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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
PACAP-ir fibers in these regions. In addition, galanin 3.1. PACAP immunoreactivity in the mudpuppy cardiac
labeled both postganglionic neurons and SIF cells, with no ganglion
evidence for PACAP-ir in these cells. A few, sporadic examples of fibers that appeared to label for galanin and
PACAP-immunoreactivity PACAP-ir was examined PACAP were observed Fig. 3. However, the majority of
using antibodies to either the long form of PACAP, galanin- and PACAP-immunoreactivity did not show co-
PACAP-38, or to the truncated form, PACAP-27. PACAP- localization.
ir was observed with the PACAP-38-specific antibody in Previous studies found PACAP in mammalian sympa-
nerve fibers coursing throughout the ganglia, as well is in thetic preganglionic fibers and postganglionic cells bodies
pericellular complexes surrounding postganglionic neurons within sympathetic ganglia [4,9]. To determine if PACAP
Fig. 1. The PACAP-27 antibody showed no specific was found in sympathetic postganglionic fibers within the
staining in three separate preparations, even at higher cardiac ganglia of the mudpuppy, we examined the degree
concentrations Fig. 1. No evidence of cell bodies with of co-localization of PACAP with tyrosine hydroxylase
PACAP-ir to either antibody was observed in any of the TH, a marker for catecholaminergic fibers. In three
preparations. The specificity of the PACAP-38 antibody in different preparations, there was no evidence of co-locali-
the mudpuppy was examined by pre-absorption with either zation of PACAP-ir fibers with TH-immunoreactivity data
5 mM PACAP-38 peptide or 5 mM PACAP-27 peptide not shown. TH-immunoreactive fibers could be seen
n53 for each condition. Pre-absorption with PACAP-38 primarily in close proximity to contractile fibers in the
peptide eliminated all specific labeling, whereas pre-ab- tissue adjacent to the ganglion, with little TH-immuno-
sorption with PACAP-27 peptide did not alter the immuno- reactivity in or around the parasympathetic postganglionic
reactivity to the PACAP-38 antibody data not shown. neurons.
3.2. Co-localization of PACAP with SP, CGRP, Galanin, 3.3. Effects of total or selective axon lesions on
and Tyrosine Hydroxylase PACAP-ir
Previous studies in the mudpuppy cardiac ganglion had The results of the initial immunohistochemical studies
described several different neuropeptide inputs to this suggested that PACAP was derived from extrinsic sources.
ganglia [21]. PACAP-ir co-localized in fibers that also This hypothesis was tested by examining PACAP-ir in
contained calcitonin gene-related peptide CGRP-im- cardiac ganglion maintained in organ culture for 14 days.
munoreactivity CGRP-ir, n54 and, in separate prepara- Whole mount explants from three animals were maintained
L .K. Schoenfeld et al. Brain Research 882 2000 180 –190
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Fig. 1. PACAP immunoreactivity in the mudpuppy cardiac ganglion. Whole mount preparations containing the cardiac ganglion were labeled with antibodies to either PACAP-38 at a concentration of 1:500 or to PACAP-27 at a concentration of 1:100. PACAP-38-immunoreactivity was observed in
all preparations tested A, whereas no evidence for specific labeling with the PACAP-27 antibody was observed in three additional preparations B. PACAP-38-ir was observed in nerve fibers and pericellular complexes, but not in cells within the preparations. Calibration bar550 mm.
for 14 days at room temperature, and then examined for sources and further experiments were designed to examine
PACAP-38 and CGRP immunoreactivity. PACAP-ir in the one input to the cardiac ganglia, the vagus nerve.
cardiac ganglion was greatly reduced following prolonged Studies in mammals found evidence for PACAP in the
culture Fig. 4A, and the few PACAP-positive fibers that nodose ganglia as well as the dorsal motor nucleus of the
were observed in one preparation showed signs of degene- vagus [3,10,17]. Thus, one source of the PACAP-con-
ration. A similar pattern of reduced immunoreactivity and taining nerve fibers in the cardiac ganglia could be the
degeneration was observed in CGRP staining in these vagus nerve. This possibility was explored in the mud-
preparations as well data not shown. These results puppy by examining PACAP-ir in cardiac ganglia follow-
suggested that PACAP was derived primarily from external ing bilateral vagotomies. A 5–10 mm segment of the
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Fig. 2. Co-localization of PACAP with CGRP and SP. Cardiac ganglia were labeled with PACAP-38 antibody 1:500 and either anti-CGRP 1:500 or anti-SP 1:500. Co-localization of immunoreactivity was observed in numerous nerve fibers for both CGRP A and B and SP C and D. In some cases,
fibers stained for only one of the antibodies. Panel B arrow shows a CGRP-positive nerve fiber which did not stain for PACAP, whereas Panel C arrowhead shows a PACAP-positive fiber which did not stain with SP antibody. SP-immunoreactive SIF cells can also be observed in Panel D arrow
which do not label with the PACAP antibody. The intense staining within the neurons in panels C and D is due to the autofluorescence of pigmented granules commonly observed in these preparations. Calibration bar550 mm.
vagus nerve was removed bilaterally from the animals, the preparations, there was also evidence of non-degenerated
wound was sutured, and the animals were allowed to PACAP-ir nerve fibers to some regions of the cardiac
recover for 14–21 days. Previously, Roper [22] showed ganglion in these animals Fig. 4D, suggesting that not all
that a time period of 14 days was sufficient to destroy all PACAP-ir fibers were affected by the surgery.
physiological responses to stimulation of the vagal trunk in the mudpuppy cardiac ganglia.
3.4. PACAP-ir in dorsal root ganglia, vagal sensory PACAP-ir showed a marked reduction in staining at 14
ganglia , and brainstem
days post-surgery n53 compared to the control sham surgery, n53 animals Fig. 4. However, some PACAP-ir
Based on the results of our co-localization and vag- fibers were still observed in the cardiac ganglion from
otomy studies, we hypothesized that the PACAP-contain- vagotomized animals. To insure that we had allowed
ing nerve fibers were originating from extrinsic sources, sufficient time for nerve fiber degeneration, three animals
such as primary afferent neurons and vagal afferent or were examined at 21 days post-vagotomy. Although the
efferent centers. Dorsal root ganglia DRG were isolated amount of PACAP-ir was still reduced compared to control
from fixation-perfused animals and cryostat sectioned. The
L .K. Schoenfeld et al. Brain Research 882 2000 180 –190
185
Fig. 3. Immunoreactivity for PACAP-38 and galanin in the cardiac ganglion. Preparations were labeled with the antibody to PACAP-38 1:500 and an antibody to galanin 1:500. Galanin immunoreactivity could be observed in fibers innervating contractile tissue outside the ganglia itself D and within
the postganglionic neurons and SIF cells B, arrow. Conversely, no PACAP immunoreactivity was observed in the fibers innervating the contractile tissue C or in the cells A. An occasional fiber was seen that appeared to show positive staining for both galanin and PACAP A, arrow. However, there was
relatively little evidence of co-localization of galanin and PACAP-38 in the majority of fibers in 4 different preparations. Calibration bar550 mm.
sections 16 mm were then labeled with PACAP-38 centrations within neuronal cell somas for immunohistoch-
antibody and either SP or CGRP antibody for immuno- emical detection. Sections from the vagal sensory ganglia,
histochemistry. PACAP-ir cell somas and nerve fibers were which contain cell bodies for vagal afferent neurons,
observed in the DRG Fig. 5. Virtually all of the PACAP- showed PACAP-ir cell somas and nerve fibers Fig. 6 in
ir positive neurons were also immunoreactive for either SP both untreated and colchicine-treated tissue. No evidence
or CGRP Fig. 5. However, not all SP or CGRP-ir cells of either CGRP or SP-ir was observed in these same tissue
were also positive for PACAP. In addition, PACAP-ir nerve sections data not shown. Caudal sections 16 mm of the
fibers were observed entering the dorsal horn in sections of brainstem ranging from the most caudal medulla to the
the cervical spinal cord Fig. 7. level of the VII cranial nerve root were also examined for
To examine the potential sources of PACAP in the vagus PACAP-ir. Both sagittal and horizontal sections showed
nerve, afferent and efferent centers were examined in both PACAP-ir fibers in the caudal brainstem which appeared to
untreated n54 and colchicine-treated n53 animals. innervate the region of the nucleus solitarius Fig. 7.
Colchicine treatment was used to increase peptide con- However, no PACAP-ir cell bodies were observed in any
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Fig. 4. Explant and vagotomy reduces PACAP immunoreactivity. Cardiac ganglia were maintained in organ culture for 14 days at room temperature prior to staining for PACAP-38 immunoreactivity Panel A. Ganglion explants showed a dramatic reduction in PACAP-ir. Specific lesioning of the vagus nerve
also reduced the amount of PACAP-ir in the cardiac ganglion Panel C as compared to sham-operated controls Panels B. Increasing the post-operative recovery time to 21 days showed an overall decrease in PACAP-ir, but selective regions still showed intact fibers and pericellular complexes Panel D.
Calibration bar550 mm.
brainstem sections, including the region originally iden- sequence [7]. Such evolutionary conservation would sug-
tified by Herrick [5] as the dorsal motor nucleus, in either gest that this peptide may perform a vital functions across
the control or the colchicine-treated tissue Fig. 7. species. The exact nature of this functions, though, still
remains largely unknown. However, increasing evidence from other investigators and the present study suggests that
4. Discussion one of the roles of PACAP may be in the regulation of
