High-resolution functional-magnetic-resonance-imaging (fMRI) has been used to study brain functions at increasingly finer scale, but whether fMRI can accurately reflect layer-specific neuronal activities is less well understood. The present study investigated layer-specific cerebral-blood-volume (CBV) fMRI and electrophysiological responses in the rat cortex. CBV fMRI at 40×40 µm in-plane resolution was performed on an 11.7-T scanner. Electrophysiology used a 32-channel electrode array that spanned the entire cortical depth. Graded electrical stimulation was used to study activations in different cortical layers, exploiting the notion that most of the sensory-specific neurons are in layers II–V and most of the nociceptive-specific neurons are in layers V–VI. CBV response was strongest in layer IV of all stimulus amplitudes. Current source density analysis showed strong sink currents at cortical layers IV and VI. Multi-unit activities mainly appeared at layers IV–VI and peaked at layer V. Although our measures showed scaled activation profiles during modulation of stimulus amplitude and failed to detect specific recruitment at layers V and VI during noxious electrical stimuli, there appears to be discordance between CBV fMRI and electrophysiological peak responses, suggesting neurovascular uncoupling at laminar resolution. The technique implemented in the present study offers a means to investigate intracortical neurovascular function in the normal and diseased animal models at laminar resolution
