Abstract
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.
Highlights
Renshaw cells (RCs) are inhibitory spinal interneurons that synapse onto motoneurons (MNs) and receive direct input from recurrent collaterals of motor axons as they exit the spinal c ord[1,2,3,4]
90–95% of these triple-labeled cells are found in a region we defined as the Renshaw cell area (RCA; Fig. 1B,C, see methods) and that occupies the bottom 45% of the ventral horn in these lumbar segments (Fig. 1C)
The results show that intraspinal AAV9 transduction in Calb1-destabilized EGFP/Cre fusion gene (dgCre) :: Pvalb-Flpo animals targets a large percentage of RCs in a region comprising approximately 2 segments above and below the injection site
Summary
Renshaw cells (RCs) are inhibitory spinal interneurons that synapse onto motoneurons (MNs) and receive direct input from recurrent collaterals of motor axons as they exit the spinal c ord[1,2,3,4]. Detailed analyses of RC connections, input/output properties and the dynamic behavior of RC synapses on MNs and Ia inhibitory interneurons led to several hypotheses about possible RC functions that were critically reviewed in 19965 To this day, these hypotheses remain unchanged and largely untested[6,7], in part due to the inability to manipulate RCs and isolate their activity from other network elements during motor actions. Pvalb expression in spinal interneurons, including RCs, only begins after P10 and becomes widespread later[20] None of these genes are unique to RCs, necessitating combinatorial approaches to increase targeting specificity. We discuss the advantages and disadvantages of each model with the objective that these models, or variations thereof, can be adopted to accelerate discovery of RC functions
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