doi:10.1152/japplphysiol.00621.200396:2279-2287, 2004. First published 20 February 2004; J Appl Physiol
Jeffrey L. Ardell and V. John MassariAlrich L. Gray, Tannis A. Johnson, Jean-Marie Lauenstein, Stephen S. Newton,vagal preganglionic neuronssynapse on three populations of negative chronotropicNeuropeptide Y-immunoreactive nerve terminalsParasympathetic control of the heart. III.
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, August 1, 2006; 101 (2): 413-419. J Appl PhysiolM. Waldmann, G. W. Thompson, G. C. Kember, J. L. Ardell and J. A. ArmourStochastic behavior of atrial and ventricular intrinsic cardiac neurons
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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
1 Department of Pharmacology and 3Specialized Neuroscience Research Program, Howard University
College of Medicine, Washington, District of Columbia 20059; and 2 Department 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
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-immunore-active nerve terminals synapse on three populations of negativechronotropic vagal preganglionic neurons. J Appl Physiol 96:2279–2287, 2004. First published February 20, 2004; 10.1152/jappl-physiol.00621.2003.—The vagal postganglionic control of cardiacrate is mediated by two intracardiac ganglia, i.e., the sinoatrial (SA)and posterior atrial (PA) ganglia. Nothing is known about the vagalpreganglionic neurons (VPNs) that innervate the PA ganglion or aboutthe neurochemical anatomy of central afferents that innervate theseVPNs. These issues were examined using light microscopic retrogradelabeling methods and dual-labeling electron microscopic histochem-ical and immunocytochemical methods. VPNs projecting to the PAganglion are found in a narrow column exclusively in the ventrolateralnucleus ambiguus (NA-VL). These neurons are relatively large(37.6 2.7 m by 21.3 3.4 m) with abundant cytoplasm andintracellular organelles, rare somatic and dendritic spines, rounduninvaginated nuclei, and myelinated axons. Previous physiologicaldata indicated that microinjections of neuropeptide Y (NPY) into theNA-VL cause negative chronotropic effects. The present morpholog-ical data demonstrate that NPY-immunoreactive nerve terminalsformed 18 4% of the axodendritic or axosomatic synapses and closeappositions on VPNs projecting to the PA ganglion. Three approxi-mately equal populations of VPNs in the NA-VL were retrogradelylabeled from the SA and PA ganglia. One population each projects tothe SA ganglion, the PA ganglion, or to both the SA and PA ganglia.Therefore, there are both shared and independent pathways involvedin the vagal preganglionic controls of cardiac rate. These data areconsistent with the hypothesis that the central and peripheral para-sympathetic controls of cardiac rate are coordinated by multiplepotentially redundant and/or interacting pathways and mechanisms.
FUNCTIONALLY SELECTIVE INTRINSIC cardiac ganglia found on thesurface of the heart are innervated by cardioinhibitory pregan-glionic vagal motoneurons that originate mainly in the ventro-lateral nucleus ambiguus (NA-VL) (13, 16, 24, 25, 35). Thereappears to be a cardiotopic organization of functionally selec-tive vagal preganglionic neurons (VPNs) in the brain stem (7,17, 39–41) 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 thatprojects to functionally selective chronotropic, dromotropic, orinotropic intracardiac ganglia [i.e., the sinoatrial (SA), atrio-ventricular (AV), and cranioventricular (CV) ganglia (for de-tails 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 twodifferent fluorescent retrograde tracers are simultaneously in- jected into the SA and AV ganglia or SA and CV ganglia, threeseparate 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). Fur-thermore, physiological evidence indicates that microinjectionsof excitatory amino acids into different areas of the NA-VLthat are retrogradely labeled from selected intracardiac gangliacan elicit selective changes in either cardiac rate, atrioventric-ular (AV) conduction, or left ventricular contractility (14, 17,39, 41). These data support the hypothesis that the pregangli-onic vagal controls of cardiac rate, AV conduction, and ven-tricular contractility are independently mediated by at least
three separate populations of cardioinhibitory VPNs.In a recent report, we have shown that the vagal postgan-
glionic control of cardiac rate is mediated by two separate butinterconnected intracardiac ganglia, i.e., the SA and posterioratrial (PA) ganglion (23). However, virtually nothing is knownabout the VPNs and central afferents that regulate the functionsof the PA ganglion. Because microinjections of neuropeptide Y(NPY) into the NA-VL cause bradycardia (36), in the presentreport, we 1) define the light microscopic distribution of VPNsprojecting to the PA ganglion, 2) describe the ultrastructuralcharacteristics of these neurons, 3) test the hypothesis thatNPY-immunoreactive (IR) afferent nerve terminals synapse onthe soma and dendrites of VPNs that regulate the function of
the PA ganglion, and 4) test the hypothesis that separatepopulations of VPNs project to the PA and SA ganglia.
MATERIALS AND METHODS
Retrograde Tracing Studies
The Institutional Animal Care and Use Committee of HowardUniversity reviewed and approved the experimental design of allanimal experiments. Experiments were performed on 15 mongrel cats
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: [email protected]).
The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement ”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
J Appl Physiol 96: 2279–2287, 2004.First published February 20, 2004; 10.1152/japplphysiol.00621.2003.
of either sex weighing 2.8–4.0 kg. Baseline vital signs of temperature,respiratory rate, and ECG were recorded. Cats were pretreated with0.05 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 catheterinto 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 moni-tored with a pulse-oximeter (Vet-Ox) via the lingual artery. The catwas artificially respired on a positive-pressure respirator with a tidalvolume setting between 75 and 150 ml and cycling at 12 breaths/min.The cat was given a 95% oxygen-5% carbon dioxide gas mixture tobreathe. An incision was made into the pericardium that was largeenough to expose the heart and allow for identification and access toboth the PA ganglion, located on the rostral dorsal surface of the rightatrium between the superior vena cava and the aorta, and the SAganglion, found at the junction of the superior vena cava and rightatrium 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) dis-solved in 2% DMSO in distilled water were injected into the PAganglion 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 yellowboth dissolved in 2% DMSO in ethylene glycol were, respectively,injected into the SA and PA ganglia using a counterbalanced design inthree to four parts in six animals or into the pericardial sac as a controlfor extraneous leakage of the tracer in two animals. After injectionswere made, the pericardium was closed, the muscles and skin weresutured in layers, spontaneous respiration was reestablished, fluidsalong with the potent analgesic butorphanol (0.2 mg/kg) and theantibiotic penicillin procaine G (30,000 IU/kg) were administered,and the animal was awakened from anesthesia. Postoperatively, bu-torphanol was given (0.2 mg/kg) twice daily for at least 2 days toreduce pain, and penicillin procaine G (30,000 IU/kg) was adminis-tered 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 whichthe fluorescent retrograde tracers diamidino yellow and fast blue wereused (30, 44) were killed via intravascular perfusion 10 days aftersurgery. On the day of perfusion, cats were deeply anesthetized with50 mg/kg pentobarbital sodium administered intraperitoneally andperfused intravascularly with 1,000 ml of oxygenated 0.1 M phos-phate-buffered saline containing 2,500 U of heparin (PBS-Hep), and4 liters of a phosphate-buffered solution containing 1.75% acroleinand 0.5% paraformaldehyde, as previously described in detail (41).This combination of fixatives provides reasonable ultrastructural mor-phology while preserving the antigenicity of the tissues for subsequentimmunocytochemical study. After the perfusion, the animal’s brainwas removed, and transverse serial 40-m-thick sections of themedulla were cut from the level of the spinomedullary junction to thecaudal border of the pons using a Vibratome. Brain sections were then
processed histochemically and immunocytochemically for later lightand electron microscopic analysis.
In animals in which fluorescent retrograde tracers were injectedinto the heart, cats were anesthetized as described above and thenperfused intravascularly with 1,000 ml of oxygenated PBS-Hep solu-tion, 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 thatwere 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 thenwashed three times with a 0.1 M sodium phosphate-buffered solution,pH 6.0, and then processed to reveal CTB-HRP-labeled cell bodies bya modification of the tungstate stabilized tetramethylbenzidine (TMB)method of Weinberg and Van Eyck (51) as previously described indetail (41).
Immunocytochemistry
All tissue sections were subsequently incubated for 30 min in asolution of 50% absolute ethanol in distilled water to enhance thepenetration of antibodies throughout the tissue (34), followed by threewashes with PBS. Tissues were then incubated in 0.1 M phosphate-buffered solution containing 1.0% BSA for 30 min and then incubatedin rabbit anti-NPY primary antiserum (Peninsula) diluted 1:2,000 in0.1% BSA dissolved in 0.1 M PBS overnight. The immunocytochem-ical procedure utilized to demonstrate NPY-IR sites was an avidin-biotin-based method utilizing the Vectastain Elite ABC kit as previ-ously described (41). HRP was visualized with a second glucoseoxidase reaction utilizing diaminobenzidine (DAB) as the chromogen.This reaction results in an amorphous electron dense reaction productin the electron microscope. The specificity of the NPY antisera
utilized in the present study was further characterized utilizing theimmunodot-blot method of Larsson (31).
Processing for Light Microscopy
Transmitted light microscopy. Tissue sections were mounted ontoslides, dehydrated with ethanol, cleared with xylene, and cover-slipped with Permount (Fisher Scientific). The slides were examinedunder a Nikon Microphot FXA light microscope using bright-field,dark-field, or Nomarski differential interference contrast optics. Thedistribution of retrogradely labeled VPNs was determined by record-ing the total number of labeled cells that were found in sections takenfrom the following levels of the brain stem: 1 mm caudal to the areapostrema (AP); the AP; 1 mm rostral to the AP; and 2 mm rostral tothe AP.
Incident light fluorescence microscopy. Paraformaldehyde-fixedfrozen brain stems were sectioned on a cryostat. Transverse serial40-m-thick sections of the medulla were cut from the level of thespinomedullary junction to the caudal border of the pons. Alternatesections were mounted onto glass slides. Slides were coverslippedwith a 1:1 mixture of glycerol and distilled water, and the tissues wereexamined under a Nikon FXA photomicroscope through Nikon CFIPlan Fluor objectives under UV fluorescence. The UV filters wereconfigured with an excitation filter of 365 nm and a barrier filter of 400 nm. These filters allowed for simultaneous visualization of bothfluorescent tracers. Diamidino yellow labels the nucleus and appearsyellow while fast blue labels the cytoplasm and appears blue (30, 44).
Processing for Electron Microscopy
In four animals, tissues were rinsed in PBS and post fixed in 2%
osmium tetroxide for 1 h, dehydrated through a graded series of alcohols and propylene oxide, embedded in resin (Embed 812) be-tween two sheets of plastic (Aclar: Dupont), and cured at 60 °C for48 h. Embedded tissues were examined in a light microscope, andareas of interest including the NA-VL were cut out and reembeddedin Beem capsulses. Serial ultrathin sections of the reembedded tissueswere cut on an ultramicrotome (Reichert, Ultracut S) at 75 nmthickness (silver-gold interference color), collected on uncoated cop-per mesh grids, poststained with uranyl acetate and Reynolds leadcitrate, and examined in a JEOL-JEM-1210 electron microscope at50 kV.
Two 40-m-thick tissue sections that contained the best combina-tion of morphological preservation and histochemical/immunocyto-chemical labeling were examined from each animal. From each thick
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section, five ultrathin sections separated by 8 m each were utilizedfor subsequent quantitative analysis. The spatial separation providedbetween the samples clearly prevented duplicate counts of the sameterminal in our five samples through the neuropil. The number of NPY-immunoreactive and unlabeled nerve terminals making axoso-matic or axodendritic synapses on cardioinhibitory VPNs retrogradelylabeled from the PA ganglion were recorded.
RESULTS
Transmitted Light Microscopy
After the injection of CTB-HRP into the PA ganglion, acolumn of retrogradely labeled neurons was observed bilater-ally in the ventrolateral medulla. This column extended fromthe spinomedullary junction to the caudal boundary of thefacial nucleus. No retrograde labeling was observed in thedorsal motor nucleus of the vagus (DMV). The relative numberof retrogradely labeled neurons found at different anteroposte-rior levels of the NA varied (Fig. 1). The majority of cells wasfound concentrated at the level of the AP, with a tapering in thenumber of cells found at the more extreme rostral and caudal
levels of the medulla. By comparison, when CTB-HRP wasinjected into the pericardial sac in the control animal, only onelabeled cell was found in the medulla. It was located in theintermediate zone between the NA and the DMV.
As we have previously demonstrated (38), NPY-IR neuronsand their processes are found in the ventrolateral medulla.These neurons were interspersed with retrogradely labeledVPNs projecting to the PA ganglion. Some NPY-IR processeswere noted to be in close apposition to these retrogradelylabeled VPNs (Fig. 2).
Incident Light Fluorescent Microscopy
After injections of diamidino yellow or fast blue into eitherthe SA or PA ganglia, three populations of retrogradely labeled
fluorescent neurons were identified in the NA-VL. Theseneurons contained either diamidino yellow alone, fast bluealone, or both diamidino yellow and fast blue (Fig. 3). Themean total number of retrogradely labeled cells observed inthe entire NA-VL was 311 53 (mean SE). However,
there were no statistically significant differences in the numberof labeled neurons across the three populations of neuronsthat contained these tracers (Fig. 4) [ANOVA, F (2,15) 1.84, P 0.05].
After injections of diamidino yellow or fast blue in thecontrol group, only 14.0 1.0% of the labeled neuronscontained a single fluor. By comparison, a total of 77.7 5.1%
Fig. 1. Illustrated are medullary cross sections taken at different levels of thebrain stem, modified from an atlas of the cat brain stem (33). Stars illustrate therelative 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 werefound 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 mmrostral to the area postrema. C is representative of sections found at the levelof the area postrema. D is representative of section cut 1 mm caudal to the areapostrema. AMB, compact formation of nucleus (n.) ambiguus; CU, n. cunea-tus; CX, external cuneate n.; DMV, dorsal motor n. of the vagus; dsc, dorsalspinocerebellar tract; ea, external arcuate fibers; FTG, gigantocellular tegmen-tal field; FTL, lateral tegmental field; FTM, medial tegmental field; 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 spinaltrigeminal n., parvocellular division; 5st, spinal trigeminal tract; st, solitarytract; VIN, inferior vestibular n.; VMN, medial vestibular n.
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of the neurons was labeled with one fluor in the experimentalanimals. Furthermore, the percentage of single and doublelabeled cells in the experimental group was statistically signif-icantly different from that found in the control group (P 0.0001).
Characterization of the NPY Antiserum
The sensitivity and specificity of the NPY antibody wascharacterized. Neurotransmitters and synthetic peptides used
included: porcine NPY, porcine/human NPY fragment 18–36,
human NPY fragment 1–24, peptide YY, serotonin, substanceP (SP), norepinephrine (NE), neurotensin (NT), leu-enkephalin(L-Enk), and met-enkephalin (M-Enk). The rabbit anti-NPYserum was able to recognize NPY between concentrations of 103 and 106 M and NPY fragments 18–36 and 1–24 be-tween concentrations of 103 and 105 M. This antibody did
not recognize peptide YY, serotonin, SP, NE, NT, M-Enk, orL-Enk even at concentrations as high as 1 mM.
Electron Microscopy
Retrogradely labeled neurons and their processes werereadily identified even at low scanning magnifications in theelectron microscope due to the presence of a characteristicelectron dense crystalline TMB-tungstate reaction product(Fig. 5). This reaction product was found primarily in theperikarya and proximal dendrites (Figs. 6 and 7 A); however, afew labeled distal dendrites were also detected (Fig. 7 B).Retrogradely labeled neurons were relatively large (37.6 2.7by 21.3 3.4 m) with abundant cytoplasm and intracellularorganelles (Fig. 5), rare somatic (Fig. 8 A) and dendritic (Fig.7 A) spines, and round nuclei (Fig. 5), occasionally showing aprominent nucleolus. Retrogradely labeled axons were foundto be myelinated (Fig. 8 B). In the tissues examined, no unmy-elinated axons or nerve terminals were found to contain thecrystalline TMB-tungstate reaction product.
NPY-IR perikarya, dendrites, and terminals were readilyidentified by the presence of a characteristic amorphous DABreaction product (Figs. 6, 7, A and B, and 9). They hadrelatively 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 retrogradelylabeled VPNs (Figs. 6 and 7, A and B). NPY-IR terminalscommonly contained multiple small clear vesicles and one or
more large dense core vesicles. A total of 7 2% of theterminals making synaptic contacts with retrogradely labeledneurons were NPY-IR, whereas another 11 2% were in closeapposition to these VPNs.
DISCUSSION
There are four major conclusions that have resulted from thepresent investigation. We have 1) demonstrated that VPNs thatproject to the PA ganglion are located exclusively in theNA-VL, 2) shown that the ultrastructural characteristics of these VPNs are very similar to VPNs that project to the SAganglion, 3) demonstrated that NPY-IR afferent terminals formaxosomatic and axodendritic synapses on VPNs projecting to
Fig. 2. Illustrated are a retrogradely labeled neuron (large straight arrow) andneuropeptide Y-immunoreactive (NPY-IR) neurons (curved arrows) in the
ventrolateral nucleus ambiguus (NA-VL). The boxed area is enlarged in theinset . The small straight arrows indicate NPY-IR nerve terminals in closeapposition to the retrogradely labeled neuron. Final magnifications: 220;440 for inset .
Fig. 3. There are 3 populations of neurons in the nucleus ambiguus (NA-VL)that are retrogradely labeled after the injection of one fluorescent tracer into thesinoatrial (SA) ganglion and a different retrograde tracer into the PA ganglion.Cells containing the nuclear labeling fluorescent tracer diamidino yellow areindicated with the open arrows. Curved arrows indicate cells containing thecytoplasmic labeling fluorescent tracer fast blue. Straight arrows indicate cellsdouble labeled with both the fast blue and diamidino fluorescent tracers.Original magnification, 220.
Fig. 4. There are 3 populations of vagal preganglionic neurons (VPNs) in theNA-VL, which project to the SA ganglion and the PA ganglion. There were nostatistically significant differences in the total number of retrogradely labeledneurons across the 3 groups.
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the PA ganglion, and 4) demonstrated that three separatepopulations 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 athird population projects to both ganglia. In conjunction withour companion finding that the SA and PA ganglia alsodemonstrate interconnectivity (23), these data indicate thepotential for the nervous system to exert control over cardiacrate via interdependent peripheral and central neural networks.
Combined anatomic and physiological studies previouslyconducted in our laboratory have demonstrated that cardioin-hibitory neurons in the external formation of the NA aredivided into at least three different functional categories. Thesegroups include negative chronotropic, negative dromotropic,and negative inotropic neurons (14, 37, 39, 40). The distribu-tion of these functionally distinct cardioinhibitory groups of neurons is not identical. In the present report, we have dem-onstrated that negative chronotropic VPNs that are retrogradelylabeled from the PA ganglion are distributed exclusively in theNA-VL and that the largest concentration of these neurons isfound at the level of the AP. This distribution is quite similarto that found after injections of retrograde tracers into the SA
ganglion (41). VPNs that project to the PA ganglion were alsoexamined by electron microscopy to characterize the ultra-structural characteristics of these neurons. The neurons wererelatively large with abundant cytoplasm and intracellular or-ganelles and contained a round uninvaginated nucleus oftenwith a prominent nucleolus. These neurons were found to haverare somatic or dendritic spines and an abundance of rough
endoplasmic reticulum. Furthermore, the axons of these neu-rons were myelinated. This result provides anatomic supportfor the electrophysiological observation that vagal pregangli-onic cardioinhibitory neurons in the NA have axons thatconduct action potentials in the range of B-fibers (29, 42).Furthermore, these morphological data indicate that the ultra-structural characteristics of VPNs projecting to the PA gan-glion closely match those of neurons retrogradely labeled fromthe SA ganglion (41).
Our data indicate that there are multiple similarities betweenVPNs innervating the PA ganglion and those projecting to theSA ganglion. For instance, VPNs innervating the SA and PAganglia 1) are found solely in the NA-VL, 2) have very similardistributions in the brain stem with the majority of their cellsfound at the level of the AP, 3) have similar size and ultra-structural characteristics, 4) receive synaptic inputs from
Fig. 5. A neuron (N) in the NA-VL retrogradely labeled from the PA ganglion
is readily identified by the presence of the large crystalline reaction product(large arrows). Note that the neuron has a round nucleus and abundantcytoplasm with large masses of rough endoplasmic reticulum (rer). Calibrationbar, 2 m.
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-IRnerve terminal (T). An unlabeled terminal (t) is indicated for comparison. Theboxed area is enlarged in the inset . Calibration bars: 1 m; 200 nm in inset .
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NPY-IR nerve terminals, and 5) innervate ganglia mediatingnegative chronotropic effects on the heart. With so manysimilarities, it was imperative to determine whether the PA andthe SA ganglia are innervated by the same or separate groupsof VPNs in the NA-VL. To test this important question, twodifferent retrograde tracers were injected into the SA and PAganglia, respectively, using a counterbalanced design. Neuronsretrogradely labeled with one fluor were considered to projectto the ganglia in which that fluor was injected. Neurons labeledwith 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. Onepopulation projected exclusively to the SA ganglion, the sec-ond population projected exclusively to the PA ganglion, andthe 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 foundto project to both ganglia. Blinder et al. (7, 8) found that90–97% of the neurons that were retrogradely labeled afterinjecting two fluorescent tracers into the SA and AV ganglia,or the SA and CV ganglia, respectively, were single labeled. Ina similar study, in piglets, 100% of the cells found in the
NA-VL after injections of two retrograde tracers into the AVand SA ganglia or into either the SA or AV ganglia and aventricular locus were single labeled (25). In the present data,78% of retrogradely labeled neurons were single labeled. Bycomparison, in the control animals, only 14% of labeledneurons contained a single fluor. Furthermore, the percentageof either single or double labeled cells in the experimental
group was statistically significantly different from that found inthe control group (P 0.0001). We conclude from theseresults that there was minimal leakage of tracer from itsinjection sites within the intracardiac ganglia in the experimen-tal animals and that neurons containing both fluors in theexperimental animals represent neurons that project to both theSA and PA ganglia. In summary, the data support the hypoth-esis that separate and distinct populations of VPNs innervateintrinsic cardiac ganglia that mediate AV conduction and leftventricular contractility, whereas intrinsic cardiac ganglia thatdirectly or indirectly mediate control of cardiac rate are inner-vated by three further populations of VPNs. The data furtherindicate that there is considerable redundancy in the centralneural mechanisms responsible for regulating heart rate. Ananalogous redundancy is found within the heart (23), whereintwo separate intracardiac ganglia (the SA and PA ganglia)
Fig. 7. A: a NPY-IR nerve terminal (T) makes an axodendritic synapse (thinarrows) on a spine (asterisk) of a proximal dendrite of a retrogradely labeledneuron (large arrows). An unlabeled terminal (t) is indicated for comparison.The boxed area is enlarged in the inset . Calibration bar, 500 nm, and 200 nmin 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). Anunlabeled terminal (t) is indicated for comparison. Calibration bar, 500 nm.
Fig. 8. A: an unlabeled terminal (t) synapses on a somatic spine (open arrowand asterisk) of a retrogradely labeled VPN (N) projecting to the PA ganglion.Note the characteristic crystalline reaction product (black thick arrows) andabundant rough endoplasmic reticulum (rer). Calibration bar, 500 nm. B: axonsof retrogradely labeled VPNs projecting to the PA ganglion are myelinated (M)and contain the crystalline reaction product (arrow). Unlabeled myelinatedaxons (m) are indicated for comparison. Calibration bar, 200 nm.
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interdependently mediate the vagal control of cardiac rate.Such redundancy provides a neural framework whereby heartrate can be subtly modulated at both the level of the centralnervous system and the heart. Collectively, these data implythat the central mechanisms that control cardiac rate are morecomplex than previously recognized. Further studies, however,will have to be conducted to determine the specific physiolog-ical role(s) this complex cardiac neural circuitry plays in theprecise regulation of cardiac rate.
Numerous studies utilizing retrograde or transganglionicviral tracers have reported that the DMV serves as one of thesources of VPNs innervating the heart (13, 21, 22, 48). Chenget 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 cardioinhibitoryVPNs contained within this nucleus are still uncertain. Geis etal. (21, 22) reported that electrical stimulation of cardioinhib-itory 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 chrono-tropic, dromotropic, or inotropic effects on the heart. We havepreviously reported that injections of a retrograde tracer intothe AV ganglion result in the labeling of significant numbers of neurons in the DMV (39). This suggests that some VPNs in theDMV influence AV conduction, but further physiological ex-periments are needed to refine our understanding of the role(s)
of the DMV on cardiac function(s). Injections of retrogradetracers into the SA ganglion (41) or the PA ganglion (presentdata) label neurons exclusively in the NA-VL. The presentdata therefore suggest that VPNs responsible for modulatingcardiac rate, either via the PA or SA ganglia, are not locatedin the DMV.
A number of previous studies have investigated the centraleffects of NPY on the cardiovascular system (3, 26, 36, 47, 49).Macrae and Reid (36), in one such study, showed that micro-injections of NPY into the region of the NA-VL producedbradycardia. Later, Batten (4) found that cardiac VPNs in theNA 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 andaxodendritic synapses on negative dromotropic (20) and neg-ative chronotropic (20, 32) VPNs in the NA-VL. In the presentdata, we have shown NPY-IR terminals formed 7.4 2% of the asymmetric axodendritic and axosomatic synapses detectedon VPNs retrogradely labeled from the PA ganglion. Another11 2% of the NPY terminals were in close apposition to
these VPNs but did not show a synapse in the planes of sectionthat were examined. These data support the previous morpho-logical and physiological data that indicate that NPY may playa substantial role in modulating multiple indexes of heartfunction.
In summary, VPNs projecting to the PA ganglion are foundprimarily in the NA-VL at the level of the AP. These neuronsare relatively large with abundant cytoplasm and intracellularorganelles, rare somatic and dendritic spines, and round nuclei;have myelinated axons; and receive axodendritic and axoso-matic synaptic inputs from NPY-IR nerve terminals. There arestatistically three equal populations of vagal preganglionicneurons in the NA-VL that mediate an effect on cardiac rate(via the SA and PA ganglia). One population projects to onlythe SA ganglion, a second population projects to only the PAganglion, and a third population projects to both the SA and PAganglia. Therefore, there are both shared and independentpathways involved in the vagal preganglionic controls of car-diac rate. These data are consistent with the hypothesis that theneural control of cardiac rate is coordinated by interdependentcentral and peripheral mechanisms. The present data indicatethat the neuronal circuits that mediate vagal control of cardiacrate 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 butunavoidable side effects. Thus, for example, sympathomimeticdrugs that enhance myocardial contractility in congestive heartfailure not uncommonly also cause an undesirable tachycardia.This is often the case because the same receptor that providesthe desired therapeutic action also mediates undesirable sideeffects. Unlike the heart, the brain contains a diverse array of potential neurotransmitters and receptors that could potentiallyinfluence cardiac or cardiovascular functions. One of the goalsof our research efforts has been to determine whether a newgeneration of drugs may be developed that can target function-ally selective neurons in the brain to elicit selective changes invarious parameters of cardiac performance. One approach toachieving this goal would be to determine whether there are
qualitative differences in the distribution of immunocytochem-ically characterized nerve terminals synapsing on functionallyselective VPNs. In this effort, we have demonstrated thatsubstance P-immunoreactive nerve terminals synapse on neg-ative chronotropic VPNs but not on negative dromotropic ornegative inotropic VPNs (6, 40, 41). Correspondingly, micro-injections of substance P into the NA-VL selectively inducebradycardia (40). These data indicate that centrally actingneurokinin 1 receptor agonists could potentially be useful inthe treatment of certain arrhythmias such as atrial fibrillationbecause they would induce bradycardia without undesirableactions on AV conduction or left ventricular contractility. Inthe present report, we have shown that NPY-IR terminals
Fig. 9. A NPY-IR neuron (N) in the NA-VL is readily identified by thepresence of dense amorphous diaminobenzidine (DAB) reaction product(curved arrows) found throughout the cytoplasm. Note the invaginations of thenuclear envelope (straight arrows). Calibration bar, 2 m.
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synapse on negative chronotropic VPNs retrogradely labeledfrom the PA ganglion. NPY-IR terminals have also been foundto 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 neuro-transmitter involved in modulating multiple vagally mediatedcardiac effects. At first glance, it would also suggest that
centrally acting NPY agonists would not be useful as selectivetools 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 serveas postsynaptic receptors on a specific functional category of cardioinhibitory VPNs. Thus it is still possible that agonists forselective NPY receptor subtypes could mediate selective ef-fects on cardiac function. Further ultrastructural and physio-logical experiments will be required to clarify the potentialroles of NPY and its receptors in the modulation of cardioin-hibitory 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 HeartAssociation to V. J. Massari and from the NHLBI (R01-HL-58140) to J. L.Ardell. Additional funding was provided by the Gustavus and Louise PfeifferResearch Foundation to A. L. Gray and by the Specialized NeuroscienceResearch Program (1U54-NS-39407; M. A. Haxhiu, Principal Investigator).
REFERENCES
1. Ardell JL and Randall WC. Selective vagal innervation of sinoatrial andatrioventricular nodes in canine heart. Am J Physiol Heart Circ Physiol
251: H764–H773, 1986.2. Balasubramaniam AA. Neuropeptide Y family of hormones: receptor
subtypes and antagonists. Peptides 18: 445–457, 1997.3. Barraco RA, Ergene E, Dunbar JC, and el-Ridi MR. Cardiorespiratory
response patterns elicited by microinjections of neuropeptide Y in thenucleus tractus solitarius. Brain Res Bull 24: 465–485, 1990.
4. Batten TF. Immunolocalization of putative neurotransmitters innervatingautonomic regulating neurons (correction of neurones) of cat ventralmedulla. Brain Res Bull 37: 487–506, 1995.
Lin YC. Selective vagal postganglionic innervation of the sinoatrial andatrioventricular nodes in the nonhuman primate. J Auton Nerv Syst 26:27–36, 1989.
6. Blinder KJ, Dickerson LW, Gray AL, Lauenstein JM, Newsome JT,Rodak DJ, Fleming TJ, Gatti PJ, Gillis RA, and Massari VJ. Controlof negative inotropic vagal preganglionic neurons in the dog: synapticinteractions with substance P afferent terminals in the nucleus ambiguus?
Brain Res 810: 251–256, 1998.7. Blinder KJ, Gatti PJ, Johnson TA, Lauenstein JM, Coleman WP,
Gray AL, and Massari VJ. Ultrastructural circuitry of cardiorespiratoryreflexes: there is a monosynaptic path between the nucleus of the solitarytract and vagal preganglionic motoneurons controlling atrioventricularconduction in the cat. Brain Res 785: 143–157, 1998.
8. Blinder KJ, Johnson TA, and Massari VJ. Negative inotropic vagalpreganglionic neurons in the nucleus ambiguus of the cat: neuroanatomicalcomparison with negative chronotropic neurons utilizing dual retrogradetracers. Brain Res 804: 325–330, 1998.
9. Bluemel KM, Wurster RD, Randall WC, Duff MJ, and O’Toole MF.
Parasympathetic postganglionic pathways to the sinoatrial node. Am J Physiol Heart Circ Physiol 259: H1504–H1510, 1990.
10. Carlson MD, Geha AS, Hsu J, Martin PJ, Levy MN, Jacobs G, andWaldo AL. Selective stimulation of parasympathetic nerve fibers to thehuman sinoatrial node. Circulation 85: 1311–1317, 1992.
11. Cheng Z, Powley TL, Schwaber JS, and Doyle FJ. Projections of thedorsal motor nucleus of the vagus to cardiac ganglia of rat atria: ananterograde tracing study. J Comp Neurol 410: 320–341, 1999.
12. Chiou CW, Eble JN, and Zipes DP. Efferent vagal innervation of thecanine atria and sinus and atrioventricular nodes. The third fat pad.Circulation 95: 2573–2584, 1997.
13. Ciriello J and Calaresu FR. Medullary origin of vagal preganglionicaxons to the heart of the cat. J Auton Nerv Syst 5: 9–22, 1982.
14. Dickerson LW, Honey KW, Fleming TJ, Panico WH, Gatti PJ,Massari VJ, and Gillis RA. Negative inotropic effects produced byL-glutamate activation of regions in the ventral lateral nucleus ambiguus of the dog. Soc Neurosci Abstr 24: 1029, 1998.
Kenzie JC, and Gillis RA. Parasympathetic neurons in the cranial medialventricular fat pad on the dog heart selectively decrease ventricularcontractility without effect on sinus rate or AV conduction. J Auton Nerv
Syst 70: 129–141, 1998.16. Ford TW, Bennett JA, Kidd C, and McWilliam PN. Neurones in the
dorsal motor vagal nucleus of the cat with nonmyelinated axons projectingto the heart and lungs. Exp Physiol 75: 459–473, 1990.
17. Gatti PJ, Johnson TA, and Massari VJ. Can neurons in the nucleusambiguus selectively regulate cardiac rate and atrio-ventricular conduc-tion? J Auton Nerv Syst 57: 123–127, 1996.
18. Gatti PJ, Johnson TA, McKenzie JC, Lauenstein JM, Gray AL, and
Massari VJ. Vagal control of left ventricular contractility is selectivelymediated by a cranioventricular intracardiac ganglion in the cat. J Auton
Nerv Syst 66: 138–144, 1997.19. Gatti PJ, Johnson TA, Phan P, Jordan IK, Coleman W, and Massari
VJ. The physiological and anatomical demonstration of functionallyselective parasympathetic ganglia located in discrete fat pads on the felinemyocardium. J Auton Nerv Syst 51: 255–259, 1995.
20. Gatti PJ, Lauenstein JM, Johnson TA, and Massari VJ. There aresynaptic interactions between vagal negative dromotropic ambigual neu-rons and neuropeptide-Y like immunoreactive nerve terminals. Soc Neu-
rosci Abstr 24: 1030, 1998.21. Geis GS, Kozelka JW, and Wurster RD. Organization and reflex control
of vagal cardiomotor neurons. J Auton Nerv Syst 3: 437–450, 1981.22. Geis GS and Wurster RD. Cardiac responses during stimulation of the
dorsal motor nucleus and nucleus ambiguus in the cat. Circ Res 46:606–611, 1980.
23. Gray AL, Johnson TA, Ardell JL, and Massari VJ. Parasympatheticcontrol of the heart. II. A novel interganglionic cardiac circuit mediatesneural control of heart rate. J Appl Physiol 96: 2273–2278, 2004.
24. Hopkins DA. The dorsal motor nucleus of the vagus nerve and the nucleusambiguus: structure and connections. In: Cardiogenic Re fl exes, edited byHainsworth R, McWilliams PN, and Mary DASG. Oxford, UK: OxfordUniv. Press, 1987, p. 185–203.
25. Hopkins DA, Gootman PM, Gootman N, and Armour JA. Anatomy of
medullary and peripheral autonomic neurons innervating the neonatalporcine heart. J Auton Nerv Syst 64: 74–84, 1997.
26. Hu Y and Dunbar JC. Intracerebroventricular administration of NPYincreases sympathetic tone selectively in vascular beds. Brain Res Bull 44:97–103, 1997.
27. Ingenhoven N and Beck-Sickinger AG. Molecular characterization of the ligand-receptor interaction of neuropeptide Y. Curr Med Chem 6:1055–1066, 1999.
28. Johnson TA, Gray AL, Lauenstein J-M, Newton SS, and Massari VJ.
Parasympathetic control of the heart. I. An interventriculo-septal ganglionis the major source of the vagal intracardiac innervation of the ventricles.
J Appl Physiol 96: 2265–2272, 2004.29. Jordan D, Khalid ME, Schneiderman N, and Spyer KM. The location
and properties of preganglionic vagal cardiomotor neurones in the rabbit.P fl u gers Arch 395: 244–250, 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 specificity
and sensitivity screening of antisera against multiple antigens. J Histo-
chem Cytochem 29: 408–410, 1981.32. Lauenstein JM, Johnson TA, Newton SS, and Massari VJ. There are
synaptic interactions between negative chronotropic vagal preganglionicneurons 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 fluorescencehistochemical and radioautographic study. Brain Res 168: 43–56, 1979.
34. Llewellyn-Smith IJ and Minson JB. Complete penetration of antibodiesinto vibratome sections after glutaraldehyde fixation and ethanol treat-ment: light and electron microscopy for neuropeptides. J Histochem
Cytochem 40: 1741–1749, 1992.
2286 CENTRAL CONTROL OF CARDIAC RATE
J Appl Physiol • VOL 96 • JUNE 2004 • www.jap.org
8/3/2019 Alrich L. Gray et al- Parasympathetic control of the heart. III. Neuropeptide Y-immunoreactive nerve terminals syna…
control of left ventricular contractility in the dog heart: synaptic interac-
tions of negative inotropic vagal preganglionic neurons in the nucleusambiguus with tyrosine hydroxylase immunoreactive terminals. Brain Res
802: 205–220, 1998.
38. Massari VJ, Hornby PJ, Friedman EK, Milner TA, Gillis RA, and
Gatti PJ. Distribution of neuropeptide Y-like immunoreactive perikarya
and processes in the medulla of the cat. Neurosci Lett 115: 37–42, 1990.
39. Massari VJ, Johnson TA, and Gatti PJ. Cardiotopic organization of the
nucleus ambiguus? An anatomical and physiological analysis of neurons
regulating atrio-ventricular conduction. Brain Res 679: 227–240, 1995.
40. Massari VJ, Johnson TA, Gillis RA, and Gatti PJ. What are the roles
of substance P and neurokinin-1 receptors in the control of negative
chronotropic and negative dromotropic vagal motoneurons? A physiolog-
ical and ultrastructural analysis. Brain Res 693: 133–147, 1996.
41. Massari VJ, Johnson TA, Llewellyn-Smith IJ, and Gatti PJ. Substance
P neurons synapse upon negative chronotropic vagal motoneurons. Brain
Res 660: 275–287, 1994.
42. McAllen RM and Spyer KM. Two types of vagal preganglionic mo-toneurones projecting to the heart and lungs. J Physiol 282: 353–364,
1978.
43. Moore CT, Blinder KJ, Johnson TA, and Massari VJ. Vagal pregan-glionic control of left ventricular contractility is modulated monosynapti-cally by neuropeptide Y (NPY) immunoreactive afferents to the nucleusambiguus (NA). Soc Neurosci Abstr 27: 170.110, 2001.
44. Novikova L, Novikov L, and Kellerth JO. Persistent neuronal labelingby retrograde fluorescent tracers: a comparison between fast blue, fluoro-gold and various dextran conjugates. J Neurosci Methods 74: 9–15, 1997.
45. Randall WC and Ardell JL. Selective parasympathectomy of automaticand conductile tissues of the canine heart. Am J Physiol Heart Circ Physiol
248: H61–H68, 1985.46. Randall WC, Randall DC, and Ardell JL. Autonomic regulation of
myocardial contractility. In: Re fl ex Control of the Circulation, edited byZucker IH and Gilmore JP. Boston, MA: CRC, 1991, p. 39–65.
47. Scott NA, Webb V, Boublik JH, Rivier J, and Brown MR. Thecardiovascular actions of centrally administered neuropeptide Y. RegulPept 25: 247–258, 1989.
48. Standish A, Enquist LW, Escardo JA, and Schwaber JS. Centralneuronal circuit innervating the rat heart defined by transneuronal trans-port of pseudorabies virus. J Neurosci 15: 1998–2012, 1995.
49. Tseng CJ, Mosqueda-Garcia R, Appalsamy M, and Robertson D.Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ Res
64: 55–61, 1989.50. Wallick DW and Martin PJ. Separate parasympathetic control of heart
rate and atrioventricular conduction of dogs. Am J Physiol Heart CircPhysiol 259: H536–H542, 1990.
51. Weinberg RJ and van Eyck SL. A tetramethylbenzidine/tungstate reac-tion for horseradish peroxidase histochemistry. J Histochem Cytochem 39:1143–1148, 1991.
