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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(A) Injection sites

(1) General remarks

As illustrated in Fig. 1B and D showing adjacent sections stained either with the ABC-HRP complex (Fig. 1B) and streptavidin-HRP (Fig. 1D), the aspect of the pressure and iontophoretic injection sites depended on the reagents used for the third step of the immunohistochemical procedure. Indeed, when using the ABC complex, whatever the secondary biotinylatod antiserum to goat, the center of the site was darkly stained while the periphery only contained a moderate to small quantity of granular material. In contrast, using streptavidin-HRP in the same conditions, the center of the site was less intensively labeled while the periphery appeared darker, containing a high density of granular material including immunostained fibers emanating from the site in all directions (Fig. 1D). Despite these differences, the diameter of the site remained approximately identical with both systems of detection.

On the other hand, we observed that the diameter and the aspect of the sites did not significantly change with different times of survival (24 h, 48 h, 10, 15 and 18 days) or the addition of a 48-h colchicine treatment.

We determined also that injections of the same volume of CTb resulted in sites of slightly variable shapes and diameters, clearly depending on the concentration of cell bodies and the presence of blood vessels and fiber bundles in the injected region.

(2) Pressure injections

The CTb pressure injection sites obtained when using a Hamilton syringe were generally round or ovoid, less frequently with a droplet shape when the tracer slightly leaked along the needle track. The diameter of 0.2, 0.1 and 0.05 µl CTb injection sites was commonly in the range of 800-1200 um, 600-1000 µm and 600-800 µm respectively. Fig. 1A illustrates a representative 0.2 µl pressure injection site in the ventrolateral part of the periaqueductal gray.

To determine whether the tissue was damaged at the level of the site, we counterstained with Cresyl violet unlabeled sections adjacent to those immunostained for CTb. After a typical injection of 0.2 µl of CTb giving a site of 800-1200 µm in diameter, we always detected a 500-800 µm diameter lesion of cell bodies in the central core of the sites. At the center of this lesion, the fibers were also totally destroyed in an area exactly corresponding to the mechanical injury made by the 400 µm diameter needle of the Hamilton Syringe. Fig. 1C illustrates the extent of the tissue necrosis at the level of 0.2 µl site in the periaqueductal gray shown in Fig. 1B and D on a Cresyl violet-counterstained section.

(3) lontophoretic injections

First, after iontophoretic injections of CTb in the initial buffer (1% in 0.5 M Tris-HCI, pH 7.5), we found only a trace of CTb immunoreactivity at the level of the site and no retrograde labeling. In sharp contrast, iontophoretic application of a 1% desalted and buffer exchange solution of CTb in 0.1 M PB (pH 6.0) through micropipettes with a 12-25 µm tip diameter using 2 uA pulsed positive current for 15 and 30 min gave respectively CTb injection sites of 200-300 µm and 400-600 µm in diameter. Using the same CTb solution but with a 5 uA pulsed current for 15 and 30 min, we obtained injection sites of 300-400 µm and 600-800 µm in diameter respectively. The iontophoretic sites were generally round or ovoid with no leak of the tracer along the pipette track. Fig. 2A and B illustrates a typical iontophoretic injection site (2 uA, 30 min) in the nucleus reticularis parvicellularis (Pc) of the medulla oblongata. A larger site (800 µm in diameter) in the nucleus raphe dorsalis obtained after a 60-min injection time with a 2 uA current parameter is shown in Fig. 3A.

To determine whether tissue damage occurred at the level of the sites in the Pc, we made a serotonin immunoreaction and then counterstained with Cresyl violet sections adjacent to those revealed by CTb. The 5 uA current produced an electrolytic lesion of 100-150 µm diameter at the center of the injection site while, as shown in Fig. 2D, with 2 uA current we hardly detected the 20 µm trace of the micropipette track. Moreover, we observed no visible destruction of serotonin fibers nor of any counterstained cell bodies despite their large number in the site.

Finally, it must be stated that the current of 5 uA but not 2 uA caused severe instability of the impedance of the CTb-filled micropipettes.

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