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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This report is an attempt to demonstrate that unconjugated CTb is a versatile and sensitive retrograde tracer when recognized by immunohistochemistry. Any evaluation of the quality of a retrograde tracer must include at least the following considerations:

(1) the incorporation and retrograde transport by all cell types without transsynaptic labeling;

(2) the ability to reveal the neuron afferents to a small injection limited to the cell group of interest;

(3) the degree of uptake and transport by damaged and undamaged axons of passage;

(4) the compatibility with immunohistochemistry of neuroactive substances.

With regard to these considerations, using unconjugated CTb as a retrograde tracer, the following was demonstrated:

(1) in all pathways investigated, we never observed the absence of a well-known afferent projection, indicating that CTb is taken up and retrogradely transported aspecifically by all the neurons of the central nervous system;

(2) pressure and furthermore iontophoretic applications of a small volume of CTb give rise to small injection sites limited to cell groups of interest associated with the labeling of all the known afferent projections;

(3) CTb is taken up only by damaged fibers of passage, a drawback largely overcome by iontophoretic application with 20 µm micropipettes;

(4) the use of a 4% paraformaldehyde fixative, the absence of decrease of labeling after long survival and colchicine treatment, the easy and straightforward identification of double labeled neurons containing black granules contrasting well with the brown reaction product allowed us to combine immunocytochemical detection of CTb with the immunohistochemistry of multiple neuroactive substances to determine the histochemical nature of afferent projections.

Moreover, this technique has the advantage of the permanence of the preparation. Indeed, it is possible to stock sections before and after staining in PBST-A or mounted on slides for years without any loss of immunoreactivity. Moreover, camera lucida drawings can be easely made at low magnification to accurately localize the retrogradely labeled cells.

These results complete those obtained by us 14 15 34 36,58-60 and Ericson et al. 13. In agreement with these previous studies, we confirmed that CTb is a very sensitive retrograde tracer extensively filling the dendrites of the retrogradely labeled cells. In addition to these studies, we demonstrated that CTb is taken up and transported in damaged but not in intact fibers of passage, can be iontophoretically applied and that the sensitivity of the tracer is further enhanced when using streptavidin-HRP immunohistochemistry. It must also be noted that, in contrast with Ericson et al. 13, we found that the CTb immunoreactivity in the retrogradely labeled cells was granular rather than homogeneous even on vibratome sections and that we observed no regional difference in the intensity of the retrograde labeling.

It would be of interest to compare the sensitivity of unconjugated CTb recognized by immunohistochemistry with that of CTb-HRP recognized with TMB. However, it is not possible because the optimal fixative conditions are very different for these two systems of detection. Nevertheless, Shapiro and Miselis 49, after restricted injections of CTb-HRP in the area postrema, described an intense retrograde labeling with an extensive filling of the dendrites of the retrogradely labeled cells as well as an extensive anterograde labeling of fibers, indicating the high sensitivity of the TMB method. It remains to be determined whether CTb-HRP, like CTb, can be iontophoretically applied. In any case, the use of CTb-HRP, like WGA-HRP (see Introduction), will always be limited by the impossibility of efficiently combining it with the immunohistochemical detection of neuroactive substances.

To overcome the problems inherent to the direct histochemical detection of HRP, unconjugated WGA 32 as well as WGA-HRP coupled to gold particles 2 38 43 (WGA-apoHRP-gold) and biotin 50 have also been successfully used as retrograde tracers in combination with immunostaining of second antigens. However, we previously found that CTb is a much more sensitive retrograde tracer than WGA 34 35. Indeed, injections of 0.2 and 0.1 µl of WGA at the same concentration and volume as CTb in the same structures resulted in sites twice as large in diameter than those of CTb (approximately 2000 µm and 1500 µm respectively). Moreover, even with such larger sites, the number of retrogradely labeled cells in the same structures was twice as small 34 35. In addition, in the case of WGA, numerous heavily labeled cells and dendrites surrounded the site making it difficult to determine its extent and the proximate afferents (unpublished results). In contrast, the limits of the CTb injections sites were very sharp, thus making the study of local afferent connections possible. Taken together, these data show that CTb is a much more sensitive retrograde tracer than WGA in the CNS and this prompted us to develop the use of this tracer for studying pathways in the CNS of the cat 14 15 34-36 58-60.

In agreement with these results, Behzadi et al. 5 recently reported that CTb recognized by immunohistochemistry is a much more sensitive tracer in the CNS than WGA-HRP revealed by the TMB method. Indeed, these authors found that the injection sites were less sharply demarcated from the surrounding tissue and the number of retrogradely labeled neurons was markedly inferior when using WGA-HRP instead of CTb.

Comparing CT-HRP and WGA-HRP effectiveness as retrograde tracers in the peripheral nervous system, Wan et al. 57 found, after tongue injections of the same volume at the same concentration of these two conjugates, not only that the number of retrogradely labeled cells in the hypoglossal nucleus but also the extent of the site in the tongue were larger in the case of CT-HRP. In addition, Horikawa and Powell 21 reported that, after injections of CTb, WGA (recognized by immunohistochemistry) and their HRP conjugates (revealed by cobalt DAB) in the whiskerpad of rats, the largest number of retrogradely labeled neurons in the facial nucleus was observed with WGA followed by CTb and WGA-HRP.

Taken together, these findings suggest that CTb is a much more sensitive retrograde tracer than WGA and WGA-HRP in the central nervous system but not in the peripheral nervous system. The superior sensitivity of CTb over WGA and WGA-HRP in the CNS may be due to the presence of a greater number of receptors (monogangliosides) to CTb than WGA 52 (N-acetylglucosamine) at the level of the nerve terminals of the CNS, a property not shared by those of the peripheral nervous system.

It remains to be determined whether unconjugated CTb is also a more sensitive and versatile retrograde tracer than the conjugate WGA-Apo-HRP-gold. Indeed, the coupling of a gold particle to WGA-Apo-HRP transforms the characteristic of the tracer. In particular, the diameter of the injection sites is dramatically reduced when using this complex 2 38. The retrogradely labeled cells contain distinct round black deposits sharper than those obtained with CTb and therefore with a higher contrast against the brown deposit obtained after immunohistochemistry of neuroactive substances 2 38. However, the round deposits are not always restricted to the retrogradely labeled neurons, in particular around the site, thus making the study of local afferent connections and the identification of single- and double-labeled cells difficult (refs. 2, 38 and our unpublished results). Moreover, the complex cannot be iontophorized and the pressure injections with micropipettes and Hamilton syringes are often followed by a tissue necrosis and a leak along the track leading to sites with a cigar shape unsuitable for studying deep brain nuclei (refs. 37, 43 and our unpublished data).

The other class of retrograde tracers is composed of the fluorescent probes introduced by Kuypers et al. in 1977 28. The extensive use of these tracers in the last years is ascribable to a number of decisive advantages: they require minimal tissue processing, can be combined to demonstrate collateralizations of neurons 29 55 and coupled with immunohistochemistry to determine the neurotransmitter content of identified pathways 6 19 30 47 51 56. However, many fluorescent tracers are (a) inferior or equal in sensitivity to free HRP visualized with TMB 1 23, (b) retrogradely transported by damaged but also undamaged fibers of passage 1 23 30 47, (c) leak from retrogradely labeled neurons during the immunohistochemical procedure 19 47 51 56 and the microscope observations 24 and (d) they cannot be iotophorized 10. Even recently introduced Fluoro-Gold and fluorescent latex microspheres have same of these drawbacks. Fluoro-Gold is only as sensitive as WGA-HRP visualized with TMB 44 and therefore (see above and Behzadi et al. 5) much less sensitive than unconjugated CTb. In addition, FluoroGold is also subject to fading under microscope observations and after immunohistochemical procedure 25 44 48.

Rhodamine latex microspheres show little diffusion and consequently produce small sharply defined injection sites and in addition resist fading 26. However, in contrast with Fast blue, WGA and CTb, massive injections of rhodamine microspheres in whiskerpad of rats did not label any neurons in the facial nucleus, thus indicating the low sensitivity of this tracer 21.

These data indicate that CTb is a more versatile and sensitive retrograde tracer than WGA and the fluorescent ones.

Another decisive advantage of our technique is that CTb is also anterogradely transported allowing us to directly identify the efferents as well as the afferents of the region or nucleus studied. Indeed, we demonstrated that CTb is a very powerful anterograde tracer transported via a slow mechanism with an estimated rate of 6-8 mm/day. We further showed that when using strep tavidin-HRP immunohistochemistry, the morphology of the CTb labeled axons and axon terminals was distinctly visible including boutons en passant, fine collateral branches and various terminal specializations just like PHAL 16. One of the drawbacks of CTb is that the tracer is also visualized in lesioned axons, making the interpretation of the data delicate. However, this artefactual labeling is dramatically reduced by using iontophoresis. Nevertheless, other important PHAL advantages are the preferential transport in the anterograde direction and the labeling of fibers directly traced from the population of neurons that was completely filled by the tracer 16.

The only serious drawback of our technique may be that visualization of transported CTb requires the sandwiching of antibodies and streptavidin-HRP and therefore is time-consuming and expensive. But we feel the improvement to be worth the effort. Moreover, this apparent disadvantage can be turned to a decisive one. Indeed, although not addressed in the present study, it is possible to:

- use many different immunohistochemical techniques to reveal CTb, e.g. streptavidin or a second IgG conjugated to gold or a fluorochrome. Such procedures may be combined for example with immunofluorescence of neuroactive substances using two different fluorochromes;

- combine the use of CTb with that of WGA (using the chromogen DAB and DAB-nickel or two different fluorescent labeled IgG or streptavidin) or fluorescent tracers to determine the collaterals of neurons. We recently successfully used a combination of WGA and CTb recognized with DAB and DAB-nickel to identify the collaterals from the hypothalamus to the neurointermediate lobe of the hypophysis and the median eminence 60.

Finally, it is possible to make electrophysiological recordings from the injection pipette to define structures prior to injections.

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