Brain Research 882 (2000) 180–190www.elsevier.com/ locate /bres

Research report

Pituitary adenylate cyclase-activating polypeptide innervation of themudpuppy cardiac ganglion

b a a ,*Linda K. Schoenfeld , Jennifer A. Souder , Jean C. HardwickaBiology Department, Ithaca College, Ithaca, NY 14850, USA

bNeuroscience Department, Case Western Reserve University, Cleveland, OH 44106, USA

Accepted 22 August 2000

Abstract

The presence and potential origin of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) was determined incardiac ganglia of the mudpuppy, Necturus maculosus. Although PACAP has been implicated in the regulation of cardiac function inseveral mammalian species, the presence of this peptide in the autonomic nervous system (ANS) of other species is unclear. Thus, thisstudy is the first to characterize this highly conserved peptide in the ANS of a non-mammalian species. PACAP-immunoreactivity wasobserved in nerve fibers throughout the mudpuppy cardiac ganglia and often was co-localized with the sensory neuropeptides substance Pand calcitonin gene-related peptide. Removal of all extrinsic inputs to the ganglia by organ culture eliminated PACAP-immunoreactivityin the cardiac ganglia, whereas bilateral vagotomies only partially reduced PACAP-labeling. PACAP-immunoreactive neurons wereobserved in both high thoracic dorsal root ganglia and in vagal sensory ganglia. While no PACAP-positive neurons were observed incaudal medulla brainstem regions, PACAP-containing nerve fibers were found in the region of the nucleus solitarius. These results suggestthat, in the mudpuppy, PACAP is found primarily in visceral afferent fibers, originating from cells in either the dorsal root ganglia orvagal sensory ganglia. Based on their anatomic localization, these afferent fibers may function to transmit important sensory informationto cardiovascular centers in the brain as well as serving as local reflex inputs to modulate postganglionic parasympathetic output withinthe cardiac ganglion itself. 2000 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Peptides: anatomy and physiology

Keywords: PACAP; Parasympathetic; Afferent; Brainstem; Immunohistochemistry

1. Introduction the superior cervical ganglion [9], and the ciliary ganglion[4] as well as parasympathetic ganglia such as the otic and

Pituitary adenylate cyclase-activating polypeptide sphenopalatine ganglia [17] and in cardiac ganglia [1].(PACAP), originally isolated from ovine hypothalamus PACAP-containing cell bodies have also been found in[8,16], is a member of the VIP/secretin /glucagon family of neurons of the dorsal root ganglia and the nodose gangliapeptides. Following its initial discovery in the brain, in rats [3,17], implying a role for the peptide in visceralPACAP has subsequently been localized in fibers of both afferent systems. PACAP has also been found in the centralthe sympathetic and parasympathetic branches of the nervous system of several non-mammalian species, includ-autonomic nervous system [1,3,4,9,10,17]. PACAP is ing amphibians [11,24] and teleosts [12]. In these studies,found in two forms, a 38 amino acid peptide (PACAP-38) PACAP peptides and/or receptors were found in a varietyor the C-terminally truncated 27 amino acid form of brain regions, suggesting multiple functional roles for(PACAP-27). In mammals, PACAP peptides have been this neuropeptide [11,12,24].found in fibers and neurons of sympathetic ganglia, such as The physiological function of PACAP in the autonomic

nervous system (ANS) in mammals is still largely un-known, although investigators have found evidence that*Corresponding author. Tel.: 11-607-274-3213; fax: 11-607-274-PACAP can alter cardiovascular functions such as changes1131.

E-mail address: jhardwick@ithaca.edu (J.C. Hardwick). in systemic blood pressure and cardiac contractility [2].

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02885-7

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PACAP has also been shown to have a negative chronot- peptide (CGRP) were examined with rabbit polyclonalropic action on the heart via the activation of the intracar- antibodies (Peninsula Laboratories) at concentrations ofdiac parasympathetic postganglionic neurons [6,23]. Re- 1:500. Tyrosine hydroxylase (TH) was examined with acent studies have provided evidence that PACAP acts mouse monoclonal antibody (1:50), developed bydirectly 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 forautonomic regulation in mammals is increasing, the origin 12–20 h at 48C. Following incubation in the primaryand function of PACAP-containing fibers in the ANS of antibodies, the tissue was washed in PBS and incubated forother species is less well studied. The present study was 90 min at 20–228C with fluorescently-tagged speciesundertaken 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 tissueis well characterized, both anatomically and physiological- was then washed and mounted on gelatin-coated slides andly [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 antibodiesneuron 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 48Cpeptidergic 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, thePACAP within the ANS of these amphibians will add to animals were initially anesthetized in 0.1% MS222 (Sigmaour understanding of neuropeptide modulation of cardiac Chemical Co., St. Louis, MO) and then perfused throughfunction 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 ofthe X cranial nerve (nodose ganglia) were removed and

2.1. General methods postfixed in 4% paraformaldehyde for 2–12 h at 48C. Thetissue was cryoprotected in 30% sucrose, embedded in

All experiments were performed on tissue isolated from OCT (Tissue Tek), and either used immediately or storedmudpuppies in accordance with procedures reviewed and at 2808C until sectioning. Tissue sections (16 mm) wereapproved by the Institutional Animal Care and Use Com- mounted onto gelatin-coated slides, washed with PBS, andmittee. 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 incubated10–158C. For most experiments, animals were euthanized with primary antibodies (1:800–1:1000) for 12–20 h atby 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) foradjacent 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 anHEPES-buffered saline solution (120 mM NaCl, 2.5 mM Olympus BX50 fluorescent microscope. Slides of imagesKCl, 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 wastissue 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 theeither PACAP-38 or PACAP-27 (Peninsula Laboratories) at medium changed daily. Following culture, the wholea 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.

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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 thoughtvagus nerve, and a 5–10 mm segment was removed on to be derived from sensory afferent ganglia [19,21]. Ineach side [22]. For sham surgeries, the vagus nerve was addition, SP-ir is also found in a subpopulation of smallvisualized but not disturbed, and the wound was sutured. intensely fluorescent (SIF) cells within the cardiac gangliaAll 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 PACAPfollowing the surgery. The animals were then euthanized with SP in nerve fibers was not absolute. In addition toand 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 stainedvagus nerve remained lesioned. positively only for SP. There were also sporadic examples

of fibers which demonstrated immunoreactivity only for2.4. Colchicine treatments PACAP (Fig. 2C,D). The same patterns were observed for

PACAP and CGRP, with some fibers showing evidence ofTo 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 withthree 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 thedescribed [13]. Prior to the colchicine injection, animals parasympathetic postganglionic neurons of the mudpuppywere first anesthetized in 0.1% MS222, injected with cardiac ganglion, as well as a subpopulation of SIF cellscolchicine solution, and then maintained in 10–158C [14,20]. In four preparations labeled with antibodies toaquaria for 3 days prior to euthanasia and tissue collection both galanin and PACAP, there was very little evidence ofas described above. co-localization of the two peptides (Fig. 3). GAL-ir fibers

could be observed coursing through areas outside theganglia itself, presumably innervating muscle fibers in

3. Results these regions (Fig. 3), however, there was no evidence ofPACAP-ir fibers in these regions. In addition, galanin

3.1. PACAP immunoreactivity in the mudpuppy cardiac labeled both postganglionic neurons and SIF cells, with noganglion evidence for PACAP-ir in these cells. A few, sporadic

examples of fibers that appeared to label for galanin andPACAP-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 bodiespericellular 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 thestaining in three separate preparations, even at higher cardiac ganglia of the mudpuppy, we examined the degreeconcentrations (Fig. 1). No evidence of cell bodies with of co-localization of PACAP with tyrosine hydroxylasePACAP-ir to either antibody was observed in any of the (TH), a marker for catecholaminergic fibers. In threepreparations. 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 (data5 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 thepeptide 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 postganglionicreactivity 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 onand Tyrosine Hydroxylase PACAP-ir

Previous studies in the mudpuppy cardiac ganglion had The results of the initial immunohistochemical studiesdescribed 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 incontained 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

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Fig. 1. PACAP immunoreactivity in the mudpuppy cardiac ganglion. Whole mount preparations containing the cardiac ganglion were labeled withantibodies 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 inall 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 examinePACAP-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 theculture (Fig. 4A), and the few PACAP-positive fibers that nodose ganglia as well as the dorsal motor nucleus of thewere 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 thedegeneration 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) oranti-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 pigmentedgranules commonly observed in these preparations. Calibration bar550 mm.

vagus nerve was removed bilaterally from the animals, the preparations, there was also evidence of non-degeneratedwound was sutured, and the animals were allowed to PACAP-ir nerve fibers to some regions of the cardiacrecover for 14–21 days. Previously, Roper [22] showed ganglion in these animals (Fig. 4D), suggesting that not allthat 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 inthe 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 brainstemdays post-surgery (n53) compared to the control (shamsurgery, 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 orwere examined at 21 days post-vagotomy. Although the efferent centers. Dorsal root ganglia (DRG) were isolatedamount of PACAP-ir was still reduced compared to control from fixation-perfused animals and cryostat sectioned. The

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Fig. 3. Immunoreactivity for PACAP-38 and galanin in the cardiac ganglion. Preparations were labeled with the antibody to PACAP-38 (1:500) and anantibody to galanin (1:500). Galanin immunoreactivity could be observed in fibers innervating contractile tissue outside the ganglia itself (D) and withinthe 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 wasrelatively 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) inir positive neurons were also immunoreactive for either SP both untreated and colchicine-treated tissue. No evidenceor CGRP (Fig. 5). However, not all SP or CGRP-ir cells of either CGRP or SP-ir was observed in these same tissuewere also positive for PACAP. In addition, PACAP-ir nerve sections (data not shown). Caudal sections (16 mm) of thefibers were observed entering the dorsal horn in sections of brainstem ranging from the most caudal medulla to thethe 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 showednerve, afferent and efferent centers were examined in both PACAP-ir fibers in the caudal brainstem which appeared tountreated (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 priorto staining for PACAP-38 immunoreactivity (Panel A). Ganglion explants showed a dramatic reduction in PACAP-ir. Specific lesioning of the vagus nervealso reduced the amount of PACAP-ir in the cardiac ganglion (Panel C) as compared to sham-operated controls (Panels B). Increasing the post-operativerecovery 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 function(s) acrossthe control or the colchicine-treated tissue (Fig. 7). species. The exact nature of this function(s), though, still

remains largely unknown. However, increasing evidencefrom other investigators and the present study suggests that

4. Discussion one of the roles of PACAP may be in the regulation ofautonomic function across species.

Pituitary adenylate cyclase-activating polypeptide is a In mammals, PACAP has been found in autonomichighly conserved peptide across species [7], with the ganglia in both the periphery and in regions of themajority of species showing no alteration in amino acid brainstem associated with autonomic control [3,10,17].sequence. Only the urochordate Chelyosoma productum (or Physiologically, PACAP has previously been demonstratedsea squirt) has a single amino acid difference in the first 27 to play a modulatory role in cardiovascular function byamino acids from the mammalian PACAP 27 amino acid stimulation of the parasympathetic postganglionic neurons

L.K. Schoenfeld et al. / Brain Research 882 (2000) 180 –190 187

Fig. 5. Co-localization of PACAP with SP and CGRP in dorsal root ganglion neurons. Sections (16 mm) of dorsal root ganglia from high thoracic regionswere stained with the antibody to PACAP-38 (1:800) and either SP (1:800) or CGRP (1:800). PACAP-ir cell somas were observed in the ganglia whichwere also immunoreactive for either CGRP (panel A and B) or SP (panel C and D). A CGRP-ir nerve fiber is also present (B, arrow) which is alsoimmunoreactive for PACAP. Occasional neurons were observed that were immunoreactive for CGRP alone (B, arrowhead). Calibration bar550 mm.

[1,6,23] as well as by direct stimulatory actions on the PACAP innervation of the cardiac ganglion in the mud-cardiac muscle fibers [2]. This study provides anatomical puppy appears to derive solely from extrinsic sources, withevidence that PACAP may serve as a visceral afferent no evidence of endogenous PACAP production within theneuromodulator of cardiac function in amphibians as well. ganglia. This differs from results found in the guinea pig,

In the mudpuppy, PACAP can be found in afferent fibers in which PACAP-positive cell bodies and PACAP mRNAand neurons innervating the parasympathetic postgan- could be detected within the ganglia itself [1].glionic neurons of the cardiac ganglion. These PACAP-ir Previous studies in mammals had found PACAP in bothfibers were found as pericellular complexes surrounding parasympathetic preganglionic efferent fibers and afferentparasympathetic postganglionic neurons and in varicosities fibers of the vagus nerve [3,10,17]. Thus, one potentialthroughout the ganglion. This finding would imply that extrinsic source of PACAP to the mudpuppy cardiacPACAP can be released in close proximity to the neurons, ganglion is via the vagus nerve. This possibility was testedpresumably to act as modulators of their activity. The by lesioning the vagus nerve and examining PACAPresults from the immunohistochemical experiments suggest immunoreactivity 2–3 weeks later. Following vagotomy,that it is the longer form of PACAP, PACAP 38, which is PACAP-ir in the cardiac ganglion was significantly re-predominantly found in these fibers. The inability to detect duced. Many of the PACAP-positive fibers still observed atPACAP 27 suggests that either PACAP 27 is not expressed this point appeared to be undergoing degeneration and thein these fibers, or that the amounts are too low to be total amount of staining was less than sham-operateddetected by standard immunohistochemical techniques. controls. However, it was also possible to observe intact

Lesioning of all fiber inputs extrinsic to the cardiac PACAP-ir fibers and pericellular complexes as long as 21ganglion by organ explant and culture, resulted in the days post vagotomy. Thus, although a significant portiondegeneration of virtually all of the PACAP-ir fibers. Thus, of the PACAP appears to be carried in the vagus nerve,

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Fig. 6. PACAP immunoreactive neurons in the vagal sensory ganglia. Sections (16 mm) of the ganglia of the X cranial nerve (vagal sensory ganglia) werestained with the antibody to PACAP-38 (1:800). Immunoreactive cell somas were observed throughout the ganglia, although not all neurons stainedpositively for PACAP. Calibration bar550 mm in Panel B, and 100 mm in Panel A.

there is a small, but significant amount of PACAP which PACAP-ir fibers innervating regions of the nucleuswas unaffected by the lesions, and therefore, must origi- solitarius, a major afferent input center for control ofnate from other sources. cardiovascular function. No PACAP-ir cell somas were

The co-localization of the PACAP with SP and CGRP in observed in the nucleus solitarius. Nor were PACAP-ir cellthe mudpuppy cardiac ganglion suggest that one alternative bodies observed in the sections from the caudal medulla,source of PACAP is from fibers originating in the dorsal which would include the regions of the dorsal motorroot ganglia. This hypothesis was supported by the co- nucleus and the nucleus ambiguous, where the majority oflocalization of PACAP-ir cells within sections of the dorsal vagal motor fibers to the heart originate. These results wereroot ganglia with either SP or CGRP. In addition, PACAP- observed in both untreated and colchicine-treated animalsir fibers could be observed entering the dorsal horn of the to determine if the lack of staining in control animals wasspinal cord. due to low cytoplasmic concentrations of the peptide in the

The lack of co-localization of PACAP with tyrosine cell somas. Colchicine treatment did not result in anyhydroxylase in the cardiac ganglion suggests that PACAP difference in peptide levels. This does not provide defini-is not found in sympathetic postganglionic fibers innervat- tive proof that PACAP is not found in vagal efferenting the cardiac ganglion. However, since no other studies nuclei, but does suggest that PACAP is localized primarilyhave yet looked at the potential localization of PACAP in in visceral afferent fibers of the vagus in the mudpuppy.other sympathetic fibers or autonomic neurons in am- Further studies are necessary to insure that no PACAP-phibians, it is not possible to conclude that PACAP is not containing cells are present in the vagal motor nucleus.found in the sympathetic nervous system in these animals. This result does appear to differ from that observed in

The PACAP-containing nerve fibers carried in the vagus mammals, where PACAP was found in both sensorynerve could be of either motor or sensory origins. The neurons and in a small population of efferent vagalexact nature of the input was explored by examining the neurons [3,10].sources of these two different vagal fiber populations. The In summary, the results of this study demonstrate thatvagal afferent fibers originate primarily from the nodose the evolutionarily-conserved neuropeptide, PACAP, may(vagal sensory) ganglia. Sections of this ganglia were play an important role in autonomic function acrossexamined for PACAP staining and both PACAP-ir cell species, and in particular, appears to play a role in thebodies and fibers were observed. The vagal motor outputs control of cardiac function in both mammals and am-originate from nuclei in the caudal brainstem. Sagittal and phibians. Although the physiological effects of PACAP inhorizontal sections of mudpuppy brainstem showed this system have yet to be explored, this study suggests

L.K. Schoenfeld et al. / Brain Research 882 (2000) 180 –190 189

Fig. 7. PACAP immunoreactivity in sections of spinal cord and brainstem. Horizontal sections (16 mm) from the cervical spinal cord (A) and caudalbrainstem (C) were labeled with antibody to PACAP-38 (1:800). Longitudinal sections of brainstem were also examined (B). PACAP-ir nerve fibers can befound entering the dorsal horn of the spinal cord (A) and innervating the region of the nucleus solitarius (B, arrow). No evidence for PACAP-ir cell somasor fibers were observed in the region of the dorsal motor nucleus in a colchicine-treated preparations (C, arrow). PACAP-ir fibers were observed in thedorsal regions of the brainstem sections in the region where the vagus nerve enters /exits. Calibration bar5100 mm in A and B, 50 mm in C. Abbreviations:NS5nucleus solitarius; 4th ven5fourth ventricle; nuc.V5nucleus of cranial nerve V; nuc. X motor5motor nucleus of cranial nerve X; root X motor5rootsof motor fibers from cranial nerve X. Nuclei and fiber tract localization from Herrick [5].

that PACAP is positioned to function as an important in part by NIH AREA grant R15 HL60619. The tyrosinesensory peptide in the regulation of cardiac function in the hydroxylase antibody developed by LeDourain and Zillermudpuppy. was obtained from the Developmental Studies Hybridoma

Bank developed under the auspices of the NICHD andmaintained by The University of Iowa, Department ofBiological Sciences, Iowa City, IA 52242.

Acknowledgements

We would like to thank Reshma Kumar, Kristen Sager Referencesand Meghan Rossi for their technical assistance in thisproject. We would also like to thank Rodney Parsons for [1] K.M. Braas, V. May, S.A. Harakall, J.C. Hardwick, R.L. Parsons,critical review of the manuscript. This study was supported Pituitary adenylate cyclase-activating polypeptide expression and

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