Parasympathetic control of the heart. III.

Neuropeptide Y-immunoreactive nerve terminals synapse on three populations of negative chronotropic vagal preganglionic neurons
Alrich L. Gray, Tannis A. Johnson, Jean-Marie Lauenstein, Stephen S. Newton, Jeffrey L. Ardell and V. John Massari
J Appl Physiol 96:2279-2287, 2004. First published 20 February 2004; doi:10.1152/japplphysiol.00621.2003 You might find this additional info useful... This article cites 47 articles, 16 of which can be accessed free at: http://jap.physiology.org/content/96/6/2279.full.html#ref-list-1 This article has been cited by 1 other HighWire hosted articles Stochastic behavior of atrial and ventricular intrinsic cardiac neurons M. Waldmann, G. W. Thompson, G. C. Kember, J. L. Ardell and J. A. Armour J Appl Physiol, August 1, 2006; 101 (2): 413-419. [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://jap.physiology.org/content/96/6/2279.full.html Additional material and information about Journal of Applied Physiology can be found at: http://www.the-aps.org/publications/jappl

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J Appl Physiol 96: 22792287, 2004. First published February 20, 2004; 10.1152/japplphysiol.00621.2003.

Parasympathetic control of the heart. III. Neuropeptide Y-immunoreactive nerve terminals synapse on three populations of negative chronotropic vagal preganglionic neurons
Alrich L. Gray,1 Tannis A. Johnson,1 Jean-Marie Lauenstein,1 Stephen S. Newton,1 Jeffrey L. Ardell,2 and V. John Massari1,3
Department 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 nal form 24 October 2003
1

Gray, Alrich L., Tannis A. Johnson, Jean-Marie Lauenstein, Stephen S. Newton, Jeffrey L. Ardell, and V. John Massari. Parasympathetic control of the heart. III. Neuropeptide Y-immunoreactive nerve terminals synapse on three populations of negative chronotropic vagal preganglionic neurons. J Appl Physiol 96: 22792287, 2004. First published February 20, 2004; 10.1152/japplphysiol.00621.2003.The vagal postganglionic control of cardiac rate is mediated by two intracardiac ganglia, i.e., the sinoatrial (SA) and posterior atrial (PA) ganglia. Nothing is known about the vagal preganglionic neurons (VPNs) that innervate the PA ganglion or about the neurochemical anatomy of central afferents that innervate these VPNs. These issues were examined using light microscopic retrograde labeling methods and dual-labeling electron microscopic histochemical and immunocytochemical methods. VPNs projecting to the PA ganglion are found in a narrow column exclusively in the ventrolateral nucleus ambiguus (NA-VL). These neurons are relatively large (37.6 2.7 m by 21.3 3.4 m) with abundant cytoplasm and intracellular organelles, rare somatic and dendritic spines, round uninvaginated nuclei, and myelinated axons. Previous physiological data indicated that microinjections of neuropeptide Y (NPY) into the NA-VL cause negative chronotropic effects. The present morphological data demonstrate that NPY-immunoreactive nerve terminals formed 18 4% of the axodendritic or axosomatic synapses and close appositions on VPNs projecting to the PA ganglion. Three approximately equal populations of VPNs in the NA-VL were retrogradely labeled from the SA and PA ganglia. One population each projects to the SA ganglion, the PA ganglion, or 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 central and peripheral parasympathetic controls of cardiac rate are coordinated by multiple potentially redundant and/or interacting pathways and mechanisms. intracardiac ganglia; retrograde transport; nucleus ambiguus; ultrastructure; posterior atrial ganglion
FUNCTIONALLY SELECTIVE INTRINSIC cardiac ganglia found on the surface of the heart are innervated by cardioinhibitory preganglionic vagal motoneurons that originate mainly in the ventrolateral nucleus ambiguus (NA-VL) (13, 16, 24, 25, 35). There appears to be a cardiotopic organization of functionally selective vagal preganglionic neurons (VPNs) in the brain stem (7, 17, 3941) that is analogous to the regional organization of functionally selective vagal postganglionic neurons in the heart (1, 5, 9, 10, 12, 15, 18, 19, 23, 45, 46, 50). For example, the

intramedullary distribution within the NA-VL of VPNs that projects to functionally selective chronotropic, dromotropic, or inotropic intracardiac ganglia [i.e., the sinoatrial (SA), atrioventricular (AV), and cranioventricular (CV) ganglia (for details and references, see Refs. 23 and 28)] is not identical. Neuroanatomic data further indicate that separate populations of VPNs project to the SA, AV, or CV ganglia. Thus, when two different uorescent retrograde tracers are simultaneously injected into the SA and AV ganglia or SA and CV ganglia, three separate populations of vagal preganglionic chronotropic, dromotropic, and inotropic neurons were found in the NA-VL, and very few double-labeled neurons were found (7, 8). Furthermore, physiological evidence indicates that microinjections of excitatory amino acids into different areas of the NA-VL that are retrogradely labeled from selected intracardiac ganglia can elicit selective changes in either cardiac rate, atrioventricular (AV) conduction, or left ventricular contractility (14, 17, 39, 41). These data support the hypothesis that the preganglionic vagal controls of cardiac rate, AV conduction, and ventricular contractility are independently mediated by at least three separate populations of cardioinhibitory VPNs. 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) dene 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.
MATERIALS AND METHODS

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Retrograde Tracing Studies 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
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Address for reprint requests and other correspondence: V. John Massari, Dept. of Pharmacology, Howard Univ. College of Medicine, 520 W St. N.W., Washington, DC 20059 (E-mail: vmassari@howard.edu). http://www.jap.org

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of either sex weighing 2.84.0 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. Isourane 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 articially 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 identication 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 l 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 l 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 l of a 2% solution of fast blue and 10 l 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, uids 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 uorescent 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 xatives provides reasonable ultrastructural morphology while preserving the antigenicity of the tissues for subsequent immunocytochemical study. After the perfusion, the animals brain was removed, and transverse serial 40- m-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 uorescent 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 postxed 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 80C. CTB-HRP Histochemistry Free-oating sections of brain tissue extracted from animals that were previously injected with the retrograde tracer CTB-HRP were
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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 modication 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 phosphatebuffered 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 specicity 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 Scientic). The slides were examined under a Nikon Microphot FXA light microscope using bright-eld, dark-eld, 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 uorescence microscopy. Paraformaldehyde-xed frozen brain stems were sectioned on a cryostat. Transverse serial 40- m-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 uorescence. The UV lters were congured with an excitation lter of 365 nm and a barrier lter of 400 nm. These lters allowed for simultaneous visualization of both uorescent 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 postxed 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 60C 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- m-thick tissue sections that contained the best combination of morphological preservation and histochemical/immunocytochemical labeling were examined from each animal. From each thick
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section, ve ultrathin sections separated by 8 m each were utilized for subsequent quantitative analysis. The spatial separation provided between the samples clearly prevented duplicate counts of the same terminal in our ve 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.
RESULTS

there were no statistically signicant 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]. After injections of diamidino yellow or fast blue in the control group, only 14.0 1.0% of the labeled neurons contained a single uor. By comparison, a total of 77.7 5.1%

Transmitted Light Microscopy 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. 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). Incident Light Fluorescent Microscopy After injections of diamidino yellow or fast blue into either the SA or PA ganglia, three populations of retrogradely labeled uorescent neurons were identied 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,

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Fig. 1. Illustrated are medullary cross sections taken at different levels of the brain stem, modied from an atlas of the cat brain stem (33). Stars illustrate the relative number of retrogradely labeled cells within the external formation of the nucleus ambiguus found at the corresponding level after the injection of beta subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) into the posterior atrial (PA) ganglion. Note that the majority of neurons were found at the level of the area postrema (C). A is representative of sections cut 2 mm rostral to the area postrema. B is representative of sections cut 1 mm rostral to the area postrema. C is representative of sections found at the level of the area postrema. D is representative of section cut 1 mm caudal to the area postrema. AMB, compact formation of nucleus (n.) ambiguus; CU, n. cuneatus; CX, external cuneate n.; DMV, dorsal motor n. of the vagus; dsc, dorsal spinocerebellar tract; ea, external arcuate bers; FTG, gigantocellular tegmental eld; FTL, lateral tegmental eld; FTM, medial tegmental eld; GR, n. gracilis; IN, n. intercalatus; IOD, dorsal accessory inferior olivary n.; IOM, medial accessory inferior olivary n.; IOP, principal inferior olivary n.; LR, lateral reticular n.; ml, medial lemniscus; mlf, medial longitudinal fasciculus; mrs, medial reticulospinal tract; 12n, 12th nerve; 12N, hypoglossal n.; P, pyramidal tract; PH, n. prepositus hypoglossi; PR, paramedian reticular n.; rb, restiform body; RO, n. raphe obscurus; RP, n. raphe pallidus; SL, lateral n. of the solitary tract; SM, medial n. of the solitary tract; 5SP, alaminar spinal trigeminal n., parvocellular division; 5st, spinal trigeminal tract; st, solitary tract; VIN, inferior vestibular n.; VMN, medial vestibular n. J Appl Physiol VOL
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Fig. 4. There are 3 populations of vagal preganglionic neurons (VPNs) in the NA-VL, which project to the SA ganglion and the PA ganglion. There were no statistically signicant differences in the total number of retrogradely labeled neurons across the 3 groups.

Fig. 2. Illustrated are a retrogradely labeled neuron (large straight arrow) and neuropeptide Y-immunoreactive (NPY-IR) neurons (curved arrows) in the ventrolateral nucleus ambiguus (NA-VL). The boxed area is enlarged in the inset. The small straight arrows indicate NPY-IR nerve terminals in close apposition to the retrogradely labeled neuron. Final magnications: 220; 440 for inset.

human NPY fragment 124, 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 1836 and 124 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 identied even at low scanning magnications 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 m) 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. NPY-IR perikarya, dendrites, and terminals were readily identied 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.
DISCUSSION

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of the neurons was labeled with one uor 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 specicity of the NPY antibody was characterized. Neurotransmitters and synthetic peptides used included: porcine NPY, porcine/human NPY fragment 1836,

Fig. 3. There are 3 populations of neurons in the nucleus ambiguus (NA-VL) that are retrogradely labeled after the injection of one uorescent tracer into the sinoatrial (SA) ganglion and a different retrograde tracer into the PA ganglion. Cells containing the nuclear labeling uorescent tracer diamidino yellow are indicated with the open arrows. Curved arrows indicate cells containing the cytoplasmic labeling uorescent tracer fast blue. Straight arrows indicate cells double labeled with both the fast blue and diamidino uorescent tracers. Original magnication, 220. J Appl Physiol VOL

There are four major conclusions that have resulted from the present investigation. We have 1) demonstrated that VPNs that project to the PA ganglion are located exclusively in the NA-VL, 2) shown that the ultrastructural characteristics of these VPNs are very similar to VPNs that project to the SA ganglion, 3) demonstrated that NPY-IR afferent terminals form axosomatic and axodendritic synapses on VPNs projecting to
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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-bers (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

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Fig. 5. A neuron (N) in the NA-VL retrogradely labeled from the PA ganglion is readily identied by the presence of the large crystalline reaction product (large arrows). Note that the neuron has a round nucleus and abundant cytoplasm with large masses of rough endoplasmic reticulum (rer). Calibration bar, 2 m.

the PA ganglion, and 4) demonstrated that three separate populations of VPNs participate in the central regulation of cardiac rate. One population projects only to the PA ganglion; another population projects only to the SA ganglion; and a third population projects to both ganglia. In conjunction with our companion nding that the SA and PA ganglia also demonstrate interconnectivity (23), these data indicate the potential for the nervous system to exert control over cardiac rate via interdependent peripheral and central neural networks. 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
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Fig. 6. A neuron (N) in the NA-VL retrogradely labeled from the PA ganglion (large arrows) receives an axosomatic synapse (thin arrow) from a NPY-IR nerve terminal (T). An unlabeled terminal (t) is indicated for comparison. The boxed area is enlarged in the inset. Calibration bars: 1 m; 200 nm in inset. www.jap.org

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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 uor. Furthermore, the percentage of either single or double labeled cells in the experimental group was statistically signicantly 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 uors 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)

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Fig. 7. A: a NPY-IR nerve terminal (T) makes an axodendritic synapse (thin arrows) on a spine (asterisk) of a proximal dendrite of a retrogradely labeled neuron (large arrows). An unlabeled terminal (t) is indicated for comparison. The boxed area is enlarged in the inset. Calibration bar, 500 nm, and 200 nm in the inset. B: a NPY-IR nerve terminal (T) makes an axodendritic synapse on a distal dendrite (D) of a retrogradely labeled neuron (large arrows). An unlabeled terminal (t) is indicated for comparison. Calibration bar, 500 nm.

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 uor were considered to project to the ganglia in which that uor 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 uorescent 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 9097% of the neurons that were retrogradely labeled after injecting two uorescent 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
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Fig. 8. A: an unlabeled terminal (t) synapses on a somatic spine (open arrow and asterisk) of a retrogradely labeled VPN (N) projecting to the PA ganglion. Note the characteristic crystalline reaction product (black thick arrows) and abundant rough endoplasmic reticulum (rer). Calibration bar, 500 nm. B: axons of retrogradely labeled VPNs projecting to the PA ganglion are myelinated (M) and contain the crystalline reaction product (arrow). Unlabeled myelinated axons (m) are indicated for comparison. Calibration bar, 200 nm. www.jap.org

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Fig. 9. A NPY-IR neuron (N) in the NA-VL is readily identied by the presence of dense amorphous diaminobenzidine (DAB) reaction product (curved arrows) found throughout the cytoplasm. Note the invaginations of the nuclear envelope (straight arrows). Calibration bar, 2 m.

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 specic 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, specic 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 ndings 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 signicant numbers of neurons in the DMV (39). This suggests that some VPNs in the DMV inuence AV conduction, but further physiological experiments are needed to rene 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 bers immunoreactive for NPY. Recently, our laboratory has shown in a series of ultrastructural
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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 inuence 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 brillation 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
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CENTRAL CONTROL OF CARDIAC RATE 13. Ciriello J and Calaresu FR. Medullary origin of vagal preganglionic axons to the heart of the cat. J Auton Nerv Syst 5: 922, 1982. 14. Dickerson LW, Honey KW, Fleming TJ, Panico WH, Gatti PJ, Massari VJ, and Gillis RA. Negative inotropic effects produced by L-glutamate activation of regions in the ventral lateral nucleus ambiguus of the dog. Soc Neurosci Abstr 24: 1029, 1998. 15. Dickerson LW, Rodak DJ, Fleming TJ, Gatti PJ, Massari VJ, McKenzie JC, and Gillis RA. Parasympathetic neurons in the cranial medial ventricular fat pad on the dog heart selectively decrease ventricular contractility without effect on sinus rate or AV conduction. J Auton Nerv Syst 70: 129141, 1998. 16. Ford TW, Bennett JA, Kidd C, and McWilliam PN. Neurones in the dorsal motor vagal nucleus of the cat with nonmyelinated axons projecting to the heart and lungs. Exp Physiol 75: 459473, 1990. 17. Gatti PJ, Johnson TA, and Massari VJ. Can neurons in the nucleus ambiguus selectively regulate cardiac rate and atrio-ventricular conduction? J Auton Nerv Syst 57: 123127, 1996. 18. Gatti PJ, Johnson TA, McKenzie JC, Lauenstein JM, Gray AL, and Massari VJ. Vagal control of left ventricular contractility is selectively mediated by a cranioventricular intracardiac ganglion in the cat. J Auton Nerv Syst 66: 138144, 1997. 19. Gatti PJ, Johnson TA, Phan P, Jordan IK, Coleman W, and Massari VJ. The physiological and anatomical demonstration of functionally selective parasympathetic ganglia located in discrete fat pads on the feline myocardium. J Auton Nerv Syst 51: 255259, 1995. 20. Gatti PJ, Lauenstein JM, Johnson TA, and Massari VJ. There are synaptic interactions between vagal negative dromotropic ambigual neurons and neuropeptide-Y like immunoreactive nerve terminals. Soc Neurosci Abstr 24: 1030, 1998. 21. Geis GS, Kozelka JW, and Wurster RD. Organization and reex control of vagal cardiomotor neurons. J Auton Nerv Syst 3: 437450, 1981. 22. Geis GS and Wurster RD. Cardiac responses during stimulation of the dorsal motor nucleus and nucleus ambiguus in the cat. Circ Res 46: 606611, 1980. 23. Gray AL, Johnson TA, Ardell JL, and Massari VJ. Parasympathetic control of the heart. II. A novel interganglionic cardiac circuit mediates neural control of heart rate. J Appl Physiol 96: 22732278, 2004. 24. Hopkins DA. The dorsal motor nucleus of the vagus nerve and the nucleus ambiguus: structure and connections. In: Cardiogenic Reexes, edited by Hainsworth R, McWilliams PN, and Mary DASG. Oxford, UK: Oxford Univ. Press, 1987, p. 185203. 25. Hopkins DA, Gootman PM, Gootman N, and Armour JA. Anatomy of medullary and peripheral autonomic neurons innervating the neonatal porcine heart. J Auton Nerv Syst 64: 7484, 1997. 26. Hu Y and Dunbar JC. Intracerebroventricular administration of NPY increases sympathetic tone selectively in vascular beds. Brain Res Bull 44: 97103, 1997. 27. Ingenhoven N and Beck-Sickinger AG. Molecular characterization of the ligand-receptor interaction of neuropeptide Y. Curr Med Chem 6: 10551066, 1999. 28. Johnson TA, Gray AL, Lauenstein J-M, Newton SS, and Massari VJ. Parasympathetic control of the heart. I. An interventriculo-septal ganglion is the major source of the vagal intracardiac innervation of the ventricles. J Appl Physiol 96: 22652272, 2004. 29. Jordan D, Khalid ME, Schneiderman N, and Spyer KM. The location and properties of preganglionic vagal cardiomotor neurones in the rabbit. Pugers Arch 395: 244250, 1982. 30. Kobbert C, Apps R, Bechmann I, Lanciego JL, Mey J, and Thanos S. Current concepts in neuroanatomical tracing. Prog Neurobiol 62: 327 351, 2000. 31. Larsson LI. A novel immunocytochemical model system for specicity and sensitivity screening of antisera against multiple antigens. J Histochem Cytochem 29: 408410, 1981. 32. Lauenstein JM, Johnson TA, Newton SS, and Massari VJ. There are synaptic interactions between negative chronotropic vagal preganglionic neurons and neuropeptide Y (NPY)-immunoreactive nerve terminals. Soc Neurosci Abstr 25: 1951, 1999. 33. Leger L, Wiklund L, Descarries L, and Persson M. Description of an indolaminergic cell component in the cat locus coeruleus: a uorescence histochemical and radioautographic study. Brain Res 168: 4356, 1979. 34. Llewellyn-Smith IJ and Minson JB. Complete penetration of antibodies into vibratome sections after glutaraldehyde xation and ethanol treatment: light and electron microscopy for neuropeptides. J Histochem Cytochem 40: 17411749, 1992. www.jap.org

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 rst glance, it would also suggest that centrally acting NPY agonists would not be useful as selective tools to inuence 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 specic 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.
GRANTS 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). REFERENCES 1. Ardell JL and Randall WC. Selective vagal innervation of sinoatrial and atrioventricular nodes in canine heart. Am J Physiol Heart Circ Physiol 251: H764H773, 1986. 2. Balasubramaniam AA. Neuropeptide Y family of hormones: receptor subtypes and antagonists. Peptides 18: 445457, 1997. 3. Barraco RA, Ergene E, Dunbar JC, and el-Ridi MR. Cardiorespiratory response patterns elicited by microinjections of neuropeptide Y in the nucleus tractus solitarius. Brain Res Bull 24: 465485, 1990. 4. Batten TF. Immunolocalization of putative neurotransmitters innervating autonomic regulating neurons (correction of neurones) of cat ventral medulla. Brain Res Bull 37: 487506, 1995. 5. Billman GE, Hoskins RS, Randall DC, Randall WC, Hamlin RL, and Lin YC. Selective vagal postganglionic innervation of the sinoatrial and atrioventricular nodes in the nonhuman primate. J Auton Nerv Syst 26: 2736, 1989. 6. Blinder KJ, Dickerson LW, Gray AL, Lauenstein JM, Newsome JT, Rodak DJ, Fleming TJ, Gatti PJ, Gillis RA, and Massari VJ. Control of negative inotropic vagal preganglionic neurons in the dog: synaptic interactions with substance P afferent terminals in the nucleus ambiguus? Brain Res 810: 251256, 1998. 7. Blinder KJ, Gatti PJ, Johnson TA, Lauenstein JM, Coleman WP, Gray AL, and Massari VJ. Ultrastructural circuitry of cardiorespiratory reexes: there is a monosynaptic path between the nucleus of the solitary tract and vagal preganglionic motoneurons controlling atrioventricular conduction in the cat. Brain Res 785: 143157, 1998. 8. Blinder KJ, Johnson TA, and Massari VJ. Negative inotropic vagal preganglionic neurons in the nucleus ambiguus of the cat: neuroanatomical comparison with negative chronotropic neurons utilizing dual retrograde tracers. Brain Res 804: 325330, 1998. 9. Bluemel KM, Wurster RD, Randall WC, Duff MJ, and OToole MF. Parasympathetic postganglionic pathways to the sinoatrial node. Am J Physiol Heart Circ Physiol 259: H1504H1510, 1990. 10. Carlson MD, Geha AS, Hsu J, Martin PJ, Levy MN, Jacobs G, and Waldo AL. Selective stimulation of parasympathetic nerve bers to the human sinoatrial node. Circulation 85: 13111317, 1992. 11. Cheng Z, Powley TL, Schwaber JS, and Doyle FJ. Projections of the dorsal motor nucleus of the vagus to cardiac ganglia of rat atria: an anterograde tracing study. J Comp Neurol 410: 320341, 1999. 12. Chiou CW, Eble JN, and Zipes DP. Efferent vagal innervation of the canine atria and sinus and atrioventricular nodes. The third fat pad. Circulation 95: 25732584, 1997. J Appl Physiol VOL

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