mainly from the deepest layers of cortex, while the work of Histed et al. is effectively limited to investigating the antidromic activation of lateral projection neurons of the superficial cortex. The apparent sparse activation is a consequence of the pattern of axonal projections based on activating a volume of axons while imaging cell bodies transecting a single plane through the cortex. This creates the false impression that the distribution of activated neurons is sparse and nonfocal. We recommend how two-photon calcium imaging, which is superb for the study of individual and groups of neurons, might be more effectively used to ascertain how electrical stimulation affects the brains of mammals. This is a timely topic since investigators are using electrical microstimulation in animals to develop prosthetic devices to restore sensory and motor functions in disabled patients.