Media 2 - Motor neurons in L6-S1 spinal cord projecting to the rat major pelvic ganglion

Retrograde labelling of motor and premotor neurons in the lumbosacral (L6-S1) spinal cord that project to the major pelvic ganglia (MPG). Microinjection of the retrograde tracer cholera toxin subunit B (CTB) into the MPG of a male rat was taken up by sacral preganglionic neurons in the intermediolateral column (IML). Somatic motor neurons were also retrogradely labelled with CTB, those innervating the urethral rhabdosphincter (ExU9), as well as motor neurons innervating the tail and levator ani (Tail9).

Visualization of motor neurons was achieved through whole tissue immunolabelling and clearing using a modified form of iDISCO for spinal cord. Motor neurons were immunolabelled via choline acetyltransferase (ChAT), while retrogradely-filled neurons were immunolabelled for CTB. Large volume imaging was performed on the light sheet microscope with a 12x lens (Ultramicroscope II, Miltenyi Biotec). Dataset was visualized in Imaris (Bitplane).