Elsevier

Neuroscience

Volume 87, Issue 1, 30 June 1998, Pages 275-288
Neuroscience

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

https://doi.org/10.1016/S0306-4522(98)00061-XGet rights and content

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.

Section snippets

Surgical procedures and tissue preparation

Experiments were performed on 16 adult male Sprague–Dawley rats (ALAB, Stockholm, Sweden) weighing 230–450 g. All efforts were made to minimize animal suffering and reduce the number of animal used. The experiments were carried out in accordance with the policy of the Society for Neuroscience on the use of animals in neuroscience research, and approved by the local animal research ethical committee. The animals were anaesthetized by an i.p. injection of chloral hydrate (30 mg/100 g body weight). A

Transganglionic transport of lectin–horseradish peroxidase conjugates

In the L6 and S1 spinal cord segments, HRP histochemical and lectin-immunofluorescence histochemical labelling was observed in all cases following injections of the lectin–HRP conjugates into the bladder wall. No labelling was found in the T13 or L1–2 spinal cord segments. There were no differences in the extent of labelling found by the two histochemical methods, although the HRP-histochemistry seemed to be more sensitive, showing a higher density of labelling than the

Discussion

Results from the present study show that CTB–, WGA– and B4–HRP were transported retrogradely to the T13–L2 and L6–S1 DRGs, and transganglionically to the L6–S1 spinal cord following injections into the bladder wall. Bladder afferent neurons in the L6–S1 DRGs, which had taken up and transported these lectin conjugates, were predominantly RT97-negative (81%). In addition, a large number of neurons that transported CTB–HRP also contained the neuropeptides CGRP (71%) and SP (36%).

Conclusion

The results of this study show that CTB–HRP is an effective retrograde and transganglionic tracer for visceral afferents from the urinary bladder, superior to both WGA–HRP and B4–HRP. It is transported mostly by neurons with unmyelinated fibres, containing the neuropeptides SP and CGRP. This is contrary to the situation for somatic primary afferent neurons in which CTB–HRP is transported almost selectively by myelinated fibres practically lacking these peptides.

Acknowledgements

This work was supported by the Swedish Medical Research Council, proj. no. 553. Dr Brita Robertson is greatly acknowledged for valuable advice and helpful discussions.

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