Matthew R. Stoyek

Intrinsic and extrinsic innervation of the heart in zebrafish (Danio rerio)

In the vertebrate heart the intracardiac nervous system is the final common pathway for autonomic control of cardiac output, but the neuroanatomy of this system is not well understood. In this study we investigated the innervation of the heart in a model vertebrate, the zebrafish. We used antibodies against acetylated tubulin, human neuronal protein C/D, choline acetyltransferase, tyrosine hydroxylase, neuronal nitric oxide synthase, and vasoactive intestinal polypeptide to visualize neural elements and their neurotransmitter content. Most neurons were located at the venous pole in a plexus around the sinoatrial valve; mean total number of cells was 197 ± 23, and 92% were choline acetyltransferase positive, implying a cholinergic role. The plexus contained cholinergic, adrenergic, and nitrergic axons and vasoactive intestinal polypeptide‐positive terminals, some innervating somata. Putative pacemaker cells near the plexus showed immunoreactivity for hyperpolarization‐activated cyclic nucleotide‐gated channel 4 (HCN4) and the transcription factor Islet‐1 (Isl1). The neurotracer neurobiotin showed that extrinsic axons from the left and right vagosympathetic trunks innervated the sinoatrial plexus proximal to their entry into the heart; some extrinsic axons from each trunk also projected into the medial dorsal plexus region. Extrinsic axons also innervated the atrial and ventricular walls. An extracardiac nerve trunk innervated the bulbus arteriosus and entered the arterial pole of the heart to innervate the proximal ventricle. We have shown that the intracardiac nervous system in the zebrafish is anatomically and neurochemically complex, providing a substrate for autonomic control of cardiac effectors in all chambers. J. Comp. Neurol. 523:1683–1700, 2015.

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.

Zebrafish heart as a model to study the integrative autonomic control of pacemaker function

The cardiac pacemaker sets the hearts 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.

Adrenergic control of swimbladder deflation in the zebrafish (Danio rerio)

SUMMARY Many teleosts actively regulate buoyancy by adjusting gas volume in the swimbladder. In physostomous fishes such as the zebrafish, a connection is maintained between the swimbladder and the oesophagus via the pneumatic duct for the inflation and deflation of this organ. Here we investigated the role of adrenergic stimulation of swimbladder wall musculature in deflation of the swimbladder. Noradrenaline (NA), the sympathetic neurotransmitter (dosage 10−6 to 10−5 mol l−1), doubled the force of smooth muscle contraction in isolated tissue rings from the anterior chamber, caused a doubling of pressure in this chamber in situ, and evoked gas expulsion through the pneumatic duct, deflating the swimbladder to approximately 85% of the pre-NA volume. These effects were mediated by β-adrenergic receptors, representing a novel role for these receptors in vertebrates. No effects of adrenergic stimulation were detected in the posterior chamber. In a detailed examination of the musculature and innervation of the swimbladder to determine the anatomical substrate for these functional results, we found that the anterior chamber contained an extensive ventral band of smooth muscle with fibres organized into putative motor units, richly innervated by tyrosine hydroxylase-positive axons. Additionally, a novel arrangement of folds in the lumenal connective tissue in the wall of the anterior chamber was described that may permit small changes in muscle length to cause large changes in effective wall distensibility and hence chamber volume. Taken together, these data strongly suggest that deflation of the zebrafish swimbladder occurs primarily by β-adrenergically mediated contraction of smooth muscle in the anterior chamber and is under the control of the sympathetic limb of the autonomic nervous system.

Intrinsic regulation of sinoatrial node function and the zebrafish as a model of stretch effects on pacemaking

Excitation of the heart occurs in a specialised region known as the sinoatrial node (SAN). Tight regulation of SAN function is essential for the maintenance of normal heart rhythm and the response to (patho-)physiological changes. The SAN is regulated by extrinsic (central nervous system) and intrinsic (neurons, peptides, mechanics) factors. The positive chronotropic response to stretch in particular is essential for beat-by-beat adaptation to changes in hemodynamic load. Yet, the mechanism of this stretch response is unknown, due in part to the lack of an appropriate experimental model for targeted investigations. We have been investigating the zebrafish as a model for the study of intrinsic regulation of SAN function. In this paper, we first briefly review current knowledge of the principal components of extrinsic and intrinsic SAN regulation, derived primarily from experiments in mammals, followed by a description of the zebrafish as a novel experimental model for studies of intrinsic SAN regulation. This mini-review is followed by an original investigation of the response of the zebrafish isolated SAN to controlled stretch. Stretch causes an immediate and continuous increase in beating rate in the zebrafish isolated SAN. This increase reaches a maximum part way through a period of sustained stretch, with the total change dependent on the magnitude and direction of stretch. This is comparable to what occurs in isolated SAN from most mammals (including human), suggesting that the zebrafish is a novel experimental model for the study of mechanisms involved in the intrinsic regulation of SAN function by mechanical effects.

A simple automated system for appetitive conditioning of zebrafish in their home tanks

HighlightsAn automated associative conditioning paradigm for zebrafish was developed.Groups of fish rapidly learned auditory and visual associations with food.Learned associations were retained for at least 2 days after conditioning.Memories can be demonstrated when testing fish both individually and in groups.The paradigm can be easily adapted for zebrafish of different ages. ABSTRACT We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. Arduino microprocessors were used to control the delivery of auditory or visual stimuli to groups of adult or juvenile zebrafish in their home tanks in a conventional zebrafish facility. An automatic feeder dispensed precise amounts of food immediately after the conditioned stimuli, or at variable delays for controls. Responses were recorded using inexpensive cameras, with the video sequences analysed with ImageJ or Matlab. Fish showed significant conditioned responses in as few as 5 trials, learning that the conditioned stimulus was a predictor of food presentation at the water surface and at the end of the tank where the food was dispensed. Memories of these conditioned associations persisted for at least 2 days after training when fish were tested either as groups or as individuals. Control fish, for which the auditory or visual stimuli were specifically unpaired with food, showed no comparable responses. This simple, low‐cost, automated system permits scalable conditioning of zebrafish with minimal human intervention, greatly reducing both variability and labour‐intensiveness. It will be useful for studies of the neural basis of learning and memory, and for high‐throughput screening of compounds modifying those processes.

Skeletal Stiffening in an Amphibious Fish Out of Water is a Response to Increased Body Weight

ABSTRACT Terrestrial animals must support their bodies against gravity, while aquatic animals are effectively weightless because of buoyant support from water. Given this evolutionary history of minimal gravitational loading of fishes in water, it has been hypothesized that weight-responsive musculoskeletal systems evolved during the tetrapod invasion of land and are thus absent in fishes. Amphibious fishes, however, experience increased effective weight when out of water – are these fishes responsive to gravitational loading? Contrary to the tetrapod-origin hypothesis, we found that terrestrial acclimation reversibly increased gill arch stiffness (∼60% increase) in the amphibious fish Kryptolebias marmoratus when loaded normally by gravity, but not under simulated microgravity. Quantitative proteomics analysis revealed that this change in mechanical properties occurred via increased abundance of proteins responsible for bone mineralization in other fishes as well as in tetrapods. Type X collagen, associated with endochondral bone growth, increased in abundance almost ninefold after terrestrial acclimation. Collagen isoforms known to promote extracellular matrix cross-linking and cause tissue stiffening, such as types IX and XII collagen, also increased in abundance. Finally, more densely packed collagen fibrils in both gill arches and filaments were observed microscopically in terrestrially acclimated fish. Our results demonstrate that the mechanical properties of the fish musculoskeletal system can be fine-tuned in response to changes in effective body weight using biochemical pathways similar to those in mammals, suggesting that weight sensing is an ancestral vertebrate trait rather than a tetrapod innovation. Highlighted Article: Did weight responsiveness evolve during the tetrapod invasion of land or is it ubiquitous among vertebrates? Skeletal stiffening in amphibious fish out of water supports the latter hypothesis.

Distribution and chronotropic effects of serotonin in the zebrafish heart

Several lines of evidence suggest that serotonin (5-HT) has a regulatory role in cardiovascular function from embryogenesis through adulthood. However, the reported actions of 5-HT are often contradictory and include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction. Clarifying such cardiac effects requires further research and may benefit from utilizing a model simpler than the mammalian hearts traditionally used in these studies. In the present study, we describe the cardiac distribution and chronotropic responses of 5-HT in the zebrafish heart. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the involvement of 5-HT pathways, and to compare neural and direct myocardial pathways of biological action. Immunohistochemical methods revealed 5-HT in endocardial cells, glial-like cells, and intracardiac neurons in the atrium. Electrocardiogram (ECG) recordings combined with the administration of pharmacological agents demonstrated that 5-HT acted predominantly through direct myocardial pathways resulting in a reduction of heart rate. Overall, the results of this study contribute significant advances in the establishment of the zebrafish as a new model for studies of the role of 5-HT in autonomic cardiac control.

The in vitro zebrafish heart as a model to investigate the chronotropic effects of vapor anesthetics

In addition to their intended clinical actions, all general anesthetic agents in common use have detrimental intrasurgical and postsurgical side effects on organs and systems, including the heart. The major cardiac side effect of anesthesia is bradycardia, which increases the probability of insufficient systemic perfusion during surgery. These side effects also occur in all vertebrate species so far examined, but the underlying mechanisms are not clear. The zebrafish heart is a powerful model for studying cardiac electrophysiology, employing the same pacemaker system and neural control as do mammalian hearts. In this study, isolated zebrafish hearts were significantly bradycardic during exposure to the vapor anesthetics sevoflurane (SEVO), desflurane (DES), and isoflurane (ISO). Bradycardia induced by DES and ISO continued during pharmacological blockade of the intracardiac portion of the autonomic nervous system, but the chronotropic effect of SEVO was eliminated during blockade. Bradycardia evoked by vagosympathetic nerve stimulation was augmented during DES and ISO exposure; nerve stimulation during SEVO exposure had no effect. Together, these results support the hypothesis that the cardiac chronotropic effect of SEVO occurs via a neurally mediated mechanism, while DES and ISO act directly upon cardiac pacemaker cells via an as yet unknown mechanism.

Data on horizontal and vertical movements of zebrafish during appetitive conditioning

This article provides supporting data for the research article “A simple automated system for appetitive conditioning of zebrafish in their home tanks” (J.M. Doyle, N. Merovitch, R.C. Wyeth, M.R. Stoyek, M. Schmidt, F. Wilfart, A. Fine, R.P. Croll, 2016) [1]. In that article, we described overall movements of zebrafish toward a food source as a response to auditory or visual cues as conditioned stimuli in a novel learning paradigm. Here, we describe separate analyses of the vertical and horizontal components of the learned response. These data provide evidence that the conditioning might result from both classical conditioning of an innate response of zebrafish to move to the surface in response to food cues and secondary conditioning of the fish to associate a food presentation with a specific location in the tank. Movement data from the twenty trial acquisition period and probe trials from 2–32 days post conditioning are included.