Material and methods
I. Retrograde Tracing experiments
Male Sprague-Dawley rats (Taconic Farms Inc. or IFFA Credo) weighing 250-350g were anesthetized with chloral hydrate (400 mg/kg, intraperitoneal) or Nembutal (60 mg/kg, intraperitoneal) and placed in a stereotaxic apparatus. A scalp incision was made, a hole was drilled in the skull overlying the LC, and the dura was reflected. The skull was placed at a 12ºangle (nose tilted down) to avoid rupturing the overlying transerse sinus; coordinates were: 3.7 mm caudal to real lambda, 1.2 mm lateral to midline and 6.0 mm ventral from skull.
A) Preparation of cholera-toxin B
To obtain reliable iontophoretic injections, it was necessary to replace the original buffer lyophilized with CTb by a phosphate buffer (PB) at pH 6.0 41. For this purpose, 1mg of lyophilized CTb was reconstituted with 1ml of 0.1M PB (pH 6.0) and then desalted, buffer exchanged and concentrated to 1% by two repeated 1h 30 ultrafiltrations (from 1ml to 0.1ml) at 7000 rpm with a centricon-10 microconcentrator (Amicon, U.S.A).
B) Cholera-toxin B injection protocol
Glass capillary tubes (1.5 or 1mm O.D.) were heated, pulled and the tips broken to 3-5 µm diameter under microscopic control. These micropipettes were backfilled with the 1% solution of CTb in 0.1M PB (pH 6.0). Electrophysiological recordings from these pipettes with a good signal to noise ratio aided in localizing LC by its distinctive spontaneous or foot pinch-evoked discharge characteristics 5*. To eject CTb, a pulsed positive current (4 or 7s on, 4 or 7s off, 0.5-2µA, 5-15 min) was applied (Midgard CS-4 or Finntronics constant current source) to avoid heating or clogging the tip. At the end, the pipettes were left in place for 10-15 min to prevent leakage of the tracer along the pipette track. Animals were allowed to survive 1-7 days before perfusion. Using different injection times and currents we obtained either large (n = 6, 1-2µA, 5-15 min) or small ( n = 8, 0.5µA, 5-15 min) injections centered in the LC. Control iontophoretic injections (0.5µA, 10 min) were also made in nuclei adjacent to LC including Barrington's nucleus (Bar, n= 3), mesencephalic trigeminal nucleus (5Me, n = 2), the peri-5Me nucleus (n=3) and the medial vestibular nucleus (MVe, n = 1).
The animals were deeply anesthetized and perfused through the ascending aorta, initially with 200 ml of Ringer's lactate solution with 0.1% heparine, followed by 1,000 ml of an ice-cold fixative in 0.1M PB (pH 7.4) containing 4% paraformaldehyde (PF), 0-0.25% glutaraldehyde and 0.2% picric acid (PA). After removal from the skull, the brains were postfixed overnight at 4 °C in 0.1 M PB containing 2% PF and 0.2% PA. The brains were then rinsed and cryoprotected by immersion in 0.1 M PB containing 30% sucrose for 48-72 hours at 4°C. Afterwards, these specimens were rapidly frozen with CO2 gas and coronal 20-25 µm thick sections were cut on a cryostat. The free-floating sections were then incubated in CTb antiserum or stocked before staining in 0.1 M PB saline (NaCl, 0.9%) containing 0.3% Triton X-100 (PBST) and 0.1% sodium azide (PBST-AZ, pH = 7.4).
D) Immunohistochemistry of cholera-toxin B
Immunohistochemical detection of CTb was carried out by sequential incubations of free-floating sections according to Hsu et al. 31, method slightly modified 41. The sections were first submitted to a long incubation over 3-4 days at 4 ºC in PBST-AZ with the "goat" CTb antiserum (List Biological Laboratories) at a 1:40,000 dilution, with gentle stirring. Then, they were rinsed 2 x 30 minutes in PBST and incubated for 90 min at room temperature or overnight at 4°C in the biotinylated "donkey" anti-goat immunoglobulin (1:2,000 in PBST, Jackson Immunoresearch Lab) followed after 2 x 30 min rinses in PBST by streptavidin-HRP (1:40,000 in PBST, Jackson Immunoresearch Lab). Finally, after 2 x 30 min rinses in PBST, the sections were immersed in 0.02% 3,3'-diaminobenzidine-4HCl (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 two washes in PBST-AZ. Finally, the sections were mounted on gelatin-coated glass slides, dried, dehydrated and coverslipped with Depex or Permount.
II. Anterograde tracing experiments
A) Injection protocol
Phaseolus vulgaris-leucoagglutinin (PHAL) was injected in certain structures found to contain cells retrogradely labeled from the LC and, in most cases, CTb was also injected contralaterally in the same structure. A stereotaxic surgical method similar to the retrograde experiments was used, but the injections were made with a flat skull and the aid of brain surface landmarks for stereotaxic orientation. PHA-L (Vector lab., 2.5% in 0.01 M PBS) was iontophoretically injected 24. Cellular recordings through the injection pipette (10-20 µm tips) aided in localizing target sites. Injections were made with 5 µA of pulsed current (4 or 7 seconds on, 4 or 7 seconds off) for 30 min. For CTb, iontophoretic injections were made thru 3-5µm micropipettes using a 2µA pulsed positive current for 15-30 min. Animals survived for 7-15 days and were then deeply anesthetized and perfused. The brains were then posfixed and cut into frontal sections as described above for the retrograde experiments. Injections were made in the infralimbic cortex (PHAL, n = 1), the area 1 of the frontal cortex and the adjacent hindlimb region of the primary somatosensory area (PHAL, n = 2), the preoptic area located dorsal to the supraoptic nucleus (CTb, n = 2, PHAL, n = 2), the dorsal hypothalamic area (CTb, n = 2, PHAL, n = 2), the perifornical area (CTb, n = 2, PHAL, n = 2), the lateral hypothalamic area laterodorsal to the fornix (CTb, n = 2, PHAL, n = 2), the lateral hypothalamic area dorso-medial to the subthalamic nucleus (CTb, n=1, PHAL, n=1), the mesencephalic reticular formation (B9 serotoninergic group) (PHAL, n = 2), the ventrolateral part of the periaqueductal gray (CTb, n = 2, PHAL, n = 3), the nucleus raphe dorsalis (CTb, n = 3), the laterodorsal tegmental nucleus of Castaldi (CTb, n = 3), the nucleus Kölliker-Fuse (CTb, n = 1, PHAL, n = 1), the nucleus raphe magnus (CTb, n = 5) and the lateral paragigantocellular nucleus (CTb, n=1, PHAL, n= 4).
B) Immunohistochemical procedures
The immunohistochemical detection of CTb was carried out as described above for the retrograde experiments. For PHAL immunohistochemistry, the sections were first submitted to a long incubation over 3-4 days at 4 °C in PBST-AZ with a "rabbit" PHA-L antiserum (DAKO) at a 1:5,000 dilution, with gentle stirring. Then, they were rinsed 2 x 30 min. in PBST and incubated for 90 min. at room temperature or overnight at 4 °C in the biotinylated "donkey" anti-rabbit immunoglobulin (1:2,000, Jackson Immunoresearch Lab.) followed after 2 x 30 min rinses in PBST by streptavidin-HRP (1:40,000, Jackson Immunoresearch Lab.). Finally, as for CTb staining, after 2 x 30 min rinses in PBST, the sections were immersed in 0.02% 3,3'-diaminobenzidine-4HCl (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-AZ. For double labeled sections, CTb-stained sections were incubated for 4 days at 4°C in rabbit antiserum to tyrosine hydroxylase (TH, 1:10,000, Institut Jacques Boy). After washes, the sections were placed sequentially for 90 min at room temperature in donkey anti-rabbit IgG (1:400, DAKO) and rabbit peroxidase-antiperoxidase (PAP, 1:400, DAKO). Sections were then reacted with 0.025% DAB containing 0.006% H2O2 in Tris-HCl buffer for 15-30 min. Following this procedure, the CTb or PHAL anterogradely labeled fibers were blue-black, whereas the cytoplasm of noradrenergic neurons were brown (Fig. 21). All sections were then mounted on gelatin coated glass slides, dried, dehydrated and coverslipped with Depex.