L .K. Schoenfeld et al. Brain Research 882 2000 180 –190
181
PACAP has also been shown to have a negative chronot- peptide CGRP were examined with rabbit polyclonal
ropic action on the heart via the activation of the intracar- antibodies Peninsula Laboratories at concentrations of
diac parasympathetic postganglionic neurons [6,23]. Re- 1:500. Tyrosine hydroxylase TH was examined with a
cent studies have provided evidence that PACAP acts mouse
monoclonal antibody
1:50, developed
by directly on guinea pig intracardiac parasympathetic neu-
LeDourain and Ziller and obtained from the Developmen- rons to produce an increase in neuronal excitability [1].
tal Studies Hybridoma Bank at the University of Iowa. Although evidence for the involvement of PACAP in
Tissue was incubated in primary antibody solutions for autonomic regulation in mammals is increasing, the origin
12–20 h at 48C. Following incubation in the primary and function of PACAP-containing fibers in the ANS of
antibodies, the tissue was washed in PBS and incubated for other species is less well studied. The present study was
90 min at 20–228C with fluorescently-tagged species undertaken to explore the origins of PACAP in the ANS,
specific secondary antibodies donkey anti-guinea pig-in- specifically the parasympathetic cardiac ganglion, of an
docarbocyanine Cy3, donkey anti-rabbit-fluorescein iso- amphibian system.
thiocyanate FITC, or donkey anti-mouse-FITC, all at The mudpuppy Necturus maculosus
cardiac ganglion 1:400, Jackson ImmunoResearch Laboratories. The tissue
is well characterized, both anatomically and physiological- was then washed and mounted on gelatin-coated slides and
ly [15,21]. The source and actions of many of the coverslipped with Citifluor Marivac Ltd., Halifax NS,
neuropeptides affecting parasympathetic postganglionic Canada. For preabsorption studies, the primary antibodies
neuron activity have been well described [21], and com- were incubated with 5 mM of either PACAP-38 or PACAP-
plimentary mammalian studies suggest that many of these 27 peptide Peninsula Laboratories for 12–20 h at 48C
peptidergic modulators are conserved across species. Thus, prior to the addition of the tissue.
additional information concerning the localization of For immunohistochemical analysis of tissue sections, the
PACAP within the ANS of these amphibians will add to animals were initially anesthetized in 0.1 MS222 Sigma
our understanding of neuropeptide modulation of cardiac Chemical Co., St. Louis, MO and then perfused through
function across species. the conus arteriosus with 150 ml of ice cold calcium-free
saline solution, followed by 150 ml of 4 paraformal- dehyde solution. Brain, dorsal root ganglia from the
2. Materials and methods cervical and high thoracic T1–T3 regions, and ganglia of
the X cranial nerve nodose ganglia were removed and 2.1. General methods
postfixed in 4 paraformaldehyde for 2–12 h at 48C. The tissue was cryoprotected in 30 sucrose, embedded in
All experiments were performed on tissue isolated from OCT Tissue Tek, and either used immediately or stored
mudpuppies in accordance with procedures reviewed and at 2808C until sectioning. Tissue sections 16 mm were
approved by the Institutional Animal Care and Use Com- mounted onto gelatin-coated slides, washed with PBS, and
mittee. Animals were obtained from commercial suppliers incubated in PBS with 0.03 Triton X-100 and 1 goat
Lembergers, Oshkosh, WI and maintained in aquaria at serum at room temperature. Sections were then incubated
10–158C. For most experiments, animals were euthanized with primary antibodies 1:800–1:1000 for 12–20 h at
by brainstem transection and pithing. The parasympathetic 48C. Sections were washed and incubated with fluorescent-
cardiac ganglion, which lies in a connective tissue sheath ly labeled species-specific secondary antibodies 1:500 for
adjacent to the heart, was removed as previously described 60–90 min at room temperature, washed, and coverslipped
[15] and pinned in a Sylgard-lined petri dish containing a with Citifluor. Immunofluorescence was viewed with an
HEPES-buffered saline solution 120 mM NaCl, 2.5 mM Olympus BX50 fluorescent microscope. Slides of images
KCl, 3.6 mM CaCl , 5.0 mM HEPES, pH 7.3. The tissue were scanned into Adobe Photoshop and minimally cor-
2
was dissected to remove overlying connective tissue and rected for brightness and contrast.
fixed in 4 paraformaldehyde in 0.1 M Millonigs buffer, pH 7.2 overnight at 48C.
2.3. Organ culture and vagotomies 2.2. Immunohistochemistry
For organ explants, whole mount preparations contain- ing the cardiac ganglion were removed from the animal
For whole mount preparations, following fixation, the and pinned in sterile saline solution. The saline was
tissue was washed in 0.1 M phosphate buffered saline replaced with L-15 growth medium Sigma Chemical Co.
solution PBS and then incubated in PBS containing 0.5 containing 7 fetal bovine serum and penicillin strep-
Triton X-100 and 4 goat serum. PACAP immunoreactivi- tomycin 200 units ml, Sigma Chemical Co.. The ex-
ty was examined with a guinea pig polyclonal antibody to plants were maintained at 18–208C for 14 days, and the
either PACAP-38 or PACAP-27 Peninsula Laboratories at medium changed daily. Following culture, the whole
a concentration of 1:500 and 1:100, respectively. Sub- mount preparations were fixed and processed for immuno-
stance P SP, galanin GAL, and calcitonin gene-related histochemistry as previously described.
182 L
For vagotomies, animals were anesthetized in 0.1 tions n55, substance P-like immunoreactivity SP-ir,
MS222. Bilateral, dorsal incisions were made to expose the Fig. 2. In the mudpuppy, both SP and CGRP are thought
vagus nerve, and a 5–10 mm segment was removed on to be derived from sensory afferent ganglia [19,21]. In
each side [22]. For sham surgeries, the vagus nerve was addition, SP-ir is also found in a subpopulation of small
visualized but not disturbed, and the wound was sutured. intensely fluorescent SIF cells within the cardiac ganglia
All animals both experimental and control were main- [18] Fig. 2D, arrow. However, no PACAP-ir was ob-
tained in aquaria at room temperature for 14–21 days served in any SIF cells. The co-localization of PACAP
following the surgery. The animals were then euthanized with SP in nerve fibers was not absolute. In addition to
and the tissue removed for immunohistochemistry. In fibers which showed immunoreactivity to both antibodies,
addition, the surgical site was examined to insure that the there were also a population of fibers which stained
vagus nerve remained lesioned. positively only for SP. There were also sporadic examples
of fibers which demonstrated immunoreactivity only for 2.4. Colchicine treatments
PACAP Fig. 2C,D. The same patterns were observed for PACAP and CGRP, with some fibers showing evidence of
To insure that sufficient peptide for immunohistochemi- co-localization, some fibers labeling only with CGRP Fig.
cal detection was present in cell somas within the brain, 2A,B, and a very small number labeling only with
three animals were injected with colchicine 0.5 mg in 10 PACAP data not shown.
ml of saline solution intraventricularly as previously Galanin has been found in the cell somas of both the
described [13]. Prior to the colchicine injection, animals parasympathetic postganglionic neurons of the mudpuppy
were first anesthetized in 0.1 MS222, injected with cardiac ganglion, as well as a subpopulation of SIF cells
colchicine solution, and then maintained in 10–158C [14,20]. In four preparations labeled with antibodies to
aquaria for 3 days prior to euthanasia and tissue collection both galanin and PACAP, there was very little evidence of
as described above. co-localization of the two peptides Fig. 3. GAL-ir fibers
could be observed coursing through areas outside the ganglia itself, presumably innervating muscle fibers in
3. Results these regions Fig. 3, however, there was no evidence of
