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1Department of Pharmacology and 3Specialized Neuroscience Research Program, Howard University College of Medicine, Washington, District of Columbia 20059; and 2Department of Pharmacology, East Tennessee State University, James H. Quillen College of Medicine, Johnson City, Tennessee 37614
Submitted 16 June 2003 ; accepted in final form 24 October 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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intracardiac ganglia; retrograde transport; nucleus ambiguus; ultrastructure; posterior atrial ganglion
In a recent report, we have shown that the vagal postganglionic control of cardiac rate is mediated by two separate but interconnected intracardiac ganglia, i.e., the SA and posterior atrial (PA) ganglion (23). However, virtually nothing is known about the VPNs and central afferents that regulate the functions of the PA ganglion. Because microinjections of neuropeptide Y (NPY) into the NA-VL cause bradycardia (36), in the present report, we 1) define the light microscopic distribution of VPNs projecting to the PA ganglion, 2) describe the ultrastructural characteristics of these neurons, 3) test the hypothesis that NPY-immunoreactive (IR) afferent nerve terminals synapse on the soma and dendrites of VPNs that regulate the function of the PA ganglion, and 4) test the hypothesis that separate populations of VPNs project to the PA and SA ganglia.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The Institutional Animal Care and Use Committee of Howard University reviewed and approved the experimental design of all animal experiments. Experiments were performed on 15 mongrel cats of either sex weighing 2.8–4.0 kg. Baseline vital signs of temperature, respiratory rate, and ECG were recorded. Cats were pretreated with 0.05 mg/kg of atropine to reduce secretions, followed by 22 mg/kg of ketamine and 0.2 mg/kg of acepromazine for induction of anesthesia. Cats were prepared for surgery by inserting an intravenous catheter into the brachial vein and intubating with a cuffed endotracheal tube. Isoflurane gas was then used to bring the animal to a surgical plane of anesthesia. Cardiac rate and blood oxygen concentration were monitored with a pulse-oximeter (Vet-Ox) via the lingual artery. The cat was artificially respired on a positive-pressure respirator with a tidal volume setting between 75 and 150 ml and cycling at 12 breaths/min. The cat was given a 95% oxygen-5% carbon dioxide gas mixture to breathe. An incision was made into the pericardium that was large enough to expose the heart and allow for identification and access to both the PA ganglion, located on the rostral dorsal surface of the right atrium between the superior vena cava and the aorta, and the SA ganglion, found at the junction of the superior vena cava and right atrium overlying the right pulmonary veins (23, 28). In one set of experiments (n = 6), 10 µl of a 1% solution of the beta subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) dissolved in 2% DMSO in distilled water were injected into the PA ganglion in three or four parts. In one control animal, 10 µl of a 1% solution of the CTB-HRP was injected into the pericardial sac over the area of the PA ganglion. In a second set of experiments, 10 µl of a 2% solution of fast blue and 10 µl of a 2% solution of diamidino yellow both dissolved in 2% DMSO in ethylene glycol were, respectively, injected into the SA and PA ganglia using a counterbalanced design in three to four parts in six animals or into the pericardial sac as a control for extraneous leakage of the tracer in two animals. After injections were made, the pericardium was closed, the muscles and skin were sutured in layers, spontaneous respiration was reestablished, fluids along with the potent analgesic butorphanol (0.2 mg/kg) and the antibiotic penicillin procaine G (30,000 IU/kg) were administered, and the animal was awakened from anesthesia. Postoperatively, butorphanol was given (0.2 mg/kg) twice daily for at least 2 days to reduce pain, and penicillin procaine G (30,000 IU/kg) was administered daily for at least 5 days.
Cats in which the tracer CTB-HRP was used were killed via intravascular perfusion 3 days after the day of surgery. Cats in which the fluorescent retrograde tracers diamidino yellow and fast blue were used (30, 44) were killed via intravascular perfusion 10 days after surgery. On the day of perfusion, cats were deeply anesthetized with 50 mg/kg pentobarbital sodium administered intraperitoneally and perfused intravascularly with 1,000 ml of oxygenated 0.1 M phosphate-buffered saline containing 2,500 U of heparin (PBS-Hep), and 4 liters of a phosphate-buffered solution containing 1.75% acrolein and 0.5% paraformaldehyde, as previously described in detail (41). This combination of fixatives provides reasonable ultrastructural morphology while preserving the antigenicity of the tissues for subsequent immunocytochemical study. After the perfusion, the animal's brain was removed, and transverse serial 40-µm-thick sections of the medulla were cut from the level of the spinomedullary junction to the caudal border of the pons using a Vibratome. Brain sections were then processed histochemically and immunocytochemically for later light and electron microscopic analysis.
In animals in which fluorescent retrograde tracers were injected into the heart, cats were anesthetized as described above and then perfused intravascularly with 1,000 ml of oxygenated PBS-Hep solution, followed by 4 liters of PBS containing 4% paraformaldehyde. Brains were removed and postfixed in the same solution for 2 h. Brains were then cryoprotected as previously described in detail (8). Brain stems were frozen on dry ice and stored at -80°C.
CTB-HRP Histochemistry
Free-floating sections of brain tissue extracted from animals that were previously injected with the retrograde tracer CTB-HRP were treated to remove reactive aldehydes by placing them into a 1% sodium borohydride solution for 30 min. Tissue sections were then washed three times with a 0.1 M sodium phosphate-buffered solution, pH 6.0, and then processed to reveal CTB-HRP-labeled cell bodies by a modification of the tungstate stabilized tetramethylbenzidine (TMB) method of Weinberg and Van Eyck (51) as previously described in detail (41).
Immunocytochemistry
All tissue sections were subsequently incubated for 30 min in a solution of 50% absolute ethanol in distilled water to enhance the penetration of antibodies throughout the tissue (34), followed by three washes with PBS. Tissues were then incubated in 0.1 M phosphate-buffered solution containing 1.0% BSA for 30 min and then incubated in rabbit anti-NPY primary antiserum (Peninsula) diluted 1:2,000 in 0.1% BSA dissolved in 0.1 M PBS overnight. The immunocytochemical procedure utilized to demonstrate NPY-IR sites was an avidinbiotin-based method utilizing the Vectastain Elite ABC kit as previously described (41). HRP was visualized with a second glucose oxidase reaction utilizing diaminobenzidine (DAB) as the chromogen. This reaction results in an amorphous electron dense reaction product in the electron microscope. The specificity of the NPY antisera utilized in the present study was further characterized utilizing the immunodot-blot method of Larsson (31).
Processing for Light Microscopy
Transmitted light microscopy. Tissue sections were mounted onto slides, dehydrated with ethanol, cleared with xylene, and coverslipped with Permount (Fisher Scientific). The slides were examined under a Nikon Microphot FXA light microscope using bright-field, dark-field, or Nomarski differential interference contrast optics. The distribution of retrogradely labeled VPNs was determined by recording the total number of labeled cells that were found in sections taken from the following levels of the brain stem: 1 mm caudal to the area postrema (AP); the AP; 1 mm rostral to the AP; and 2 mm rostral to the AP.
Incident light fluorescence microscopy. Paraformaldehyde-fixed frozen brain stems were sectioned on a cryostat. Transverse serial 40-µm-thick sections of the medulla were cut from the level of the spinomedullary junction to the caudal border of the pons. Alternate sections were mounted onto glass slides. Slides were coverslipped with a 1:1 mixture of glycerol and distilled water, and the tissues were examined under a Nikon FXA photomicroscope through Nikon CFI Plan Fluor objectives under UV fluorescence. The UV filters were configured with an excitation filter of 365 nm and a barrier filter of 400 nm. These filters allowed for simultaneous visualization of both fluorescent tracers. Diamidino yellow labels the nucleus and appears yellow while fast blue labels the cytoplasm and appears blue (30, 44).
Processing for Electron Microscopy
In four animals, tissues were rinsed in PBS and postfixed in 2% osmium tetroxide for 1 h, dehydrated through a graded series of alcohols and propylene oxide, embedded in resin (Embed 812) between two sheets of plastic (Aclar: Dupont), and cured at 60°C for 48 h. Embedded tissues were examined in a light microscope, and areas of interest including the NA-VL were cut out and reembedded in Beem capsulses. Serial ultrathin sections of the reembedded tissues were cut on an ultramicrotome (Reichert, Ultracut S) at 
75 nm thickness (silver-gold interference color), collected on uncoated copper mesh grids, poststained with uranyl acetate and Reynolds lead citrate, and examined in a JEOL-JEM-1210 electron microscope at 50 kV.
Two 40-µm-thick tissue sections that contained the best combination of morphological preservation and histochemical/immunocytochemical labeling were examined from each animal. From each thick section, five ultrathin sections separated by 8 µm each were utilized for subsequent quantitative analysis. The spatial separation provided between the samples clearly prevented duplicate counts of the same terminal in our five samples through the neuropil. The number of NPY-immunoreactive and unlabeled nerve terminals making axosomatic or axodendritic synapses on cardioinhibitory VPNs retrogradely labeled from the PA ganglion were recorded.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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After the injection of CTB-HRP into the PA ganglion, a column of retrogradely labeled neurons was observed bilaterally in the ventrolateral medulla. This column extended from the spinomedullary junction to the caudal boundary of the facial nucleus. No retrograde labeling was observed in the dorsal motor nucleus of the vagus (DMV). The relative number of retrogradely labeled neurons found at different anteroposterior levels of the NA varied (Fig. 1). The majority of cells was found concentrated at the level of the AP, with a tapering in the number of cells found at the more extreme rostral and caudal levels of the medulla. By comparison, when CTB-HRP was injected into the pericardial sac in the control animal, only one labeled cell was found in the medulla. It was located in the intermediate zone between the NA and the DMV.
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As we have previously demonstrated (38), NPY-IR neurons and their processes are found in the ventrolateral medulla. These neurons were interspersed with retrogradely labeled VPNs projecting to the PA ganglion. Some NPY-IR processes were noted to be in close apposition to these retrogradely labeled VPNs (Fig. 2).
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Incident Light Fluorescent Microscopy
After injections of diamidino yellow or fast blue into either the SA or PA ganglia, three populations of retrogradely labeled fluorescent neurons were identified in the NA-VL. These neurons contained either diamidino yellow alone, fast blue alone, or both diamidino yellow and fast blue (Fig. 3). The mean total number of retrogradely labeled cells observed in the entire NA-VL was 311 ± 53 (mean ± SE). However, there were no statistically significant differences in the number of labeled neurons across the three populations of neurons that contained these tracers (Fig. 4) [ANOVA, F(2,15) = 1.84, P > 0.05].
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After injections of diamidino yellow or fast blue in the control group, only 14.0 ± 1.0% of the labeled neurons contained a single fluor. By comparison, a total of 77.7 ± 5.1% of the neurons was labeled with one fluor in the experimental animals. Furthermore, the percentage of single and double labeled cells in the experimental group was statistically significantly different from that found in the control group (P < 0.0001).
Characterization of the NPY Antiserum
The sensitivity and specificity of the NPY antibody was characterized. Neurotransmitters and synthetic peptides used included: porcine NPY, porcine/human NPY fragment 18–36, human NPY fragment 1–24, peptide YY, serotonin, substance P (SP), norepinephrine (NE), neurotensin (NT), leu-enkephalin (L-Enk), and met-enkephalin (M-Enk). The rabbit anti-NPY serum was able to recognize NPY between concentrations of 10-3 and 10-6 M and NPY fragments 18–36 and 1–24 between concentrations of 10-3 and 10-5 M. This antibody did not recognize peptide YY, serotonin, SP, NE, NT, M-Enk, or L-Enk even at concentrations as high as 1 mM.
Electron Microscopy
Retrogradely labeled neurons and their processes were readily identified even at low scanning magnifications in the electron microscope due to the presence of a characteristic electron dense crystalline TMB-tungstate reaction product (Fig. 5). This reaction product was found primarily in the perikarya and proximal dendrites (Figs. 6 and 7A); however, a few labeled distal dendrites were also detected (Fig. 7B). Retrogradely labeled neurons were relatively large (37.6 ± 2.7 by 21.3 ± 3.4 µm) with abundant cytoplasm and intracellular organelles (Fig. 5), rare somatic (Fig. 8A) and dendritic (Fig. 7A) spines, and round nuclei (Fig. 5), occasionally showing a prominent nucleolus. Retrogradely labeled axons were found to be myelinated (Fig. 8B). In the tissues examined, no unmyelinated axons or nerve terminals were found to contain the crystalline TMB-tungstate reaction product.
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NPY-IR perikarya, dendrites, and terminals were readily identified by the presence of a characteristic amorphous DAB reaction product (Figs. 6, 7, A and B, and 9). They had relatively sparse cytoplasm and invaginated nuclei (Fig. 9). Numerous NPY-IR axon terminals were found in the NA-VL. Some NPY-IR terminals formed synapses on retrogradely labeled VPNs (Figs. 6 and 7, A and B). NPY-IR terminals commonly contained multiple small clear vesicles and one or more large dense core vesicles. A total of 7 ± 2% of the terminals making synaptic contacts with retrogradely labeled neurons were NPY-IR, whereas another 11 ± 2% were in close apposition to these VPNs.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Combined anatomic and physiological studies previously conducted in our laboratory have demonstrated that cardioinhibitory neurons in the external formation of the NA are divided into at least three different functional categories. These groups include negative chronotropic, negative dromotropic, and negative inotropic neurons (14, 37, 39, 40). The distribution of these functionally distinct cardioinhibitory groups of neurons is not identical. In the present report, we have demonstrated that negative chronotropic VPNs that are retrogradely labeled from the PA ganglion are distributed exclusively in the NA-VL and that the largest concentration of these neurons is found at the level of the AP. This distribution is quite similar to that found after injections of retrograde tracers into the SA ganglion (41). VPNs that project to the PA ganglion were also examined by electron microscopy to characterize the ultrastructural characteristics of these neurons. The neurons were relatively large with abundant cytoplasm and intracellular organelles and contained a round uninvaginated nucleus often with a prominent nucleolus. These neurons were found to have rare somatic or dendritic spines and an abundance of rough endoplasmic reticulum. Furthermore, the axons of these neurons were myelinated. This result provides anatomic support for the electrophysiological observation that vagal preganglionic cardioinhibitory neurons in the NA have axons that conduct action potentials in the range of B-fibers (29, 42). Furthermore, these morphological data indicate that the ultrastructural characteristics of VPNs projecting to the PA ganglion closely match those of neurons retrogradely labeled from the SA ganglion (41).
Our data indicate that there are multiple similarities between VPNs innervating the PA ganglion and those projecting to the SA ganglion. For instance, VPNs innervating the SA and PA ganglia 1) are found solely in the NA-VL, 2) have very similar distributions in the brain stem with the majority of their cells found at the level of the AP, 3) have similar size and ultrastructural characteristics, 4) receive synaptic inputs from NPY-IR nerve terminals, and 5) innervate ganglia mediating negative chronotropic effects on the heart. With so many similarities, it was imperative to determine whether the PA and the SA ganglia are innervated by the same or separate groups of VPNs in the NA-VL. To test this important question, two different retrograde tracers were injected into the SA and PA ganglia, respectively, using a counterbalanced design. Neurons retrogradely labeled with one fluor were considered to project to the ganglia in which that fluor was injected. Neurons labeled with both were considered to send projections to both ganglia. Three separate but approximately equal size populations of negative chronotropic VPNs were found in the NA-VL. One population projected exclusively to the SA ganglion, the second population projected exclusively to the PA ganglion, and the third population projected to both the SA and PA ganglia.
In analogous experiments in cats involving the injection of two fluorescent tracers into other pairs of intracardiac ganglia, only a tiny minority of the retrogradely labeled cells was found to project to both ganglia. Blinder et al. (7, 8) found that 90–97% of the neurons that were retrogradely labeled after injecting two fluorescent tracers into the SA and AV ganglia, or the SA and CV ganglia, respectively, were single labeled. In a similar study, in piglets, 100% of the cells found in the NA-VL after injections of two retrograde tracers into the AV and SA ganglia or into either the SA or AV ganglia and a ventricular locus were single labeled (25). In the present data, 78% of retrogradely labeled neurons were single labeled. By comparison, in the control animals, only 14% of labeled neurons contained a single fluor. Furthermore, the percentage of either single or double labeled cells in the experimental group was statistically significantly different from that found in the control group (P < 0.0001). We conclude from these results that there was minimal leakage of tracer from its injection sites within the intracardiac ganglia in the experimental animals and that neurons containing both fluors in the experimental animals represent neurons that project to both the SA and PA ganglia. In summary, the data support the hypothesis that separate and distinct populations of VPNs innervate intrinsic cardiac ganglia that mediate AV conduction and left ventricular contractility, whereas intrinsic cardiac ganglia that directly or indirectly mediate control of cardiac rate are innervated by three further populations of VPNs. The data further indicate that there is considerable redundancy in the central neural mechanisms responsible for regulating heart rate. An analogous redundancy is found within the heart (23), wherein two separate intracardiac ganglia (the SA and PA ganglia) interdependently mediate the vagal control of cardiac rate. Such redundancy provides a neural framework whereby heart rate can be subtly modulated at both the level of the central nervous system and the heart. Collectively, these data imply that the central mechanisms that control cardiac rate are more complex than previously recognized. Further studies, however, will have to be conducted to determine the specific physiological role(s) this complex cardiac neural circuitry plays in the precise regulation of cardiac rate.
Numerous studies utilizing retrograde or transganglionic viral tracers have reported that the DMV serves as one of the sources of VPNs innervating the heart (13, 21, 22, 48). Cheng et al. (11) have further demonstrated that when an anterograde tracer is injected into the DMV, a substantial population of labeled axons and terminals can be detected in the rat atria. However, specific functional roles of the cardioinhibitory VPNs contained within this nucleus are still uncertain. Geis et al. (21, 22) reported that electrical stimulation of cardioinhibitory neurons in the DMV exerts a negative inotropic effect, but these findings have been challenged by Ford et al. (16). Their data suggest that the DMV has no consistent chronotropic, dromotropic, or inotropic effects on the heart. We have previously reported that injections of a retrograde tracer into the AV ganglion result in the labeling of significant numbers of neurons in the DMV (39). This suggests that some VPNs in the DMV influence AV conduction, but further physiological experiments are needed to refine our understanding of the role(s) of the DMV on cardiac function(s). Injections of retrograde tracers into the SA ganglion (41) or the PA ganglion (present data) label neurons exclusively in the NA-VL. The present data therefore suggest that VPNs responsible for modulating cardiac rate, either via the PA or SA ganglia, are not located in the DMV.
A number of previous studies have investigated the central effects of NPY on the cardiovascular system (3, 26, 36, 47, 49). Macrae and Reid (36), in one such study, showed that microinjections of NPY into the region of the NA-VL produced bradycardia. Later, Batten (4) found that cardiac VPNs in the NA are surrounded by nerve fibers immunoreactive for NPY. Recently, our laboratory has shown in a series of ultrastructural studies that NPY-IR nerve terminals make axosomatic and axodendritic synapses on negative dromotropic (20) and negative chronotropic (20, 32) VPNs in the NA-VL. In the present data, we have shown NPY-IR terminals formed 7.4 ± 2% of the asymmetric axodendritic and axosomatic synapses detected on VPNs retrogradely labeled from the PA ganglion. Another 11 ± 2% of the NPY terminals were in close apposition to these VPNs but did not show a synapse in the planes of section that were examined. These data support the previous morphological and physiological data that indicate that NPY may play a substantial role in modulating multiple indexes of heart function.
In summary, VPNs projecting to the PA ganglion are found primarily in the NA-VL at the level of the AP. These neurons are relatively large with abundant cytoplasm and intracellular organelles, rare somatic and dendritic spines, and round nuclei; have myelinated axons; and receive axodendritic and axosomatic synaptic inputs from NPY-IR nerve terminals. There are statistically three equal populations of vagal preganglionic neurons in the NA-VL that mediate an effect on cardiac rate (via the SA and PA ganglia). One population projects to only the SA ganglion, a second population projects to only the PA ganglion, and a third population projects to both the SA and PA ganglia. Therefore, there are both shared and independent pathways involved in the vagal preganglionic controls of cardiac rate. These data are consistent with the hypothesis that the neural control of cardiac rate is coordinated by interdependent central and peripheral mechanisms. The present data indicate that the neuronal circuits that mediate vagal control of cardiac rate are more complex than previously recognized.
Perspectives
Drugs that directly act on the heart in the treatment of an assortment of cardiac disorders often exert undesirable but unavoidable side effects. Thus, for example, sympathomimetic drugs that enhance myocardial contractility in congestive heart failure not uncommonly also cause an undesirable tachycardia. This is often the case because the same receptor that provides the desired therapeutic action also mediates undesirable side effects. Unlike the heart, the brain contains a diverse array of potential neurotransmitters and receptors that could potentially influence cardiac or cardiovascular functions. One of the goals of our research efforts has been to determine whether a new generation of drugs may be developed that can target functionally selective neurons in the brain to elicit selective changes in various parameters of cardiac performance. One approach to achieving this goal would be to determine whether there are qualitative differences in the distribution of immunocytochemically characterized nerve terminals synapsing on functionally selective VPNs. In this effort, we have demonstrated that substance P-immunoreactive nerve terminals synapse on negative chronotropic VPNs but not on negative dromotropic or negative inotropic VPNs (6, 40, 41). Correspondingly, microinjections of substance P into the NA-VL selectively induce bradycardia (40). These data indicate that centrally acting neurokinin 1 receptor agonists could potentially be useful in the treatment of certain arrhythmias such as atrial fibrillation because they would induce bradycardia without undesirable actions on AV conduction or left ventricular contractility. In the present report, we have shown that NPY-IR terminals synapse on negative chronotropic VPNs retrogradely labeled from the PA ganglion. NPY-IR terminals have also been found to synapse on VPNs retrogradely labeled from the SA ganglion (32), the AV ganglion (20, 32), and the CV ganglion (43). These data indicate that NPY serves as an important neurotransmitter involved in modulating multiple vagally mediated cardiac effects. At first glance, it would also suggest that centrally acting NPY agonists would not be useful as selective tools to influence cardiac functions. However, the effects of NPY are mediated through at least four receptor subtypes, Y1, Y2, Y3, and Y5 (2, 27), any of which could potentially serve as postsynaptic receptors on a specific functional category of cardioinhibitory VPNs. Thus it is still possible that agonists for selective NPY receptor subtypes could mediate selective effects on cardiac function. Further ultrastructural and physiological experiments will be required to clarify the potential roles of NPY and its receptors in the modulation of cardioinhibitory VPNs.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This research was supported in part by grants from the National Heart, Lung, and Blood Institute (NHLBI) (RO1-HL-51917) and the American Heart Association to V. J. Massari and from the NHLBI (R01-HL-58140) to J. L. Ardell. Additional funding was provided by the Gustavus and Louise Pfeiffer Research Foundation to A. L. Gray and by the Specialized Neuroscience Research Program (1U54-NS-39407; M. A. Haxhiu, Principal Investigator).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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