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Renshaw cells (RCs) are one of the most studied spinal interneurons; however, their roles in motor control remain enigmatic in part due to the lack of experimental models to interfere with RC function, specifically in adults. To overcome this limitation, we leveraged the distinct temporal regulation of Calbindin (Calb1) expression in RCs to create genetic models for timed RC manipulation. We used a Calb1 allele expressing a destabilized Cre (dgCre) theoretically active only upon trimethoprim (TMP) administration. TMP timing and dose influenced RC targeting efficiency, which was highest within the first three postnatal weeks, but specificity was low with many other spinal neurons also targeted. In addition, dgCre showed TMP-independent activity resulting in spontaneous recombination events that accumulated with age. Combining Calb1-dgCre with Parvalbumin (Pvalb) or Engrailed1 (En1) Flpo alleles in dual conditional systems increased cellular and timing specificity. Under optimal conditions, Calb1-dgCre/Pvalb-Flpo mice targeted 90% of RCs and few dorsal horn neurons; Calb1-dgCre/En1-Flpo mice showed higher specificity, but only a maximum of 70% of RCs targeted. Both models targeted neurons throughout the brain. Restricted spinal expression was obtained by injecting intraspinally AAVs carrying dual conditional genes. These results describe the first models to genetically target RCs bypassing development.


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