Botulinum toxin type A induces changes in the chemical coding of substance P-immunoreactive dorsal root ganglia sensory neurons supplying the porcine urinary bladder

Abstract

Botulinum toxin (BTX) is a potent neurotoxin which blocks acetylcholine release from nerve terminals, and therefore leads to cessation of somatic motor and/or parasympathetic transmission. Recently it has been found that BTX also interferes with sensory transmission, thus, the present study was aimed at investigating the neurochemical characterization of substance P-immunoreactive (SP-IR) bladder-projecting sensory neurons (BPSN) after the toxin treatment. Investigated neurons were visualized with retrograde tracing method and their chemical profile was disclosed with double-labelling immunohistochemistry using antibodies against SP, calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase activating polypeptide (PACAP), neuronal nitric oxide synthase (nNOS), galanin (GAL), calbindin (CB), and somatostatin (SOM). In the control group (n = 6), 45% of the total population of BPSN were SP-IR. Nearly half of these neurons co-expressed PACAP or CGRP (45% and 35%, respectively), while co-localization of SP with GAL, nNOS, SOM or CB was found less frequently (3.7%, 1.8%, 1.2%, and 0.7%, respectively). In BTX-treated pigs (n = 6), toxin-injections caused a decrease in the number of SP-IR cells containing CGRP, SOM or CB (16.2%, 0.5%, and 0%, respectively) and a distinct increase in these nerve cells immunopositive to GAL (27.2%). The present study demonstrates that BTX significantly modifies the chemical phenotypes of SP-IR BPSN.

Keywords: botulinum toxin A; dorsal root ganglia; immunohistochemistry; neurotransmitters; pain; pig; sensory innervation; substance P; urinary bladder.

Scheme 1

A schematic diagram of a spinal ganglion section showing its arbitrary division into topographical domains, in which the occurrence and relative frequency of substance P (SP) containing bladder projecting sensory neurons (BPSN) were studied. Presented data were pooled from all dorsal root ganglia (DRG) sections studied, both the ipsi- and contralateral ganglia. C—central, P—peripheral, Cr—cranial and Cd—caudal domains of the DRG, M—middle region of the ganglion.

Figure 1

Representative images of substance P-positive (SP+) dorsal root ganglia (DRG)-urinary bladder-projecting neurons (UBPN) in control pigs. All images were taken separately from blue (a,d,g,j,m,p), green (b,e,h,k,n,r) and red (c,f,i,l,o,s) fluorescent channels; a–c One fast blue-positive (FB+) neuron (a, blue, arrow), which simultaneously contains SP (b, green, arrow) and pituitary adenylate cyclase activating peptide-PACAP (c, red, arrow). d–f One FB+ neuron (d, blue, arrow), which is simultaneously SP+ (e, green, arrow) and calcitonin gene-related peptide-positive (CGRP+, f, red, arrow). g–i Two FB+ neurons (g, blue, 1 short arrow, 1 long arrow), which are simultaneously SP+ (h, green, 1 short arrow) or SP-negative (h, green, 1 long arrow) and galanin-positive (GAL+, i, red, 1 short arrow) or GAL-negative (GAL-, i, red, 1 red arrow). j–l One FB+ neuron (j, blue, arrow), which simultaneously contains neuronal nitric oxide synthase-nNOS (k, green, arrow) and SP (l, red, arrow). m–o One FB+ neuron (m, blue, arrow), which is simultaneously calbindin-positive (CB+, n, green, arrow) and SP+ (o, red, arrow). p-s One FB+ neuron (p, blue, arrow), which simultaneously contains SP (r, green, arrow) and somatostatin-SOM (s, red, arrow). Bars 50 μm (a–s).

Figure 1

Representative images of substance P-positive (SP+) dorsal root ganglia (DRG)-urinary bladder-projecting neurons (UBPN) in control pigs. All images were taken separately from blue (a,d,g,j,m,p), green (b,e,h,k,n,r) and red (c,f,i,l,o,s) fluorescent channels; a–c One fast blue-positive (FB+) neuron (a, blue, arrow), which simultaneously contains SP (b, green, arrow) and pituitary adenylate cyclase activating peptide-PACAP (c, red, arrow). d–f One FB+ neuron (d, blue, arrow), which is simultaneously SP+ (e, green, arrow) and calcitonin gene-related peptide-positive (CGRP+, f, red, arrow). g–i Two FB+ neurons (g, blue, 1 short arrow, 1 long arrow), which are simultaneously SP+ (h, green, 1 short arrow) or SP-negative (h, green, 1 long arrow) and galanin-positive (GAL+, i, red, 1 short arrow) or GAL-negative (GAL-, i, red, 1 red arrow). j–l One FB+ neuron (j, blue, arrow), which simultaneously contains neuronal nitric oxide synthase-nNOS (k, green, arrow) and SP (l, red, arrow). m–o One FB+ neuron (m, blue, arrow), which is simultaneously calbindin-positive (CB+, n, green, arrow) and SP+ (o, red, arrow). p-s One FB+ neuron (p, blue, arrow), which simultaneously contains SP (r, green, arrow) and somatostatin-SOM (s, red, arrow). Bars 50 μm (a–s).

Figure 2

Representative images of substance P-positive (SP+) dorsal root ganglia (DRG)-urinary bladder-projecting neurons (UBPN) in BTX-treated animals. All images were taken separately from blue (a,d,g,j,m,p), green (b,e,h,k,n,r) and red (c,f,i,l,o,s) fluorescent channels; a–c one fast blue-positive (FB+) neuron (a, blue, arrow), which simultaneously contains SP (b, green, arrow) and pituitary adenylate cyclase activating peptide-PACAP (c, red, arrow). d–f One FB+ neuron (d, blue, arrow), which is simultaneously SP+ (e, green, arrow) and calcitonin gene-related peptide-positive (CGRP+, f, red, arrow). g–i One FB+ neuron (g, blue, arrow), which is simultaneously SP+ (h, green, arrow) and galanin-positive (GAL+, i, red, arrow). j–l One FB+ neuron (j, blue, arrow), which simultaneously contains neuronal nitric oxide synthase-nNOS (k, green, arrow) and SP (l, red, arrow). m–o One FB+ neuron (m, blue, arrow), which is simultaneously SP+ (n, green, arrow) and calbindin-negative (CB-, o, red, arrow). p-s One FB+ neuron (p, blue, arrow), which simultaneously contains SP (r, green, arrow) and somatostatin-SOM (s, red, arrow). Bars 50 μm (a–s).