Mice deficient in brain‐derived neurotrophic factor have altered development of gastric vagal sensory innervation

Vagal sensory neurons are dependent on neurotrophins for survival during development. Here, the contribution of brain‐derived neurotrophic factor (BDNF) to survival and other aspects of gastric vagal afferent development was investigated. Post‐mortem anterograde tracing with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbo‐cyanine perchlorate (DiI) was used to label selectively vagal projections to the stomach on postnatal days (P) 0, 3, 4, and 6 in wild types and heterozygous or homozygous BDNF mutants. Sampling sites distributed throughout the ventral stomach wall were scanned with a confocal microscope, and vagal axon bundles, single axons, putative mechanoreceptor precursors (intraganglionic laminar endings, IGLEs; intramuscular arrays, IMAs), and efferent terminals were quantified. Also, myenteric neurons, which are innervated by IGLEs, were stained with cuprolinic blue and counted. Quantitative comparisons across wild‐type stomach compartments demonstrated that the adult distribution of IMAs was not present at P0 but began to form by P3–6. Among all the quantified elements, at P0, only IGLE density was significantly different in homozygous mutants compared with wild types, exhibiting a 50% reduction. Also, antrum innervation appeared disorganized, and some putative IMA precursors had truncated telodendria. At P3–6, the effect on IGLEs had recovered, the disorganization of antrum innervation had partially recovered, and some IMA telodendria were still truncated. The present results suggest that gastric IGLEs are among the vagal sensory neurons dependent on BDNF for survival or axon guidance. Alternatively, BDNF deficiency may delay gastric IGLE development. Also, BDNF may contribute to IMA differentiation and patterning of antral vagal innervation. J. Comp. Neurol. 518:2934–2951, 2010. © 2010 Wiley‐Liss, Inc.     

Vagal afferent innervation of the atria of the rat heart reconstructed with confocal microscopy

We have used confocal microscopy to analyze the vagal afferent innervation of the rat heart. Afferents were labeled by injecting 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the nodose ganglia of animals with prior supranodose de-efferentations, autonomic ganglia were stained with Fluoro-gold, and tissues were examined in whole mounts. Distinctively different fiber specializations were observed in the epi-, myo-, and endocardium: Afferents to the epicardium formed complexes associated with cardiac ganglia. These ganglia consisted of four major ganglionated plexuses, two on each atrium, at junctions of the major vessels with the atria. Ganglionic locations and sizes (left > right) were consistent across animals. In addition to principal neurons (PNs), significant numbers of small intensely fluorescent (SIF) cells were located in each of these plexuses, and vagal afferents provided dense pericellular varicose endings around the SIF cells in each ganglionic plexus, with few if any terminations on PNs. In the myocardium, vagal afferents formed close contacts with cardiac muscles, including conduction fibers. In the endocardium, vagal fibers formed “flower-spray” and “end-net” terminals in connective tissue. With three-dimensional reconstruction of confocal optical sections, a novel polymorphism was seen: Some fibers had one or more collaterals ending as endocardial flower sprays and other collaterals ending as myocardial intramuscular endings. Some unipolar or pseudounipolar neurons within each cardiac ganglionic plexus were retrogradely labeled from the nodose ganglia. In conclusion, vagal afferents form a heterogeneity of differentiated endings in the heart, including structured elements which may mediate chemoreceptor function, stretch reception, and local cardiac reflexes. J. Comp. Neurol. 381:1-17, 1997. © 1997 Wiley-Liss, Inc.     

Simultaneous labeling of vagal innervation of the gut and afferent projections from the visceral forebrain with dil injected into the dorsal vagal complex in the rat.

The vagal innervation of the different layers of the rat gastrointestinal wall was identified with the fluorescent carbocyanine dye Dil, injected into the dorsal motor nucleus of the vagus (dmnX). Multiple, bilateral injections were used to label all dmnX preganglionic motoneurons, and as a consequence, most of the vagal primary afferents that terminate in the adjacent nucleus of the solitary tract (nts) were also retrogradely and transganglionically labeled. With Fluorogold used to label the enteric nervous system completely and specifically, the Dil-labeled vagal profiles could be visualized and quantified in their anatomical relation to the neurons of the myenteric and submucous ganglia. In the myenteric plexus, vagal fibers and terminals were found throughout the gastrointestinal tract as far caudal as the descending colon, but there was a general decreasing proximodistal gradient in the density of vagal innervation. All parts of the gastric myenteric plexus (fundus, corpus, antrum), as well as the proximal duodenum, were extremely densely innervated, with vagal fibers and terminals in virtually every ganglion and connective. Further caudally, both the percentage of innervated myenteric ganglia and the average density of label within the ganglia rapidly decreased, with the exception of the cecum and proximal colon, where up to 65% of the ganglia were innervated. In the gastric and duodenal submucosa very few and in the mucosa no vagal fibers and terminals were found. With both normal epifluorescence and laser scanning confocal microscopy, highly varicose or beaded terminal structures of various size and geometry could be identified. The Dil injections, which impregnated the dmnX as well as the adjacent nts, resulted in retrograde and anterograde labeling of all the previously reported forebrain connections with the dorsal vagal complex. We conclude that the myenteric plexus is the primary target of vagal innervation throughout the gastrointestinal tract, and that its innervation is more complete than previously assumed. In contrast, vagal afferent (and efferent) innervation of mucosa and submucosa seems conspicuously sparse or absent. Furthermore, the use of more focal injections of Dil offers the prospect to simultaneously identify specific subsets of vagal preganglionics and their central nervous inputs.     

Basic and clinical aspects of visceral sensation: transmission in the CNS

Pain and discomfort are the leading cause for consultative visits to gastroenterologists. Acute pain should be considered a symptom of an underlying disease, thereby serving a physiologically important function. However, many patients experience chronic pain in the absence of potentially harmful stimuli or disorders, turning pain into the primary problem rather than a symptom. Vagal and spinal afferents both contribute to the sensory component of the gut-brain axis. Current evidence suggests that they convey different elements of the complex sensory experience. Spinal afferents play a key role in the discriminatory dimension, while vagal input primarily affects the strong emotional and autonomic reactions to noxious visceral stimuli. Drugs, surgical and non-pharmacological treatments can target these pathways and provide therapeutic options for patients with chronic visceral pain syndromes.     

Ultrastructural evidence for communication between intramuscular vagal mechanoreceptors and interstitial cells of Cajal in the rat fundus.

To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach's plexus were encountered.     

Alpha-synuclein immunopositive aggregates in the myenteric plexus of the aging Fischer 344 rat

Dystrophic axons and terminals are common in the myenteric plexus and smooth muscle of the gastrointestinal (GI) tract of aged rats. In young adult rats, alpha-synuclein in its normal state is abundant throughout the myenteric plexus, making this protein–which is prone to fibrillization–a candidate marker for axonopathies in the aged rat. To determine if aggregation of alpha-synuclein is involved in the formation of age-related enteric neuropathies, we sampled the stomach, small intestine and large intestine of adult, middle-aged, and aged virgin male Fischer 344 rats stained for alpha-synuclein in both its normal and pathological states. Alpha-synuclein-positive dystrophic axons and terminals were present throughout the GI tract of middle-aged and aged rats, with immunohistochemical double labeling demonstrating co-localization within nitric oxide synthase-, calretinin-, calbindin-, or tyrosine hydroxylase-positive markedly swollen neurites. However, other dystrophic neurites positive for each of these four markers were not co-reactive for alpha-synuclein. Similarly, a subpopulation of alpha-synuclein inclusions contained deposits immunostained with an anti-tau phospho-specific Ser²⁶² antibody, but not all of these hyperphosphorylated tau-positive aggregates were co-localized with alpha-synuclein. The presence of heteroplastic and potentially degenerating neural elements and protein aggregates both positive and negative for alpha-synuclein suggests a complex chronological relationship between the onset of degenerative changes and the accumulation of misfolded proteins. Additionally, proteins other than alpha-synuclein appear to be involved in age-related axonopathies. Finally, this study establishes the utility of the aging Fischer 344 rat for the study of synucleopathies and tauopathies in the GI tract.     

Regional distribution and extrinsic innervation of intrinsic cardiac neurons in the guinea pig.

Mammalian intrinsic cardiac neurons subserve different functions in different cardiac regions, but the regional anatomical organisation of the intracardiac nervous system is not well understood. We investigated the quantitative and qualitative distribution of cholinergic and adrenergic elements, and the intracardiac pathways of extrinsic cardiac nerves, in whole-mount preparations of guinea pig atria. Protein gene product 9.5 immunoreactivity (PGP 9.5-IR) marked intracardiac neuronal elements; immunoreactions for choline acetyltransferase (ChAT-IR) and tyrosine hydroxylase (TH-IR) distinguished cholinergic and adrenergic components, respectively. Catecholamine-containing components were identified by aldehyde-induced fluorescence histochemistry. Mean total number of atrial neurons was 1510+/-251 (SE); 85% of these occurred in ganglia of < or = 20 neurons. All neuronal somata expressing PGP 9.5-IR also expressed ChAT-IR, suggesting that these neurons were cholinergic. Right (RA) and left (LA) atria had statistically similar neuronal densities (6.4+/-1.2 and 2.4+/-0.7 neurons/mm2, respectively; analysis of variance, P< or =0.05). Neurons in RA were concentrated intercavally; LA neurons were concentrated near pulmonary vein ostia. Greatest density occurred in the interatrial septum (16.3+/-4.0 neurons/mm2). No neuronal somata expressed TH-IR or contained detectable amines but these elements were expressed by somata of small cells (mean total 124+/-33) throughout the atria, primarily associated with ganglia. Amine- and TH- containing varicosities were also present in ganglia, representing potential sites for adrenergic modulation of ganglionic neurotransmission. Branches of extrinsic cardiopulmonary and vagus nerves were distributed to all parts of both atria. The organisation of the intracardiac nervous system revealed in this study will facilitate further investigations of regional autonomic control of the heart.     

Development of enteric and vagal innervation of the zebrafish (Danio rerio) gut

The autonomic nervous system develops following migration and differentiation of precursor cells originating in the neural crest. Using immunohistochemistry on intact zebrafish embryos and larvae we followed the development of the intrinsic enteric and extrinsic vagal innervation of the gut. At 3 days postfertilization (dpf), enteric nerve cell bodies and fibers were seen mainly in the middle and distal intestine, while the innervation of the proximal intestine was scarcer. The number of fibers and cell bodies gradually increased, although a large intraindividual variation was seen in the timing (but not the order) of development. At 11-13 dpf most of the proximal intestine received a similar degree of innervation as the rest of the gut. The main intestinal branches of the vagus were similarly often already well developed at 3 dpf, entering the gut at the transition between the proximal and middle intestine and projecting posteriorly along the length of the gut. Subsequently, fibers branching off the vagus innervated all regions of the gut. The presence of several putative enteric neurotransmitters was suggested by using markers for neurokinin A (NKA), pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide, serotonin (5-hydroxytryptamine, 5-HT), and calcitonin gene-related peptide (CGRP). The present results corroborate the belief that the enteric innervation is well developed before the onset of feeding (normally occurring around 5-6 dpf). Further, the more detailed picture of how development proceeds at stages previously not examined suggests a correlation between increasing innervation and more regular and elaborated motility patterns.     

Primary afferent projections from dorsal and ventral roots to autonomic preganglionic neurons in the cat sacral spinal cord: light and electron microscopic observations

HRP applied to cut dorsal and ventral roots of the cat sacral spinal cord labeled afferent axons with swellings in close apposition to labeled preganglionic neurons (PGNs) in the sacral parasympathetic nucleus. Electron microscopy allowed characterization of synaptic contacts between afferents and PGNs. The results suggest that both the dorsal and ventral root afferents can directly activate autonomic preganglionic neurons.     

Development of the vagal innervation of the gut: steering the wandering nerve

Background The vagus nerve is the major neural connection between the gastrointestinal tract and the central nervous system. During fetal development, axons from the cell bodies of the nodose ganglia and the dorsal motor nucleus grow into the gut to find their enteric targets, providing the vagal sensory and motor innervations respectively. Vagal sensory and motor axons innervate selective targets, suggesting a role for guidance cues in the establishment of the normal pattern of enteric vagal innervation. Purpose This review explores known molecular mechanisms that guide vagal innervation in the gastrointestinal tract. Guidance and growth factors, such as netrin‐1 and its receptor, deleted in colorectal cancer, extracellular matrix molecules, such as laminin‐111, and members of the neurotrophin family of molecules, such as brain‐derived neurotrophic factor have been identified as mediating the guidance of vagal axons to the fetal mouse gut. In addition to increasing our understanding of the development of enteric innervation, studies of vagal development may also reveal clinically relevant insights into the underlying mechanisms of vago‐vagal communication with the gastrointestinal tract.     

Structure, afferent innervation, and transmitter content of ganglia of the guinea pig gallbladder: relationship to the enteric nervous system

Although a well-developed plexus of nerves and ganglia is known to be present in the wall of the gallbladder, little has previously been learned about the function or organization of this innervation. The current study was undertaken in order to evaluate the hypothesis that the ganglionated plexus of the gallbladder is analogous to elements of the enteric nervous system (ENS). The ganglionated plexus of the gallbladder was found to resemble closely the submucosal plexus of the small intestine in its organization into two irregular anastomosing and interwoven networks of ganglia, in the numbers of neurons per ganglion, and in the manifestation of histochemically demonstrable acetylcholinesterase activity in virtually all ganglion cells. In common with enteric ganglia, laminin immunoreactivity was observed to be excluded from the interiors of gallbladder ganglia, which were surrounded by a periganglionic laminin-immunoreactive sheath. As in the submucosal plexus, intrinsic substance P-, vasoactive intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive neurons were seen in the ganglionated plexus of the gallbladder. Extrinsic nerves in the gallbladder that degenerated following chemical sympathectomy with 6-hydroxydopamine (6-OHDA), and which contained NPY, tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH) immunoreactivities, formed a perivascular plexus closely associated with blood vessels. Endogenous catecholamines could also be demonstrated in these perivascular nerves by aldehyde-induced histofluorescence. In addition to perivascular nerves, paravascular nerve bundles were observed that were loosely associated with vessels, did not degenerate following administration of 6-OHDA, and contained NPY immunoreactivity. Other paravascular nerves, probably visceral sensory axons, coexpressed substance P and calcitonin-gene-related peptide (CGRP) immunoreactivities. The ganglionated plexus of the gallbladder resembled enteric ganglia in having intrinsic 5-hydroxytryptamine (5-HT)-immunoreactive cells and highly varicose nerve fibers. The 5-HT-immunoreactive gallbladder axons were, like those of the gut, resistant to 6-OHDA, and separate from fibers that expressed TH immunoreactivity. Differences between the ganglionated plexus of the gallbladder and enteric ganglia of the small intestine included in the gallbladder are 1) the presence of TH-immunoreactive cells that contain an endogenous catecholamine, but not DBH; 2) DBH-immunoreactive neurons, some of which coexpress substance P immunoreactivity, but which contain neither a catecholamine nor TH immunoreactivity; 3) an apparent absence of CGRP-immunoreactive cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in nitrergic innervation of defunctionalized rat colon after diversion colostomy

Abstract  After 45 days of complete diversion colostomy in male Wistar rats, morphometry of soma and nuclei of NADPH diaphorase positive cells of the myenteric plexus was evaluated. There was a significant ( P  < 0.0001) diminution in the area, perimeter and volume-weighted mean volume of soma and nuclei of nitrergic myenteric neurones in the defunctionalized colon. In addition, there was a significant reduction in the neuronal density of the myenteric neurones, and increased distance between the ganglia. In addition, there was myenteric glial atrophy. Atrophy of colonic myenteric neurones was accompanied by significant reduction ( P  < 0.001) in the volume fraction of the muscularis externa, the prime targets of these neurones. The disturbances in the microecology of the colon may jeopardize the finely orchestrated functioning of the components of the Enteric nervous system (ENS) leading to colonic dysfunction. Our observations, by extrapolation, may explain the bowel dysmotility in humans after restoration of colonic continuity after colostomy.     

Immunohistochemical identification of neurons in ganglia of the guinea pig sphincter of oddi

The sphincter of Oddi is a smooth muscle sphincter that regulates the flow of bile into the duodenum. To identify potential chemical coding in sphincter of Oddi neurons, immunohistochemistry and histochemistry were employed to assay for putative neurotransmitters and related synthetic enzymes in wholemount preparations, with and without colchicine treatment. Immunoreactivities for enkephalin-endorphin (ENK-END), substance P (SP), nitric oxide synthase, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin generelated peptide (CGRP) were demonstrated within the ganglionated plexus. Roughly half of the neurons in the sphincter of Oddi expressed immunoreactivity for both SP and ENK-END, but not for nitric oxide synthase. About 25% of the neurons expressed nitric oxide synthase immunoreactivity as well as NADPH-diaphorase activity. This contingent of neurons was made up of two subgroups: one that expressed immunoreactivity for VIP, the other for NPY. Neurons that expressed CGRP immunoreactivity were sparse in sphincter of Oddi ganglia; however, many axons immunoreactive for both CGRP and SP were present in the ganglionated plexus. The CGRP/SP fibers are probably visceral afferents that may influence ganglionic output through axon reflex circuits. These results, along with studies of the actions of these neuroactive compounds on sphincter tone, support the view that ganglia of the sphincter of Oddi are largely comprised of excitatory (SP/ENK-END-immunoreactive) and inhibitory (nitric oxide synthase/VIP- or NPY-immunoreactive) neurons, and that sphincter of Oddi tone is controlled by the regulation of the outputs of these two groups of cells. © 1995 Wiley-Liss, Inc.     

VR1-, VRL-1- and P2X3 receptor-immunoreactive innervation of the rat temporomandibular joint

Immunohistochemistry for vanilloid receptor subtype 1 (VR1), vanilloid receptor 1-like receptor (VRL-1) and P2X3 receptor was performed in the rat temporomandibular joint (TMJ). Blood vessels in the articular disk and capsule, the synovial membrane and the fibrous tissue around the condylar process were innervated by VR1- or P2X3 receptor-immunoreactive (ir) nerve fibers. However, VRL-1-immunoreactivity (ir) could not be detected in the TMJ. Retrograde tracing and immunohistochemical methods revealed that 25%, 41% and 52% of TMJ neurons in the trigeminal ganglion (TG) exhibited VR1-, VRL-1- and P2X3 receptor-ir, respectively. VR1-ir TMJ neurons were mostly small to medium-sized, whereas VRL-1- and P2X3 receptor-ir TMJ neurons were predominantly medium-sized to large. In addition, 73%, 28% and 44% of VR1-, VRL-1- and P2X3 receptor-ir TMJ neurons, respectively, coexpressed calcitonin gene-related peptide (CGRP)-ir. The present study suggests that the TMJ has abundant nociceptors which respond to vanilloid compounds, protons, heat and extracellular ATP.     

Projections to the cardioinhibitory region of the nucleus ambiguus of rat

The nucleus ambiguus is a brainstem structure which sends projections through the vagus nerve to the viscera, primarily heart, lung, and gut. The anatomical relationship between the nucleus ambiguus and other brain structures has not been elucidated nor has the cardiac region been identified physiologically in rats. We have attempted to clarify which areas of the nucleus ambiguus are cardioinhibitory and to determine other regions of the brain which send direct projections to this physiologically identified cardiac region. Stimulating electrodes were positioned stereotaxically in the medulla of anesthetized rats. Small currents were passed through the electrodes to locate regions in the ventrolateral medulla which slowed heart rate. In each rat, the area found was small (less than 200 μm in diameter), very specific, and located in the rostral portion of the nucleus ambiguus. Micro-quantities of horseradish peroxidase were then iontophoretically ejected into this brainstem area; 24–72 hours following the HRP injection, the rats were processed for HRP reaction product using the tetramethybenzidine method. The major brain area which sent projections to the rostral nucleus ambiguus was the ipsilateral medial subnucleus of the solitary tract. A few labeled cells were found in the ipsilateral ventrolateral subnucleus of the solitary tract, parabrachial complex, the paraventricular nucleus of the hypothalamus, and the contralateral nucleus ambiguus. Control injections in reticular areas surrounding the rostral nucleus ambiguus showed no label in the medial solitary nucleus.     

Localization of cholecystokininlike immunoreactivity in the rat spinal cord,with particular reference to the autonomic innervation of the pelvic organs

The distribution of cholecystokinin in the spinal cord was investigated by immunohistochemistry. Throughout the length of the spinal cord cholecystokinin immunoreactivity was found in laminae I and II, in the spinal re-ticular nucleus, and in the surroundings of the central canal. On the basis of the cholecystokinin pattern lamina II could be divided into a dorsal and ventral part. In the lumbar and sacral spinal cord additional terminal fields of cholecystokinin immunoreactive boutons unique to these levels were found. They corresponded to the intermediolateral nucleus and to the medial lumbar sympathetic nucleus dorsal to the central canal in the first and second lumbar segment. Also the intermediolateral nucleus in L₆–S₁ received a dense cholecystokinin positive input. Moreover, the area surrounding the central canal in L₆–S₁, contained many cholecystokinin immunoreactive structures. Combined retrograde tracing and immunocytochemistry revealed that the two cholecystokinin terminal fields characteristic for L₁–L₂ and that sur-rounding the intermediolateral nucleus in L₆–S₁ were situated corresponding to preganglionic neurons innervating pelvic organs through the hypo-gastric nerve or the pelvic nerves. It thus appears that the unique lumbosacral cholecystokinin is related to nuclei influencing pelvic structures, pointing to a special need for regulation of the organs involved in evacuation and sexual functions. Moreover, it is demonstrated that the caudal part of the spinal sympathetic system differs from the more cranial part with respect to type of afferent connections. The origin of the spinal cholecystokinin was investigated and it was found that neither complete transection of the spinal cord nor ipsilateral sectioning of three or four dorsal roots induced visible changes in the cholecystokinin staining pattern. Treatment of the caudal spinal cord with colchicine revealed the presence of cholecystokinin immunoreactive neurons in the intermediate gray, at the lateral border of the dorsal horn, in the dorsal horn proper, and in the substantia gelatinosa. These findings indicate that the majority of spinal cholecystokinin has a spinal origin.     

Regional innervation of the heart in the goldfish,Carassius auratus: A confocal microscopy study

The intracardiac nervous system represents the final common pathway for autonomic control of the vertebrate heart in maintaining cardiovascular homeostasis. In teleost fishes, details of the organization of this system are not well understood. Here we investigated innervation patterns in the heart of the goldfish, a species representative of a large group of cyprinids. We used antibodies against the neuronal markers zn‐12, acetylated tubulin, and human neuronal protein C/D, as well as choline acetyltransferase, tyrosine hydroxylase, nitric oxide synthetase, and vasoactive intestinal polypeptide (VIP) to detect neural elements and their transmitter contents in wholemounts and sections of cardiac tissue. All chambers of the heart were innervated by choline acetyltransferase‐positive axons, implying cholinergic regulation; and by tyrosine hydroxylase‐containing axons, implying adrenergic regulation. The mean total number of intracardiac neurons was 713 ± 78 (SE), nearly half of which were cholinergic. Neuronal somata were mainly located in a ganglionated plexus around the sinoatrial valves. Somata were contacted by cholinergic, adrenergic, nitrergic, and VIP‐positive terminals. Putative pacemaker cells, identified by immunoreactivity for hyperpolarization activated, cyclic nucleotide‐gated channel 4, were located in the base of the sinoatrial valves, and this region was densely innervated by cholinergic and adrenergic terminals. We have shown that the goldfish heart possesses the necessary neuroanatomical substrate for fine, region‐by‐region autonomic control of the myocardial effectors that are involved in determining cardiac output. J. Comp. Neurol. 522:456–478, 2014. © 2013 Wiley Periodicals, Inc.