Introduction the superior cervical ganglion [9], and the ciliary ganglion
Brain Research 882 2000 180–190 www.elsevier.com locate bres
Research report
Pituitary adenylate cyclase-activating polypeptide innervation of the mudpuppy cardiac ganglion
b a
a ,
Linda K. Schoenfeld , Jennifer A. Souder , Jean C. Hardwick
a
Biology Department , Ithaca College, Ithaca, NY 14850, USA
b
Neuroscience 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 in cardiac ganglia of the mudpuppy, Necturus maculosus. Although PACAP has been implicated in the regulation of cardiac function in
several mammalian species, the presence of this peptide in the autonomic nervous system ANS of other species is unclear. Thus, this study is the first to characterize this highly conserved peptide in the ANS of a non-mammalian species. PACAP-immunoreactivity was
observed in nerve fibers throughout the mudpuppy cardiac ganglia and often was co-localized with the sensory neuropeptides substance P and calcitonin gene-related peptide. Removal of all extrinsic inputs to the ganglia by organ culture eliminated PACAP-immunoreactivity
in the cardiac ganglia, whereas bilateral vagotomies only partially reduced PACAP-labeling. PACAP-immunoreactive neurons were observed in both high thoracic dorsal root ganglia and in vagal sensory ganglia. While no PACAP-positive neurons were observed in
caudal medulla brainstem regions, PACAP-containing nerve fibers were found in the region of the nucleus solitarius. These results suggest that, in the mudpuppy, PACAP is found primarily in visceral afferent fibers, originating from cells in either the dorsal root ganglia or
vagal sensory ganglia. Based on their anatomic localization, these afferent fibers may function to transmit important sensory information to cardiovascular centers in the brain as well as serving as local reflex inputs to modulate postganglionic parasympathetic output within
the 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 ganglia
peptides. Following its initial discovery in the brain, in rats [3,17], implying a role for the peptide in visceral
PACAP has subsequently been localized in fibers of both afferent systems. PACAP has also been found in the central
the 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 variety
or 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 changes
1131. E-mail address
: jhardwickithaca.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. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 8 5 - 7
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