Sensory Neuron Development in Mouse Coccygeal Vertebrae and Its Relationship to Tail Biopsies for Genotyping

Abstract

A common method of genotyping mice is via tissue obtained from tail biopsies. However, there is no available information on the temporal development of sensory neurons in the tail and how their presence or absence might affect the age for performing tail biopsies. The goals of this study were to determine if afferent sensory neurons, and in particular nociceptive neurons, are present in the coccygeal vertebrae at or near the time of birth and if not, when they first can be visualized on or in those vertebrae. Using toluidine blue neuronal staining, transmission electron microscopy, and calcitonin-related gene peptide immunostaining, we found proximal to distal maturation of coccygeal nerve growth in the C57BL/6J mouse. Single nerve bundles were first seen on postpartum day (PPD) 0. On PPD 3 presumptive nociceptive sensory nerve fibers were seen entering the vertebral perichondrium. Neural development continued through the last time point (PPD 7) but at no time were neural fibers seen entering the body of the vertebrae. The effect of age on the development of pain perception in the neonatal mouse is discussed.

DNA from tissues of young mice is optimal for genotyping

“…In a recent study, it has been suggested that in a neonatal mouse or rat, particularly prior to approximately 12 d of age, a nociceptive stimulus may not result in the conscious perception of pain due to the lack of a competent pain pathway at this age [18]. At 14-17 d after birth, mice have less ossified tails and samples can be smaller than those from adult mice [6].…”

A highly efficient strategy to determine genotypes of genetically-engineered mice using genomic DNA purified from hair roots

“…Finally these procedures, alone or in combination, can alter the overall well-being of mice, 32,33 and are also widely considered to be painful. [34][35][36][37]…”

Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High‐Throughput Genotyping Protocol for Any Type of Mutation

“…Table shows the age(s) at which each tissue sampling method can be used. Sampling should, where possible, be done on young animals for the reasons listed below: DNA from tissues of young mice is more optimal for genotyping than that from older animals (Picazo & García‐Olmo, ). In a newborn mouse (particularly before 12 days of age), discomfort due to the sampling is reduced because the sampled tissue is not fully ossified and because the nociceptive stimulus may not result in the conscious perception of pain due to the lack of a competent pain pathway at this age (Hankenson, Garzel, Fischer, Nolan, & Hankenson, ; Silverman & Hendricks, ; Wever, Geessink, Brouwer, Tillema, & Ritskes‐Hoitinga, ). If genotyping is completed before weaning, extra animals or those of nondesired genotype can be sacrificed before separation of young animals into different cages. …”

“…Current Protocols in Mouse Biology not fully ossified and because the nociceptive stimulus may not result in the conscious perception of pain due to the lack of a competent pain pathway at this age (Hankenson, Garzel, Fischer, Nolan, & Hankenson, 2008;Silverman & Hendricks, 2014;Wever, Geessink, Brouwer, Tillema, & Ritskes-Hoitinga, 2017).…”