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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Materials and Methods

Eighty adult eats of both sexes weighing 2.5-4.0 kg were used. The animals were deeply anesthetized with pentobarbital (25 mg/kg, i.v.)

(A) Retrograde labeling with cholera toxin B subunit (CTb)

(1) Preparation of the tracers

For pressure injeetions, 1 mg of lyophilized cholera toxin B subunit (CTb, List Biological Lab) was diluted in 100 µl of distilled water to a final concentration of 1% in the original buffer containing 0.5 M Tris-HCI (pH 7.5), 2 M NaCl, 0.03 M NaCl and 0.01 M EDTA. For iontophoretic injections, it appeared necessary to replace the original buffer by a phosphate buffer (PB) at pH 6.0. For this purpose, 1 mg of lyophilized CTb was reconstituted with 1 ml of 0.1 M PB (pH 6.0) and then desalted, buffer exchanged and concentrated to 1% by two repeated 1 h 30 min ultrafiltrations (from 1 ml to 0.1 ml) at 7000 rpm with a centricon-10 microconcentrator (Amicon, U.S.A.).

(2) Pressure injections

0.05, 0.1 or 0.2 µl of 1% CTb was injected stereotaxically with a 5-µl Hamilton syringe into several brainstem nuclei such as the nuclei reticularis magnocellularis and parvicellularis, the nucleus paragigantocellularis lateralis, the nuclei raphe magnus, pallidus and dorsalis, the locus coeruleus complex, the pontine reticular formation, the trigeminal motor nucleus, the facial nucleus, the posterior and anterior hypothalamic areas. After injection of CTb over 10-15 min, the needle was left in place for about 15 min.

(3) Iontophoretic injections

Glass capillary tubes (1 mm o.d., Clark Electromedical Instruments) were heated, pulled and the tips cut back to 12-25 µm diameter under microscopic control. Using vacuum lines, these micropipettes were filled with the 1% solution of desalted CTb in 0.1 M PB (pH 6.0) just prior to the injections. The pipettes were then lowered stereotaxically into the regions under study, such as the nuclei raphe dorsalis, reticularis parvicellularis, paragigantocellularis lateralis and the posterior and anterior hypothalamic areas. A pulsed positive current (7 s on, 7 s off) was then applied using a Midgard CS-4 to avoid electrocoagulation. We tested the effect of different intensities of current (2,5 µA) as well as duration of injections (15, 30 min) on the diameter of the sites. At the end, the pipettes were left in place for 10-20 min to avoid leakage of the tracer along the pipette track.

(4) Colchicine treatment

In order to inject colchicine, two guide cannulae were implanted in the lateral and fourth ventricles just after the injection of CTb. Twenty-four hours later, the animals were treated with colchicine (200 ug in 20 µl of 0.9% saline for each ventricle) through the guide cannula by means of an injection cannula connected to a 50-µl Hamilton syringe. The animals were perfused 24 and 48 h after the colchicine administration. As controls, 10 cats did not receive colchicine treatment and were allowed to survive 24 h, 48 h, 10, 15 and 18 days after CTb pressure or iontophoretic applications.

(5) Perfusion and histology

The animals were deeply anesthetized and perfused through the ascending aorta, initially with one liter of Ringer's lactate solution containing 0.1% heparine, followed by 2.500 ml of an ice-cold fixative in 0.1 M PB (pH = 7.4). Four different perfusion regiments were tested:

(a) 2-4% paraformaldehyde (PF), 0-0.1% glutaraldehyde (GLU) and 0.2% picric acid (PA);
(b) 4% PF, 0.25% GLU and 0.2% PA;
(c) 4% PF, 0.1% GLU and 0.2% carbodiimide;
(d) 5% GLU and 0.2% PA.

After removal from the skull, the brains were cut into several blocks, and postfixed overnight at 4 °C in 0.1 M PB containing 2% PF and 0.2% PA (fixation a,b,c) or 2.5% GLU and 0.2% PA (fixation d). The blocks were then rinsed and cryoprotected by immersion in 0.1 M PB containing 30% sucrose for 48-72 h at 4 °C. Afterwards, these specimens were rapidly frozen with CO2 gas and coronal 20 µm thick sections were cut on a cryostat. In a few cases, vibratome sections 40-100 µm thick were obtained using a sliding microtome (Oxford Instruments). The free-floating sections were then incubated in CTb antiserum or stocked up to 4 years before staining m 0.1 M phosphate-buffered saline containing 0.3% Triton X-100 (PBST) and 0.1% sodium azide (PBST-AZ, pH = 7.4).

(B) Immunohistochemistry of CTb

Immunohistochemical detection of CTb was carried out by sequential incubations of free-floating sections using the modified technique of Hsu 22 or the classical PAP method. We used two different incubation times under gentle stirring in the goat CTb antiserum (List Biological Laboratories):

(1) a short incubation overnight at room temperature at 1:20 000 dilution in PBST in order to visualize the injection site the day after cutting;

(2) a long incubation over 3-4 days at 4 ° C in PBST-AZ with a 1:40 000 dilution for double labeling experiments.

After incubation in CTb antiserum, the free floating sections were rinsed 2 x 30 min in PBST and then successively incubated for 90 min at room temperature or overnight at 4 °C in the linking antibodies and the streptavidin-HRP, ABC-HRP complex or goat PAP using one of the following protocols:

(a) donkey anti-goat immunoglobulin (1:1,000, Jackson Immunoresearch Lab.) followed after 2 x 30 min rinses in PBST by goat PAP (1:1,000, Jackson Immunoresearch Lab.)

(b) biotinylated swine anti-goat immunoglobulin (1:1,000, Tago) followed after 2 x 30 min rinses in PBST by the ABC-HRP complex (1:500-1,000, Vector Lab.);

(c) biotinylated donkey anti-goat immunoglobulin (1:2.-4,000, Jackson Immunoresearch Lab.) followed after 2 x 30 min rinses in PBST by the ABC-HRP complex (1:500-1,000, Vector Lab.);

(d) biotinylated donkey anti-goat immunoglobulin (1:2-4,000, Jackson Immunoresearch Lab.) followed after 2 x 30 min rinses in PBST by streptavidin-HRP (1:40,000, Jackson Immunoresearch Lab.).

Finally, in all cases, after 2 x 30 min rinses in PBST, the free floating sections were immersed in 0.02% 3,3'-diaminobenzidine4HCl (DAB, Sigma) containing 0.003% H2O2 and 0.6% nickel ammonium sulfate in 0.05 M Tris-HCl buffer (pH = 7.6) for 10-15 min at room temperature. The reaction was terminated by extensive washes in PBST.

(C) Immunohistochemistry of neurotransmitters and their synthesing enzymes

To determine the histochemical nature of the retrogradely labeled cells, the CTb-pretreated sections were incubated for 4-6 days at 4°C in "rabbit" antiserum diluted in PBST-AZ to either:

- tyrosine hydroxylase (TH, 1:10,000, Institut Jacques Boy, France).
- serotonin (5-HT,1:20,000, a gift of Pr. Kimura, Japan).
- methionin-enkephalin (M-Enk, 1:5,000, UCB, Belgium).
- substance P (SP,1:5,000, Biogenex, U.S.A.).
- corticotropin-releasing factor (CRF, 1:5,000, UCB,Belgium).
- neurophysin (NP, 1:80,000, DAKO, Denmark).
- somatostatin (SRIF,1:5,000, DAKO, Denmark).
- neurotensin (NT, 1:40,000, Milab, Sweden).
- a-melanocyte stimulating hormone (a-MSH, 1:40,000, UCB, Belgium).
- cholecystokinin (CCK, 1:5,000, UCB, Belgium).
- galanin (GAL, 1:20,000, Peninsula, U.K.).
- choline-acetyltransferase (ChAT, 1:5,000, Chemicon, U.S.A.).
- u-amino acid decarboxylase (AADC, 1:20,000, a gift of Dr. Weber, France).

After 2 x 30 min washes in PBST, the sections were placed for 90 min at room temperature or overnight at 4 °C in swine (1:400, DAKO) or donkey (1:800, Jackson Immunoresearch Lab.) antirabbit immunoglobulin diluted in PBST. They were then rinsed 2 x 30 min in PBST and immersed in rabbit peroxidase-antiperoxidase (PAP, DAKO, Jackson Immunoresearch Lab.) diluted 1:400-1000 in PBST for 90 min at room temperature. After 2 x 30 min rinses in PBST, the sections were reacted with 0.025% DAB containing 0.006% H2O2 in 0.05 M Tris-HCI buffer for 15-30 min at room temperature.

Finally, the sections were mounted on gelatin-coated glass slides, dried, dehydrated and coverslipped with Depex.

The CTb reaction products obtained by DAB-nickel histochemical procedure consisted of black punctate granules in the cell soma and dendrites, whereas for the neuroactive substances the immunohistochemical reaction product revealed using DAB appeared as a homogeneous light-brown staining of the cell bodies.

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