Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin B subunit, wheatgerm agglutinin and isolectin B4 from Griffonia simplicifolia I in primary afferent neurons innervating the rat urinary bladder

Abstract

In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6–S1 spinal cord segments and in the T13–L2 and L6–S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit–horseradish peroxidase was injected. Cholera toxin B subunit–horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV–VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin–horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit–horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I–horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit–horseradish peroxidase than with wheat germ agglutinin– or isolectin B4–horseradish peroxidase. In the L6–S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with myelinated fibres). Double labelling with other neuronal markers showed that 71%, 43% and 36% of the cholera toxin B subunit-immunoreactive cells were calcitonin gene-related peptide-, isolectin B4-binding- and substance P-positive, respectively. A few cholera toxin B subunit cells showed galanin-immunoreactivity, but none were somatostatin-, vasoactive intestinal polypeptide-, or neuropeptide Y-immunoreactive or contained fluoride-resistant acid phosphatase. The results show that cholera toxin B subunit–horseradish peroxidase is a more effective retrograde and transganglionic tracer for pelvic primary afferents from the urinary bladder than wheat germ agglutinin–horseradish peroxidase and isolectin B4–horseradish peroxidase, but in contrast to somatic nerves, it is transported mainly by unmyelinated fibres in the visceral afferents.