Extended Data Fig. 2: Developing and characterization of the neonatal intraspinal injection strategy and AAV8retro.
From: Deconstruction of a spino-brain–spinal cord circuit that drives chronic pain
a, Images of dissected spinal cord from mice injected with 3 μl (left) and 1 μl (right) of AAV8retro-mCherry. Neonatal injection of 1 μl or 3 μl of AAV8retro-mCherry led the virus infection of the entire lumbar region or both lumber and thoracic regions of the spinal cord, respectively. Inset shows the expression of mCherry was restricted largely in the dorsal horn. Scale bar: 1 mm. b, Representative images of RVM section from three independently performed experiments following spinal cord injection of AAV8retro-H2B-Ruby3 at Postnatal day 1(neonatal, P1) and injection of AAV8retro-H2B-Clover3 at P28 (adult), and Venn diagram illustrated the number of Ruby3-labelled, Clover3-labelled, and colocalized neurons in the RVM. Scale bar: 100 μm. c, Representative medulla section from three independently performed experiments following neonatal and adult spinal cord injection of AAV8retro-H2B-Ruby3 and AAV8retro-H2B-Clover3, respectively. Retrogradely labelled neurons in RVM and facial motor nucleus (FMN). Scale bar: 1 mm. d, Plasmid map shows the site for inserting the decapeptide LADQDYTKTA between N590 and T591 of the AAV8 capsid (AAV8retro). e, Representative images of retrogradely labelled RVM neurons by AAV8retro-mCherry (red), AAV2retro-eGFP (green), and merge (yellow) after co-injection of AAV8retro-mCherry and AAV2retro-eGFP into the spinal cord of P1.5 wild-type mice. Scale bar: 200 μm. GRN: gigantocellular reticular nucleus; MARN: magnocellular reticular nucleus. f, Quantification of e (n = 3). g, Representative images (left) from three independently performed experiments and quantification (right) of retrogradely labelled neurons in the prefrontal cortex (PFC) when co-inject AAV8retro-mCherry (red) and AAV2retro-eGFP (green) into the paraventricular nucleus of the thalamus (PVT) (inset). Scale bar: PVT: 100 μm; PFC: 200 μm. h, Quantification of retrogradely labelled neurons in the RVM when co-inject AAV8retro-mCherry (red) and CTB488 (green) into the adult spinal cord. i, Representative image from three independently performed experiments of retrogradely labelled neurons in the LC and axon terminals in parabrachial nucleus (PBN) when inject AAV8retro-Cre in the spinal cord of Ai14 mice. Scale bar: 500 μm. j, Representative images three independently performed experiments of retrogradely labelled neurons in the RVM when inject AAV8retro-H2B-Clover3-FLEX(LoxP)-H2B-Ruby3 in the spinal cord of Oprm1Cre/+ mice. Majority of OPRM1+ RVMSC neurons were located outside the lateral paragigantocellular nucleus (LPGi). Scale bar: 200 μm.
