Differential regional spinal transgene expression after intrathecal AAV9-UBI-GFP versus subpial AAV9-UBI-RFP delivery in rats. (a–c) Lumbar intrathecal injection of AAV9-UBI-GFP led to the preferential GFP expression in the dorsal funiculus (DF), primary afferents distributed in the dorsal and ventral gray matter (white arrows) and dorsal root ganglion neurons (a; upper-right BW insert). A subpopulation of α-motoneurons and glial cells localized in the ventral root entry zone (VRE) also show GFP expression (yellow right insert; white left insert-VRE). No or minimal GFP expression in axons localized in the lateral (LF) or ventral funiculi (VF) was seen. Similarly, no GFP expression in interneurons in the dorsal horn or in the intermediate zone could be detected (b, c). (d–f) In contrast to intrathecal injection, the lumbar subpial injection of AAV9-UBI-GFP led to widespread transgene expression. Numerous GFP expressing NeuN+ neurons localized throughout the gray matter, including interneurons and α-motoneurons, were seen (e, f). Consistent with α-motoneuronal GFP expression, high GFP expression was seen bilaterally in transversely cut motor axons in the ventral roots (d; VR). Similarly, high intensity of GFP was seen virtually in all transversely cut axons in the lateral and ventral funiculi (Scale bars: a, D-200 μm; b, c, e-50 μm; f-20 μm). CC, central canal; DF, dorsal funiculus; DH, dorsal horn; LF, lateral funiculus; VF, ventral funiculus; VH, ventral horn; VR, ventral root; VRE, ventral root entry zone.