The neuronal circuitry through which prolonged excitation is generated in the spinal dorsal horn was investigated using optical imaging of neuronal excitation in transverse slices of rat spinal cords. It is known that tetanic stimulation (20 Hz for 1 s) of the dorsal root that activates both A and C primary afferent fibres elicits slow intrinsic optical signals (IOS) in the dorsal horn, seen most intensely in the substantia gelatinosa (SG), lamina II, and that IOS expresses in part the slow synaptic response recorded intracellularly in dorsal horn neurons. We here report that the slow IOS within the SG were completely abolished after an incision was made at the border between the SG and the deeper laminae, but not after an incision within the deeper dorsal horn of the laminae III-V. The result demonstrates directly that, in order to generate prolonged excitation in the SG, the neuronal elements in the deeper dorsal horn must be intact. Thus, the afferent information might be received first by the deeper elements and then transmitted to the SG region, and/or collaboration between the SG and deeper elements is necessary to maintain prolonged excitation in the SG.