Disengaging spinal afferent nerve communication with the brain in live mice

Abstract

Our understanding of how abdominal organs (like the gut) communicate with the brain, via sensory nerves, has been limited by a lack of techniques to selectively activate or inhibit populations of spinal primary afferent neurons within dorsal root ganglia (DRG), of live animals. We report a survival surgery technique in mice, where select DRG are surgically removed (unilaterally or bilaterally), without interfering with other sensory or motor nerves. Using this approach, pain responses evoked by rectal distension were abolished by bilateral lumbosacral L5-S1 DRG removal, but not thoracolumbar T13-L1 DRG removal. However, animals lacking T13-L1 or L5-S1 DRG both showed reduced pain sensitivity to distal colonic distension. Removal of DRG led to selective loss of peripheral CGRP-expressing spinal afferent axons innervating visceral organs, arising from discrete spinal segments. This method thus allows spinal segment-specific determination of sensory pathway functions in conscious, free-to-move animals, without genetic modification.

Anatomical distribution of CGRP-containing lumbosacral spinal afferent neurons in the mouse uterine horn

“…Scale bar in panel [ (E) ; lower] applies to all images. Independent control data in panel (B) was originally published in Kyloh et al (2022) . LS, lumbosacral; DRG, dorsal root ganglia; Ipsi, ipsilateral; Contra, contralateral; CGRP, calcitonin gene-related peptide.…”

“…This procedure has recently been described in detail ( Kyloh et al, 2022 ). Mice ( N = 6) were anesthetized with inhaled isoflurane (induced at 4%; maintained at 1.5–2% in 1 L/min O 2 ) and administered s.c. 0.05 mg/kg buprenorphine (Temvet; Troy Laboratories; Glendenning; NSW, Australia).…”

“…CGRP density, nerve fiber counts, network lengths, junctions, and area of CGRP, were compared using a two-way, repeated measures ANOVA with Geisser-Greenhouse correction and Tukey’s multiple comparisons (fixed effect of region: cranial/mid/caudal uterine horn) or Šídák’s multiple comparisons (fixed effect of lesion side: ipsilateral/contralateral to DRG removal). Independent, non-surgical control data of CGRP density in the mouse uterine horn ( N = 5) was originally published in Kyloh et al (2022) , in which the tissue had been processed identically to DRG-removed animals here. These control data were compared to values from DRG-removed animals using a mixed-effects model (REML) with Tukey’s multiple comparisons.…”

“…Given the largely ipsilateral innervation to each half of the female rodent reproductive tract ( Klein and Burden, 1988 ; Nance et al, 1988 ), deafferentation in the uterine horn due to unilateral DRG removal could be compared against the contralateral side. In that study, thoracolumbar (T13-L2) DRG ablation revealed most thoracolumbar spinal afferents (∼60%) containing the sensory neuropeptide, calcitonin gene-related peptide (CGRP), innervate the cranial end of the mouse uterine horn, while a smaller proportion (∼45%) supply the caudal region ( Kyloh et al, 2022 ). These data support the topographical arrangement suggested for substance P-containing thoracolumbar spinal afferents in the rat uterine horn ( Nance et al, 1988 ), which was also deduced from functional studies on the rat female reproductive tract ( Berkley et al, 1988 , 1990 , 1993 ).…”

“…To address this, we recently developed a survival surgery in mice where subsets of DRG are removed in vivo ( Kyloh et al, 2022 ). Given the largely ipsilateral innervation to each half of the female rodent reproductive tract ( Klein and Burden, 1988 ; Nance et al, 1988 ), deafferentation in the uterine horn due to unilateral DRG removal could be compared against the contralateral side.…”