Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons
Luppi P.H., Fort P., Jouvet M.
Brain Res. 534 (1-2) pages : 209-224 (1990)


Materials and Methods

Materials and Methods


(A) Injection sites

(B) Retrograde labeling

(C) Artefactual labeling due to uptake by fibers of passage

(D) Anterograde tracing

(E) Double immunostaining technique



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In this report, we demonstrate that cholera-toxin B subunit (CTb) is a very sensitive retrograde tracer in the central nervous system when recognized by streptavidin-peroxidase immunohistochemistry. We further show that:
(1) injection of a small volume of CTb gives rise to small sharply defined injection sites limited to the cell group of interest associated with the labeling of all the known afferent projections,
(2) CTb is taken up, and anterogradely as well as retrogradely transported in damaged but not in intact fibers of passage,
(3) CTb can be applied iontophoretically, allowing us to study the afferents to small cell groups without any evidence of tissue necrosis in the sites and therefore without artefactual labeling due to uptake by damaged fibers of passage,
(4) the use of 4% paraformaldehyde fixative ideally suited for the preservation of most neural antigens, the addition of a 48 h colchicine treatment and the development of a double immunohistochemical method allow the biochemical characterization of the cell of origin of particular pathways in the CNS,
(5) CTb is also anterogradely transported with an extensive filling of axons and axon terminals and thereby opens up the possibility of identifying simultaneously the afferents as well as the efferents of the group of cells studied and finally
(6) the very long conservation of the preparation, the possibility of counterstaining it and of making camera lucida drawings allow easy and precise localization of the retrogradely labeled cells.


The introduction of HRP as a retrograde tracer by Kristensson and Olsson 27 and La Vail and La Vail 31 has greatly accelerated our knowledge of neuroanatomy. Improvements of the original technique including the use of the more sensitive chromogen TMB 39-42, the microelectrophoretic delivery technique 18 and the introduction of the HRP conjugates with wheat germ agglutinin (WGA-HRP) 11 17 53 54 57 or cholera toxin (CT-HRP) 53 54 57 have further increased the sensitivity of the technique and permit one to obtain more restricted injection sites. The remarkable increase in the number of putative transmitters has further pointed to the need for methods allowing simultaneous identification of a pathway and its neurochemical identity. For this purpose, the histochemical detection of HRP and its conjugates using DABS 8 9 33 45 or stabilized TMB 46 has been successfully coupled with the immunohistochemistry of neurochemical substances on the same sections. However, such double labeling techniques present many drawbacks. The DAB induced stabilization of the TMB product greatly decreases the method's sensitivity, especially when this procedure is coupled with the non-optimal fixation required for immunohistochemistry 44 46. The sensitivity of other chromogens like DAB is very low 3 39-42. In addition, HRP, and to a lesser extent its conjugates with WGA and CTb, are rapidly degraded in the retrogradely labeled cells 53 54 57, making delicate the coupling with a colchicine treatment essential for the optimal visualization of peptidergic cell bodies 3.

The introduction of a variety of retrogradely transported fluorescent dyes raises another possibility of biochemically characterizing the cells of origin of specific pathways in the CNS 28 30. Indeed, with such tracers, the 4% paraformaldehyde fixative ideally suited for the preservation of most neural antigens can be used 12 19 47 51 56 and the tissue can be processed for immunohistochemistry to identify the neurotransmitter contents of the retrogradely labeled cells 6 19 30 47 51 56 .Moreover it is easy to distinguish between these fluorescent dyes and immunocytochemically stained cells by simply using appropriate excitation wavelengths for each. In addition, two different dyes can be combined to demonstrate collateralization of afferents 29 55. However, the majority of these fluorescent retrograde tracers are less sensitive than HRP visualized with TMB 1 23, retrogradely transported by damaged as well as undamaged fibers of passage 1 23 30 47 and leak from the labeled neurons during the immunocytochemical procedure 19 47 51 56 and microscope observations 24. Furthermore, these dyes cannot be iontophoretically injected and application by pressure damages the tissue and increases the minimum size of the injection sites 30 44.

To overcome the limitations inherent to these fluorescent tracers and the HRP conjugates, we previously introduced the use of unconjugated cholera toxin B subunit (CTb) directly recognized by immunohistochemistry 14 15 34-36 58-60, thereby avoiding the severe drawbacks of a direct histochemical detection of HRP. We demonstrated that CTb is a very sensitive retrograde tracer 34 when visualized by the immunohistochemical ABC technique of Hsu 22. We further showed that retrograde transport of CTb can be used in conjunction with immunohistochemistry of many neurotransmitters and enzymes in colchicine-treated animals perfused with 4% paraformaldehyde in order to biochemically characterize the labeled pathways 34 35.

In this report we confirm and extend the above findings. We demonstrate that :

  • the sensitivity of CTb is further increased by using unlabeled streptavidin immunohistochemistry;
  • CTb can be iontophoretically applied, thus allowing us to determine the afferents to very small groups of cells without significant tissue damage;
  • CTb is not taken up, and retrogradely and anterogradely transported by intact fibers of passage;
  • CTb is a very efficient anterograde tracer allowing thE detailed morphology of axons and terminals to be revealed.

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