The present study investigated the origin of pituitary adenylate cyclase-activating polypeptide (PACAP) -immunoreactive (IR) fibers innervating guinea pig cardiac ganglia. Immunohistochemistry was performed on whole-mounts containing cardiac ganglia, and sections of stellate, nodose, and dorsal root ganglia (DRG, thoracic levels 1-4), and caudal medulla. In control preparations, only 4% of the cardiac neurons were PACAP-IR, although most cardiac ganglion cells were surrounded by a network of PACAP-IR fibers. After 3-7 days in explant culture, the number of PACAP-IR cardiac neurons increased approximately eightfold. However, virtually all PACAP-IR fibers surrounding the cardiac neurons had degenerated, demonstrating that the major source of the PACAP-IR fibers was extrinsic to the cardiac ganglia preparation. PACAP- and choline acetyltransferase (ChAT) immunoreactivity were colocalized in fibers within the stellate ganglia but not within neuropeptide Y (NPY) -IR cell bodies and fibers. PACAP-IR cells and fibers were present in the nodose ganglia. PACAP immunoreactivity also was present in fibers and primarily small neurons in thoracic DRGs. In situ hybridization demonstrated the presence of proPACAP mRNA within neurons in the region of the dorsal motor nucleus of the vagus and nucleus ambiguus. PACAP immunoreactivity was colocalized with ChAT immunoreactivity, but not with NPY immunoreactivity or SP immunoreactivity, in fibers surrounding neurons within cardiac ganglia. We conclude that PACAP-containing fibers innervating the postganglionic parasympathetic neurons in guinea pig cardiac ganglia are primarily preganglionic parasympathetic axons.

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.

This study was conducted to determine the origin(s) of neuronal nitric oxide synthase-immunoreactive (NOS-IR) fibers within guinea pig atrial whole-mount preparations containing the cardiac ganglia. Intrinsic NOS-IR cardiac neurons exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they were cholinergic as well as nitrergic. Comparison of control versus 72-hour explant culture preparations indicated that most of the nitrergic fibers within cardiac ganglia were extrinsic. The extrinsic NOS-IR fibers were not IR for ChAT (marker of preganglionic parasympathetic neurons), tyrosine hydroxylase (marker of catecholaminergic sympathetic postganglionic axons), or calcitonin gene-related peptide (CGRP) (marker of afferent fibers). Separate NOS-IR and ChAT-IR neurons were present within medullary regions containing the cardiovascular regulatory nuclei (nucleus ambiguus and dorsal motor nucleus of the vagus), but no cells were found that exhibited both NOS immunoreactivity and ChAT immunoreactivity. The small size and location of the medullary NOS-IR neurons suggested they were probably interneurons. Only an occasional sympathetic postganglionic cell in the stellate ganglion complex exhibited NOS immunoreactivity. NOS-IR cells were present in dorsal root ganglia (thoracic 1-5), but these typically also exhibited CGRP immunoreactivity. NOS-IR cells were also present in the nodose ganglia, but only some exhibited CGRP immunoreactivity. We concluded that virtually all the extrinsic NOS-IR nerve fibers represented an afferent fiber input that was separate from the substance P (SP)/CGRP-containing population of sensory fibers. Furthermore, much of this NOS innervation is probably derived from the nodose ganglia.

This study was conducted to establish the presence of cocaine- and amphetamine-regulated transcript peptide (CARTp) immunoreactivity in neurons and fibers within guinea pig atrial whole-mount preparations containing the intrinsic cardiac ganglia. Many cardiac ganglia, but not all, in a given whole-mount preparation, were innervated by CARTp-immunoreactive (IR) fibers. Following explant culture of whole mounts for 72 hours, the CARTp-IR fiber networks were absent, but the number of CARTp-IR neurons was increased markedly. These observations suggested that the majority of the CARTp-IR fibers in the intracardiac ganglia were derived from sources extrinsic to the heart. In control whole-mount preparations, very few CARTp-positive neurons were present. The few intrinsic CARTp-IR neurons also exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they make up a small subpopulation of cholinergic postganglionic neurons. Some CARTp-IR neurons also exhibited nitric oxide synthase (NOS) immunoreactivity, indicating that they were nitrergic as well. We compared the immunohistochemical staining patterns of CARTp-IR fibers with the staining patterns of a number of other neurotransmitters or neurotransmitter synthetic enzymes that mark specific extrinsic inputs. The CARTp-IR fibers were not immunoreactive for ChAT, tyrosine hydroxylase, calcitonin gene-related peptide, or substance P. However, virtually all CARTp-IR fibers exhibited immunoreactivity to neuronal NOS (a marker for nitric oxide-producing neurons). CARTp-IR cells and NOS-IR cells were present in the nodose ganglia. In addition, CARTp-IR neurons in the nodose also were stained positively for NADPH-diaphorase. Thus, we propose that most CARTp-IR fibers within the guinea pig intrinsic cardiac ganglia are vagal afferent fibers that also contain NOS.

1. Presynaptic function was investigated at K + -stimulated motor nerve terminals in snake costocutaneous nerve muscle preparations exposed to carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 µM), oligomycin (8 µg ml _1 ) or CCCP and oligomycin together. , but then declined. The decline occurred more rapidly in preparations treated with CCCP or CCCP and oligomycin together than in those treated with oligomycin alone.3. Staining with FM1-43 indicated that synaptic vesicle membrane endocytosis occurred at some CCCP-or oligomycin-treated nerve terminals after 120 or 180 min of K + stimulation, respectively.4. The addition of glucose to stimulate production of ATP by glycolysis during sustained K + stimulation attenuated the decline in MEPC frequency and increased the percentage of terminals stained by FM1-43 in preparations exposed to either CCCP or oligomycin.5. We propose that the decline in K + -stimulated quantal release in preparations treated with CCCP, oligomycin or CCCP and oligomycin together could result from a progressive elevation of intracellular calcium concentration ([Ca 2+ ] i ). For oligomycin-treated nerve terminals, a progressive elevation of [Ca 2+ ] i could occur as the cytoplasmic ATP/ADP ratio decreases, causing energy-dependent Ca 2+ buffering mechanisms to fail. The decline in MEPC frequency could occur more rapidly in preparations treated with CCCP or CCCP and oligomycin together because mitochondrial Ca 2+ buffering and ATP production were both inhibited. Therefore, the proposed sustained elevation of [Ca 2+ ] i could occur more rapidly.