Many teleosts including zebrafish, Danio rerio, actively regulate buoyancy with a gas-filled swimbladder, the volume of which is controlled by autonomic reflexes acting on vascular, muscular, and secretory effectors. In this study, we investigated the morphological development of the zebrafish swimbladder together with its effectors and innervation. The swimbladder first formed as a single chamber, which inflated at 1-3 days posthatching (dph), 3.5-4 mm body length. Lateral nerves were already present as demonstrated by the antibody zn-12, and blood vessels had formed in parallel on the cranial aspect to supply blood to anastomotic capillary loops as demonstrated by Tie-2 antibody staining. Neuropeptide Y-(NPY-) like immunoreactive (LIR) fibers appeared early in the single-chambered stage, and vasoactive intestinal polypeptide (VIP)-LIR fibers and cell bodies developed by 10 dph (5 mm). By 18 dph (6 mm), the anterior chamber formed by evagination from the cranial end of the original chamber; both chambers then enlarged with the ductus communicans forming a constriction between them. The parallel blood vessels developed into an arteriovenous rete on the cranial aspect of the posterior chamber and this region was innervated by zn-12-reactive fibers. Tyrosine hydroxylase- (TH-), NPY-, and VIP-LIR fibers also innervated this area and the lateral posterior chamber. Innervation of the early anterior chamber was also demonstrated by VIP-LIR fibers. By 25-30 dph (8-9 mm), a band of smooth muscle formed in the lateral wall of the posterior chamber. Although gas in the swimbladder increased buoyancy of young larvae just after first inflation, our results suggest that active control of the swimbladder may not occur until after the formation of the two chambers and subsequent development and maturation of vasculature, musculature and innervation of these structures at about 28-30 dph.

Many teleosts actively regulate buoyancy by using a gas-filled swim bladder, which is thought to be under autonomic control. Here we investigated the swim bladder in the zebrafish to determine possible mechanisms of gas-content regulation. Fluorescently labelled phalloidin revealed myocytes that appeared to form a possible sphincter at the junction of the pneumatic duct and esophagus. Myocytes also formed thick bands along the ventral surface of the anterior chamber and bilaterally along the posterior chamber. Thinner layers of myocytes were located elsewhere. Staining of peroxidase within erythrocytes revealed a putative rete and smaller blood vessels in muscle bands and elsewhere. The antibodies zn-12, a general neuronal marker, and SV2, a synaptic vesicle marker labelling presynaptic terminals, revealed widespread innervation of the swim bladder system. Widespread innervation of the swim bladder was also indicated by acetylcholinesterase histochemistry, but choline acetyltransferase-immunoreactive (-IR) somata and fibers were limited to the junction of the pneumatic duct and esophagus. In contrast, varicose tyrosine hydroxylase-IR fibers innervated muscles and blood vessels throughout the system. Neuropeptide Y-IR somata were located near the junction of the duct and esophagus and varicose fibers innervated muscles and vasculature of the posterior chamber and duct. Vasoactive intestinal polypeptide immunoreactivity was abundant throughout the anterior chamber but sparsely distributed elsewhere. Serotonin-IR fibers and varicosities were located only along blood vessels near the junction of the pneumatic duct and posterior chamber. Our results suggest that the zebrafish swim bladder is a complex and richly innervated organ and that buoyancy-regulating effectors may be controlled by multiple populations of autonomic neurons.

The cardiac pacemaker sets the heart's primary rate, with pacemaker discharge controlled by the autonomic nervous system through intracardiac ganglia. A fundamental issue in understanding the relationship between neural activity and cardiac chronotropy is the identification of neuronal populations that control pacemaker cells. To date, most studies of neurocardiac control have been done in mammalian species, where neurons are embedded in and distributed throughout the heart, so they are largely inaccessible for whole-organ, integrative studies. Here, we establish the isolated, innervated zebrafish heart as a novel alternative model for studies of autonomic control of heart rate. Stimulation of individual cardiac vagosympathetic nerve trunks evoked bradycardia (parasympathetic activation) and tachycardia (sympathetic activation). Simultaneous stimulation of both vagosympathetic nerve trunks evoked a summative effect. Effects of nerve stimulation were mimicked by direct application of cholinergic and adrenergic agents. Optical mapping of electrical activity confirmed the sinoatrial region as the site of origin of normal pacemaker activity and identified a secondary pacemaker in the atrioventricular region. Strong vagosympathetic nerve stimulation resulted in a shift in the origin of initial excitation from the sinoatrial pacemaker to the atrioventricular pacemaker. Putative pacemaker cells in the sinoatrial and atrioventricular regions expressed adrenergic β2 and cholinergic muscarinic type 2 receptors. Collectively, we have demonstrated that the zebrafish heart contains the accepted hallmarks of vertebrate cardiac control, establishing this preparation as a viable model for studies of integrative physiological control of cardiac function by intracardiac neurons.

The spontaneous activity of 175 neurons located in the ventral right atrial and ventral interventricular ganglionated plexi was recorded in eight anesthetized dogs, the hearts of which were chronically decentralized at least 2 wk before recordings were made. Ganglia were subsequently identified anatomically in the immediate vicinity of the recording sites. Spontaneous activity was correlated with the cardiac cycle in 57% of the atrial and 62% of the ventricular neurons and with the respiratory cycle in 29% of the atrial and 28% of the ventricular neurons. Spontaneous cardiovascular-related activity was recorded when systemic arterial pressure was between 80 and 175 mmHg. The activity of 23 atrial and 15 ventricular neurons was altered when discrete regions of the heart were mechanically distorted by gentle touch. These data imply that cardiac ganglionated plexi contain afferent neurons that receive inputs from limited regions of the heart. The spontaneous activity generated by intracardiac neurons was not altered when extracardiac tissues were distorted. These results demonstrate that neurons in ganglia on chronically decentralized hearts can generate spontaneous activity, a large fraction of which is correlated with cardiovascular or respiratory events.