Frank M. Smith

Structure and autonomic innervation of the swim bladder in the zebrafish (Danio rerio)

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. J. Comp. Neurol. 495:587–606, 2006.

The heart reinnervates after transplantation

BACKGROUND Whether cardiac reinnervation occurs after transplantation remains controversial. If reinnervation does occur, how sympathetic and parasympathetic efferent neurons do this remains unknown. METHODS Power spectral analysis of heart rate variability was assessed for 1 year after cardiac autotransplantation in 9 dogs. After induction of anesthesia 13 months after transplantation, cardiac and intrinsic cardiac neuronal responses elicited by both electrical stimulation of parasympathetic or sympathetic efferent neurons and systemic or local coronary artery administration of nicotine (5 microg/kg), angiotensin II (0.75 microg/kg), and tyramine (1.2 microg/kg) were studied. The transmembrane electrical properties of intrinsic cardiac neurons were studied in vitro. Ventricular tissue catecholamine content, alpha-tubulin expression, and beta-adrenergic receptor density and affinity were studied. The presence of axons crossing suture lines was sought histologically. RESULTS Nerves were identified crossing suture lines. Electrical or chemical (ie, nicotine or angiotensin II) activation of sympathetic efferent neurons enhanced cardiodynamics, as did tyramine. Stimulating vagal efferent preganglionic axons induced bradycardia in half of the dogs. Functional reinnervation did not correlate with specific power spectra derived from rate variability in the conscious state. Responding to nicotine and angiotensin II in situ, transplanted intrinsic cardiac neurons generated spontaneous activity. These neurons displayed nicotine-dependent synaptic inputs in vitro. Ventricular tissue had normal beta-adrenergic receptor affinity and density but reduced catecholamine and alpha-tubulin contents. CONCLUSIONS The intrinsic cardiac nervous system receives reduced input from extracardiac sympathetic efferent neurons after transplantation and inconsistent input from parasympathetic efferent preganglionic neurons. These heterogeneous neuronal inputs are not reflected in heart rate variability or ventricular beta-adrenergic receptor function. Transplanted angiotensin II-sensitive intrinsic cardiac neurons exert greater cardiac control than do nicotine-sensitive ones. The intrinsic cardiac nervous system remodels itself after cardiac transplantation, and this indicates that direct assessment of extracardiac and intrinsic cardiac neuronal behavior is required to fully understand cardiac control after transplantation.

Confocal Microscopic Localization of Constitutive and Heat Shock–Induced Proteins HSP70 and HSP27 in the Rat Heart

BackgroundHeat-shock treatment of rats elevates expression of heat-shock proteins, which play a role in improving the contractile recovery and reducing infarct size in hearts after ischemic injury. However, the location of these proteins in the heart is unknown. Methods and ResultsAnesthetized rats were heat-shocked by elevation of body temperature to 42°C to 42.5°C for 15 minutes, followed by 24 hours of recovery. Control and heat-shocked hearts were extirpated and perfused briefly with saline followed by 2% paraformaldehyde in PBS. Confocal immunofluorescence microscopy of control hearts revealed that HSP27 was localized in cardiomyocytes in a pattern reminiscent of Z bands and was colocalized with neuronal markers in somata and axons. No obvious change in HSP27 content or distribution occurred after heat shock. Confocal microscopy revealed little or no HSP70 in control hearts. After heat shock, HSP70 was detected neither in cardiomyocytes nor in neuronal elements within the heart, but HSP70 was abundant in small blood vessels found between the ventricular cardiomyocytes. ConclusionsHeat shock induces a cell type–specific expression of HSP70 in blood vessels but not myocytes or intrinsic cardiac neurons, suggesting that blood vessels play a primary role in myocardial protection.

From Inflation to Flotation: Contribution of the Swimbladder to Whole-Body Density and Swimming Depth During Development of the Zebrafish (Danio rerio)

Teleost fishes have body tissues that are denser than water, causing them to sink. Many teleosts therefore possess a gas-filled swimbladder that provides lift, allowing fish to attain neutral buoyancy. The importance of the swimbladder as a buoyancy aid during changing body sizes over ontogeny and its role in determining the swimming depth of fish remain unclear. In this study, we have used the zebrafish (Danio rerio) to investigate changes in the size and shape of the swimbladder during development and examine whether these changes affect the hydrostatic contribution of the swimbladder during swimming. Our results showed that swim-up behavior is critical for larvae to first inflate their swimbladder, decrease body density, and attain neutral buoyancy. Following inflation, we found a strong linear correlation between fish volume and swimbladder volume over ontogeny. This trend was supported by measures of the density of zebrafish, which was conserved within a narrow range between 1.00 +/- 0.001 and 0.996 +/- 0.001 g/cm(3) despite an increase in the swimming depth of zebrafish, which occurred upon transition to a double-chambered organ. Finally, we demonstrated that the contribution of the swimbladder keeps the fish within 1.7% of neutral buoyancy throughout larval development.

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.

Extrinsic inputs to intrinsic neurons in the porcine heart in vitro

Convergence of inputs from extrinsic cardiac nerves [vagus and cardiopulmonary (CPN)] on intrinsic cardiac neurons was investigated in the pig ( Sus scrofa). A segment of the right atrial wall containing epicardial neurons along with attached stumps of the right vagus nerve and CPN was maintained in vitro; intracellular recordings were made from 57 neurons. Three types of neuron were identified by their responses to long intracellular depolarizing current pulses: phasic [discharged 1 action potential (AP); 40%]; accommodating (discharged multiple APs decrementing in frequency during pulse; 33%); and tonic (discharged multiple APs at a high frequency; 27%). Sixty-six percent of the neurons responded with excitatory postsynaptic potentials (EPSP) to vagal nerve stimulation; two-thirds of these cells fired APs when EPSP amplitude exceeded threshold level. Postsynaptic responses to vagal nerve stimulation were mediated by nicotinic ion channels; responses were eliminated by hexamethonium. CPN stimulation produced EPSPs but no APs in 17% of the neurons. All neurons responding with postsynaptic depolarizations to CPN stimulation also received vagal inputs. Combined stimulation of the vagus nerve and CPN produced APs in all but one of these neurons. Timolol eliminated postsynaptic responses from CPN stimulation, indicating that these responses involved β-adrenergic receptors and likely resulted from activation of sympathetic postganglionic terminals. These results show that some intrinsic cardiac neurons receive convergent inputs from the CPN and vagus nerve. It is suggested that such neurons represent intraganglionic sites for sympathetic-parasympathetic interactions in neural control of the heart.

Chronic spinal cord stimulation modifies intrinsic cardiac synaptic efficacy in the suppression of atrial fibrillation

We sought to determine whether spinal cord stimulation (SCS) therapy, when applied chronically to canines, imparts long-lasting cardio-protective effects on neurogenic atrial tachyarrhythmia induction and, if so, whether its effects can be attributable to i) changes in intrinsic cardiac (IC) neuronal transmembrane properties vs ii) modification of their interneuronal stochastic interactivity that initiates such pathology. Data derived from canines subjected to long-term SCS [(group 1: studied after 3-4 weeks SCS; n = 5) (group 2: studied after 5 weeks SCS; n = 11)] were compared to data derived from 10 control animals (including 4 sham SCS electrode implantations). During terminal studies conducted under anesthesia, chronotropic and inotropic responses to vagal nerve or stellate ganglion stimulation were similar in all 3 groups. Chronic SCS suppressed atrial tachyarrhythmia induction evoked by mediastinal nerve stimulation. When induced, arrhythmia durations were shortened (controls: median of 27 s; SCS 3-4 weeks: median of 16s; SCS 5 weeks: median of 7s). Phasic and accommodating right atrial neuronal somata displayed similar passive and active membrane properties in vitro, whether derived from sham or either chronic SCS group. Synaptic efficacy was differentially enhanced in accommodating (not phasic) IC neurons by chronic SCS. Taken together these data indicate that chronic SCS therapy modifies IC neuronal stochastic inter-connectivity in atrial fibrillation suppression by altering synaptic function without directly targeting the transmembrane properties of individual IC neuronal somata.

Actions of substance P on membrane potential and ionic currents in guinea pig stellate ganglion neurons

Neuropeptides are known to modulate the excitability of mammalian sympathetic neurons by their actions on various types of K+ and Ca2+ channels. We used whole cell patch-clamp recording methods to study the actions of substance P (SP) on dissociated adult guinea pig stellate ganglion (SG) neurons. Under current-clamp conditions, SG neurons exhibited overshooting action potentials followed by afterhyperpolarizations (AHP). The K+ channel blocker tetraethylammonium (1 mM), the Ca2+ channel blocker Cd2+ (0.1-0.2 mM), and SP (500 nM) depolarized SG neurons, decreased the AHP amplitude, and increased the action potential duration. In the presence of Cd2+, the effect of SP on membrane potential and AHP was reduced. Under voltage-clamp conditions, several different K+ currents were observed, including a transient outward K+ conductance and a delayed rectifier outward K+ current ( I K) consisting of Ca2+-sensitive [ I K(Ca)] and Ca2+-insensitive components. SP (500 nM) inhibited I K. Pretreatment with Cd2+ (20-200 μM) or the high-voltage-activated Ca2+ channel blocker ω-conotoxin (10 μM) blocked SPs inhibitory effects on I K. This suggests that SP reduces I K primarily through the inhibition of I K(Ca) and that this may occur, in part, via a reduction of Ca2+ influx through voltage-dependent Ca2+ channels. SPs actions on I K were mediated by a pertussis toxin-insensitive G protein(s) coupled to NK1 tachykinin receptors. Furthermore, we have confirmed that 500 nM SP reduced an inward Cd2+- and ω-conotoxin-sensitive Ba2+ current in SG neurons. Thus the actions of SP on I K(Ca) may be due in part to a reduction in Ca2+influx occurring via N-type Ca2+channels. This study presents the first description of ionic currents in mammalian SG neurons and demonstrates that SP may modulate excitability in SG neurons via inhibitory actions on K+ and Ca2+ currents.

The contribution of the swimbladder to buoyancy in the adult zebrafish (Danio rerio): a morphometric analysis.

Many teleost fishes use a swimbladder, a gas‐filled organ in the coelomic cavity, to reduce body density toward neutral buoyancy, thus minimizing the locomotory cost of maintaining a constant depth in the water column. However, for most swimbladder‐bearing teleosts, the contribution of this organ to the attainment of neutral buoyancy has not been quantified. Here, we examined the quantitative contribution of the swimbladder to buoyancy and three‐dimensional stability in a small cyprinid, the zebrafish (Danio rerio). In aquaria during daylight hours, adult animals were observed at mean depths from 10.1 ± 6.0 to 14.2 ± 5.6 cm below the surface. Fish mass and whole‐body volume were linearly correlated (r2 = 0.96) over a wide range of body size (0.16–0.73 g); mean whole‐body density was 1.01 ± 0.09 g cm−3. Stereological estimations of swimbladder volume from linear dimensions of lateral X‐ray images and direct measurements of gas volumes recovered by puncture from the same swimbladders showed that results from these two methods were highly correlated (r2 = 0.85). The geometric regularity of the swimbladder thus permitted its volume to be accurately estimated from a single lateral image. Mean body density in the absence of the swimbladder was 1.05 ± 0.04 g cm−3. The swimbladder occupied 5.1 ± 1.4% of total body volume, thus reducing whole‐body density significantly. The location of the centers of mass and buoyancy along rostro‐caudal and dorso‐ventral axes overlapped near the ductus communicans, a constriction between the anterior and posterior swimbladder chambers. Our work demonstrates that the swimbladder of the adult zebrafish contributes significantly to buoyancy and attitude stability. Furthermore, we describe and verify a stereological method for estimating swimbladder volume that will aid future studies of the functions of this organ. J Morphol., 2008.

Electrophysiological properties of in vitro intrinsic cardiac neurons in the pig (Sus scrofa).

Physiological properties and synaptically mediated responses of 34 ganglionated plexus neurons from the right atrium of the pig heart were studied with in vitro intracellular recording techniques. Whole-cell input resistance of these neurons was lower, time constant was shorter, and threshold for directly evoked action potentials was higher than the same properties in extracardiac autonomic neurons. Long intracellular depolarizing current pulses (400-500 ms) failed to generate more than one or two action potentials. Nicotinic and non-nicotinic synapses were present on neurons in cardiac ganglia and neuronal properties could be modified by norepinephrine. Based on their physiological properties, cardiac ganglionated plexus neurons in the pig appear to represent a distinct population of autonomic neurons that may be capable of intracardiac integration of efferent information to the heart.