Central control of atrio-ventricular conduction and left ventricular contractility in the cat heart: Synaptic interactions of vagal preganglionic neurons in the nucleus ambiguus with neuropeptide Y-immunoreactive nerve terminals

In the cat, vagal postganglionic controls of heart rate, atrio-ventricular (AV) conduction and left ventricular contractility are mediated by three separate intrinsic cardiac ganglia, the sinoatrial (SA), AV and cranioventricular (CV) ganglia, respectively. The vagal preganglionic neurons (VPNs) that project to these ganglia are located in the ventrolateral nucleus ambiguus (NA-VL). We have previously shown that the VPNs projecting to the SA, AV and CV ganglia are distinct from one another. We have also demonstrated that neuropeptide Y-immunoreactive (NPY-IR) axon terminals synapse upon VPNs projecting to the SA ganglion. In the present study, we test the hypothesis that those VPNs projecting to the AV ganglion (negative dromotropic VPNs) and those projecting to the CV ganglion (negative inotropic VPNs) are innervated by NPY-IR terminals in NA-VL. A retrograde tracer was injected into the AV or CV ganglion of the cat, and the brains subsequently processed for visualization of tracer and the immunocytochemical visualization of NPY by dual labeling electron-microscopic methods. We observed that 11+/-5% of all axodendritic synapses and 8+/-6% of all axosomatic synapses upon negative inotropic VPNs were NPY-IR. Furthermore, 19+/-14% of all axodendritic synapses upon negative dromotropic VPNs were NPY-IR. A few NPY-IR axosomatic synapses upon negative dromotropic neurons were also observed. NPY-IR terminals in NA-VL occasionally formed axosomatic synapses with NPY-IR neurons and axoaxonic synapses with unlabeled terminals. These results suggest that central NPY afferents to the NA-VL modulate the vagal preganglionic control of AV conduction and left ventricular contractility.     

Enkephalins and functionally specific vagal preganglionic neurons to the heart: ultrastructural studies in the cat

In cat, distinct populations of vagal preganglionic and postganglionic neurons selectively modulate heart rate, atrioventricular conduction and left ventricular contractility, respectively. Vagal preganglionic neurons to the heart originate in the ventrolateral part of nucleus ambiguus and project to postganglionic neurons in intracardiac ganglia, including the sinoatrial (SA), atrioventricular (AV) and cranioventricular (CV) ganglia, which selectively modulate heart rate, AV conduction and left ventricular contractility, respectively. These ganglia receive projections from separate populations of vagal preganglionic neurons. The neurochemical anatomy and synaptic interactions of afferent neurons which mediate central control of these preganglionic neurons is incompletely understood. Enkephalins cause bradycardia when microinjected into nucleus ambiguus. It is not known if this effect is mediated by direct synapses of enkephalinergic terminals upon vagal preganglionic neurons to the heart. The effects of opioids in nucleus ambiguus upon AV conduction and cardiac contractility have also not been studied. We have tested the hypothesis that enkephalinergic nerve terminals synapse upon vagal preganglionic neurons projecting to the SA, AV and CV ganglia. Electron microscopy was used combining retrograde labeling from the SA, AV or CV ganglion with immunocytochemistry for enkephalins in ventrolateral nucleus ambiguus. Eight percent of axodendritic synapses upon negative chronotropic, and 12% of axodendritic synapses upon negative dromotropic vagal preganglionic neurons were enkephalinergic. Enkephalinergic axodendritic synapses were also present upon negative inotropic vagal preganglionic neurons. Thus enkephalinergic terminals in ventrolateral nucleus ambiguus can modulate not only heart rate but also atrioventricular conduction and left ventricular contractility by directly synapsing upon cardioinhibitory vagal preganglionic neurons.     

Neural control of left ventricular contractility in the dog heart: synaptic interactions of negative inotropic vagal preganglionic neurons in the nucleus ambiguus with tyrosine hydroxylase immunoreactive terminals

Recent physiological evidence indicates that vagal postganglionic control of left ventricular contractility is mediated by neurons found in a ventricular epicardial fat pad ganglion. In the dog this region has been referred to as the cranial medial ventricular (CMV) ganglion [J.L. Ardell, Structure and function of mammalian intrinsic cardiac neurons, in: J.A. Armour, J.L. Ardell (Eds.), Neurocardiology, Oxford Univ. Press, New York, 1994, pp. 95–114; B.X. Yuan, J.L. Ardell, D.A. Hopkins, A.M. Losier, J.A. Armour, Gross and microscopic anatomy of the canine intrinsic cardiac nervous system, Anat. Rec., 239 (1994) 75–87]. Since activation of the vagal neuronal input to the CMV ganglion reduces left ventricular contractility without influencing cardiac rate or AV conduction, this ganglion contains a functionally selective pool of negative inotropic parasympathetic postganglionic neurons. In the present report we have defined the light microscopic distribution of preganglionic negative inotropic neurons in the CNS which are retrogradely labeled from the CMV ganglion. Some tissues were also processed for the simultaneous immunocytochemical visualization of tyrosine hydroxylase (TH: a marker for catecholaminergic neurons) and examined with both light microscopic and electron microscopic methods. Histochemically visualized neurons were observed in a long slender column in the ventrolateral nucleus ambiguus (NA-VL). The greatest number of retrogradely labeled neurons were observed just rostral to the level of the area postrema. TH perikarya and dendrites were commonly observed interspersed with vagal motoneurons in the NA-VL. TH nerve terminals formed axo-dendritic synapses upon negative inotropic vagal motoneurons, however the origin of these terminals remains to be determined. We conclude that synaptic interactions exist which would permit the parasympathetic preganglionic vagal control of left ventricular contractility to be modulated monosynaptically by catecholaminergic afferents to the NA-VL.     

Ultrastructural circuitry of cardiorespiratory reflexes: there is a monosynaptic path between the nucleus of the solitary tract and vagal preganglionic motoneurons controlling atrioventricular conduction in the cat

We have tested the hypothesis: (1) that presumptive negative dromotropic vagal preganglionic neurons in the ventrolateral nucleus ambiguus (NA-VL) can be selectively labelled from the heart, by injecting one of two fluorescent tracers into the two intracardiac ganglia which independently control sino–atrial (SA) rate or atrioventricular (AV) conduction; i.e., the SA and AV ganglia, respectively. The NA-VL was examined for the presence of single and/or double labelled cells. Over 91% of vagal preganglionic neurons in the NA-VL projecting to either intracardiac ganglion did not project to the second ganglion. Consequently, we also tested the hypothesis: (2) that there is a monosynaptic connection between neurons of the medial, and/or dorsolateral nucleus of the solitary tract (NTS), rostral to obex, and negative dromotropic neurons in the NA-VL. An anterograde tracer was injected into the NTS, and a retrograde tracer into the AV ganglion. The anterograde marker was found in both myelinated and unmyelinated axons in the NA-VL, as well as in nerve terminals. Axo–somatic and axo–dendritic synapses were detected between terminals labelled from the NTS, and retrogradely labelled negative dromotropic neurons in the NA-VL. This is the first ultrastructural demonstration of a monosynaptic pathway between neurons in the NTS and functionally associated (negative dromotropic) cardioinhibitory neurons. The data are consistent with the hypothesis that the neuroanatomical circuitry mediating the vagal baroreflex control of AV conduction may be composed of as few as four neurons in series, although interneurons may also be interposed within the NTS.     

Control of negative inotropic vagal preganglionic neurons in the dog: synaptic interactions with substance P afferent terminals in the nucleus ambiguus?

Previous research from this laboratory has shown that substance P-immunoreactive (SP) terminals synapse upon negative chronotropic vagal preganglionic neurons (VPNs), but not upon negative dromotropic VPNs, of the ventrolateral nucleus ambiguus (NA-VL). Moreover, SP agonists injected into NA-VL cause bradycardia without decreasing AV conduction. In the current study, we have: (1) defined the electron microscopic characteristics of the SP neurons of NA-VL in dog; and (2) tested the hypothesis that SP nerve terminals synapse upon negative inotropic VPNs of NA-VL, retrogradely labeled from the cranial medial ventricular (CMV) ganglion. Numerous SP terminals and a few SP neurons were observed in the vicinity of retrogradely labeled neurons. SP terminals were observed forming synapses with unlabeled dendrites and with SP dendrites, but never with the retrogradely labeled neurons. Together, these results and earlier findings suggest that SP agonists may be able to induce bradycardia without decreasing AV conduction or ventricular contractility.     

Substance P nerve terminals synapse upon negative chronotropic vagal motoneurons

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic ganglia on the surface of the heart which are capable of selective control of sino-atrial rate, atrio-ventricular conduction, and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates heart rate (the SA ganglion). Medullary tissues were processed for the histochemical visualization of retrogradely labeled neurons and for the immunohistochemical detection of the neurotransmitter substance P (SP) by dual labeling light and electron microscopic methods. Negative chronotropic retrogradely labeled cells were found in a long slender column in the ventrolateral nucleus ambiguus (NA-VL) which enlarged somewhat at the level of the area postrema. These cells were found bilaterally, but they were asymmetrically distributed. Half the animals showed a pronounced right side predominance in retrograde labeling, while the other half of the animals showed a lesser left side predominance. These observations may help to explain some of the controversy in the literature concerning the relative influence of the right and left vagus nerves on sinus rate. Ultrastructural examination demonstrated axo-somatic and axo-dendritic contacts between SP nerve terminals and retrogradely labeled negative chronotropic NA-VL neurons. SP immunoreactivity was often associated with large dense-core vesicles in terminals forming either symmetric or asymmetric synapses. These observations provide a potential anatomical substrate for the centrally mediated bradycardia elicited by microinjections of SP into the NA. SP immunoreactive terminals were also observed to make axo-somatic, axo-dendritic, and axo-axonic synapses with unlabeled neurons in NA-VL. These data suggest that SP may also modulate the activity of other vagal preganglionic neurons.     

Cardiotopic organization of the nucleus ambiguus? An anatomical and physiological analysis of neurons regulating atrioventricular conduction

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic postganglionic vagal motoneurons on the surface of the heart which are capable of selective control of sinoatrial rate, atrioventricular conduction and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates atrioventricular conduction (the AV ganglion). Medullary tissues were processed for the histochemical detection of retrogradely labeled neurons by light and electron microscopic methods. Negative dromotropic retrogradely labeled cells were found in a long column in the ventrolateral nucleus ambiguus (NA-VL), which enlarged somewhat at the level of the area postrema, but reached its largest size rostral to the area postrema in an area termed the rostral ventrolateral nucleus ambiguus (rNA-VL). Three times as many cells were observed in the left rNA-VL as compared to the right (P < 0.025). Retrogradely labeled cells were also consistantly observed in the dorsal motor nucleus of the vagus (DMV). The DMV contained one third as many cells as the NA-VL. The right DMV contained twice as many cells as the left (P < 0.05). These data are consistent with physiological evidence that suggests that the left vagus nerve is dominant in the regulation of AV conduction, but that the right vagus nerve is also influential. While recording the electrocardiogram in paced and non-paced hearts,l-glutamate (GLU) was microinjected into the rNA-VL. Microinjections of GLU caused a 76% decrease in the rate of atrioventricular (AV) conduction (P < 0.05) and occasional second degree heart block, without changing heart rate. The effects of GLU were abolished by ipsilateral cervical vagotomy. These physiological data therefore support the anatomical inference that CNS neurons that are retrogradely labeled from the AV ganglion selectively exhibit negative dromotropic properties. Retrogradely labeled negative dromotropic neurons displayed a round nucleus with ample cytoplasm, abundant rough endoplasmic reticulum and the presence of distinctive somatic and dendritic spines. These neurons received synapses from afferent terminals containing small pleomorphic vesicles and large dense core vesicles. These terminals made both asymmetric and symmetric contacts with negative dromotropic dendrites and perikarya, respectively. In conclusion, the data presented indicate that there is a cardiotopic organization of ultrastructurally distinctive negative dromotropic neurons in the NA-VL. This central organization of parasympathetic preganglionic vagal motoneurons mirrors the functional organization of cardioinhibitory postganglionic neurons of the peripheral vagus nerve. These data are further discussed in comparison to a recent report on the light microscopic distribution and ultrastructural characteristics of negative chronotropic neurons in the NA-VL⁴². The data support the hypothesis that anatomically separated and functionally selective parasympathetic preganglionic vagal motoneurons in the NA may independently control AV conduction and cardiac rate.     

Localization of sympathetic postganglionic neurons of physiologically identified cardiac nerves in the dog

Cardiac nerves were identified physiologically and injected with horseradish peroxidase in 38 dogs. Retrogradely labeled neurons were present in the greatest number in the middle cervical ganglion, whereas fewer labeled neurons were present in the stellate ganglion. Only occasional neurons in the superior cervical ganglion were labeled, and no labelphysiologically and injected with horseradish peroxidase in 38 dogs. Retrogradely labeled neurons were present in the greatest number in the middle cervical ganglion, whereas fewer labeled neurons were present in the stellate ganglion. Only occasional neurons in the superior cervical ganglion were labeled, and no labelphysiologically and injected with horseradish peroxidase in 38 dogs. Retrogradely labeled neurons were present in the greatest number in the middle cervical ganglion, whereas fewer labeled neurons were present in the stellate ganglion. Only occasional neurons in the superior cervical ganglion were labeled, and no labeled cells were found in the T3 to T6 paravertebral ganglia or in the ganglia contralateral to the nerve injected. following injections into specific cardiac nerves, retrograde labeling was widespread within the middle cervical ganglion, and the distributions of labeled neurons from different nerves overlapped considerably. In the middle cervical ganglion there was little or no regional grouping of cells projecting to specific cardiac nerves. within the stellate ganglion, however, te cardiac-sympathetic cells were clustered primarily at the cranial pole near toe origin of the ventral and dorsal ansae. Mediastinal ganglia and ganglia located in cardiac nerves were frequently as heavily labeled as the ipsilateral stellate ganglion. The occurrence of heavy labeling in mediastinal and cardiac nerve ganglia indicates that these hitherto unreported ganglia play a significant role in cardiac neural regulation. These data imply that the organization of sympathetic neurons controlling the heart is much more complex than has previously been considered.     

The coexistence of TrkA with putative transmitter agents and calcium-binding proteins in the vagal and glossopharyngeal sensory neurons of the adult rat

The presence of the neurotrophin receptor, TrkA, in neurochemically identified vagal and glossopharyngeal sensory neurons of the adult rat was examined. TrkA was colocalized with calcitonin gene-related peptide (CGRP), parvalbumin, or calbindin D-28k in neurons of the nodose, petrosal and/or jugular ganglia. In contrast, no TrkA-immunoreactive (ir) neurons in these ganglia colocalized tyrosine hydroxylase-ir. About one-half of the TrkA-ir neurons in the jugular and petrosal ganglia contained CGRP-ir, whereas only a few of the numerous TrkA-ir neurons in the nodose ganglion contained CGRP-ir. Although 43% of the TrkA-ir neurons in the nodose ganglion contained calbindin D-28k-ir, few or no TrkA-ir neurons in the petrosal or jugular ganglia were also labeled for either calcium-binding protein. These data show distinct colocalizations of TrkA with specific neurochemicals in vagal and glossopharyngeal sensory neurons, and suggest that nerve growth factor (NGF), the neurotrophin ligand for TrkA, plays a role in functions of specific neurochemically defined subpopulations of mature vagal and glossopharyngeal sensory neurons.     

Nitric oxide synthase in vagal sensory and sympathetic neurons innervating the guinea-pig trachea

Sympathetic (stellate and superior cervical ganglion) and sensory vagal (nodose and jugular ganglion) neurons innervating the guinea-pig trachea were labelled using a retrograde neuronal tracer (Fast Blue) and tested for immunoreactivity to nitric oxide synthase (NOS) and either tyrosine hydroxylase (TH; sympathetic ganglia) or substance P (SP; vagal afferent neurons). Approx. 3% of the sympathetic neurons innervating the trachea were NOS-positive. These neurons belonged to the non-catecholaminergic phenotype. Amongst the retrogradely labelled neurons in the vagal sensory ganglia, 5–10% of retrogradely labelled neurons in the nodose (inferior vagal) ganglion, and 10–20% of those in the jugular (superior vagal) ganglion were NOS-immunoreactive. All NOS-positive vagal afferent neurons labelled with retrograde tracer were negative for substance P. Accordingly, the results of these studies provide evidence that portions of the sympathetic and sensory innervation of the guinea-pig trachea is provided by NOS-immunoreactive neurons.     

Presence of cholinergic neurons in the vagal afferent system: biochemical and immunohistochemical approaches

The presence of cholinergic fibers in the afferent vagal system of various species was shown using biochemical and immunohistochemical methods. Biochemical activity of choline acetyl transferase, the synthesizing enzyme for acetylcholine, was detected in the nodose ganglion of cat, rabbit, dog and sheep. Immunohistochemistry, using a monoclonal antibody raised against choline acetyl transferase, revealed labelled cell bodies in the nodose ganglion of the rabbit. Acetylcholine endogenous content, measured in nodose ganglia devoid of efferent fibers, was twice as high in the right ganglion as compared to the left. Enzyme transport and choline acetyl transferase activity analysis were each determined on separate peripheral vagus nerves. These results are discussed in terms of functional properties of the vagal afferent neurons, including the modulation of vagal afferent messages at the level of the nodose ganglion and the eventual control of peripheral intrinsic neurons by sensory vagal terminals.     

Galanin immunoreactivity in the mudpuppy cardiac ganglion.

The source of galanin-immunoreactive fibers in the cardiac ganglion and on cardiac muscle in mudpuppy (Necturus maculosus) has been determined utilizing immunohistochemical techniques. The galanin-immunoreactive fibers are not processes of afferent fibers originating in either the rostral four dorsal root ganglia or vagal sensory ganglia. Following colchicine treatment, all of the postganglionic parasympathetic neurons and a subpopulation of the small intrinsic neurons in the cardiac ganglion exhibit galanin immunoreactivity. The majority of the galanin-immunoreactive fibers that form complexes on the parasympathetic postganglionic neurons are derived from galanin-immunoreactive small intrinsic neurons, although some of these connections may represent collateral processes from other parasympathetic postganglionic neurons. All of the galanin-immunoreactive processes that innervate cardiac muscle are derived from postganglionic parasympathetic neurons in the cardiac ganglion.     

Superior laryngeal neurons directly excite cardiac vagal neurons within the nucleus ambiguus

The aim of this study was to test whether superior laryngeal neurons have axon collaterals that synapse upon cardiac vagal neurons. Superior laryngeal neurons were tested as likely mediators of cardio-respiratory interaction because these neurons are active in post-inspiration, co-localized with cardiac vagal neurons, and have many axon collaterals within the nucleus ambiguus. Nontoxic fluorescent tracers were utilized to identify, in vitro, both superior laryngeal neurons that innervated the crico-thyroid muscle, and cardiac vagal neurons that projected to cardiac ganglia. Co-localization of these two populations of neurons demonstrated that cardiac vagal and superior laryngeal neurons are both co-localized in the nucleus ambiguus. Simultaneous dual patch clamp recordings were used to either inject depolarizing current and evoke an action potential (current clamp configuration) or control the voltage and depolarize an identified single superior laryngeal neuron (voltage clamp configuration) while simultaneously recording from a cardiac vagal neuron. Depolarization of some, but not all, individual superior laryngeal neurons elicited post-synaptic excitatory currents in cardiac vagal neurons, indicating that at least some superior laryngeal neurons monosynaptically synapse upon cardiac vagal neurons within the nucleus ambiguus.     

Ganglionic distribution of afferent neurons innervating the canine heart and cardiopulmonary nerves

The ganglionic distribution of the perikarya of afferent axons in cardiopulmonary nerves or the heart was studied in 64 dogs by injecting horseradish peroxidase into physiologically identified cardiopulmonary nerves or different regions of the heart. In 6 additional dogs, horseradish peroxidase was injected into the aortic arch, pericardial sac, left ventricular cavity or the skin. After injections into cardiopulmonary nerves, retrogradely labeled perikarya were found in the ipsilateral nodose ganglion and the ipsilateral C7-T7 dorsal root ganglia. After injections into different regions of the heart, retrogradely labeled neurons were found in the nodose ganglia bilaterally and in the C6-T6 dorsal root ganglia bilaterally. Many more retrogradely labeled neurons were found in the nodose ganglia in comparison to the dorsal root ganglia. The largest numbers of retrogradely labeled perikarya in the dorsal root ganglia occurred in the T 2-4 ganglia following nerve or heart injections. Following injections into specific regions of the heart or individual physiologically identified cardiopulmonary nerves, regional distributions of labeled neurons could not be identified within or among ganglia with respect to the structures injected. Perikarya in dorsal root ganglia which were labeled after heart injections ranged in area from 436-3280 microns 2 (X = 1279 +/- 51 S.E.M.) while after skin injections labeled perikarya ranged in area from 224-5701 microns 2 (X = 1631 +/- 104 S.E.M.). The results show that the afferent innervation of the canine heart is provided by neurons located throughout the nodose ganglia and to a lesser degree in the C6-T6 dorsal root ganglia bilaterally. The bilateral distribution of cardiac afferent neurons raises questions regarding mechanisms underlying unilateral symptoms frequently associated with heart disease.     

ASIC3-immunoreactive neurons in the rat vagal and glossopharyngeal sensory ganglia

ASIC3-immunoreactivity (ir) was examined in the rat vagal and glossopharyngeal sensory ganglia. In the jugular, petrosal and nodose ganglia, 24.8%, 30.8% and 20.6% of sensory neurons, respectively, were immunoreactive for ASIC3. These neurons were observed throughout the ganglia. A double immunofluorescence method demonstrated that many ASIC3-immunoreactive (ir) neurons co-expressed calcitonin gene-related peptide (CGRP)- or vanilloid receptor subtype 1 (VRL-1)-ir in the jugular (CGRP, 77.8%; VRL-1, 28.0%) and petrosal ganglia (CGRP, 61.7%; VRL-1, 21.5%). In the nodose ganglion, however, such neurons were relatively rare (CGRP, 6.3%; VRL-1, 0.4%). ASIC3-ir neurons were mostly devoid of tyrosine hydroxylase in these ganglia. However, some ASIC3-ir neurons co-expressed calbindin D-28k in the petrosal (5.5%) and nodose ganglia (3.8%). These findings may suggest that ASIC3-containing neurons have a wide variety of sensory modalities in the vagal and glossopharyngeal sensory ganglia.     

Horseradish peroxidase localization of the sympathetic postganglionic neurons innervating the cat heart

The localization of the sympathetic postganglionic neurons innervating the cat heart has been investigated by using retrograde axonal transport of horseradish peroxidase (HRP). HRP was injected into the subepicardial layers of 4 different cardiac regions. The animals were sacrificed 72-96 h later and fixed by perfusion via the left ventricle. The paravertebral sympathetic ganglia from the superior cervical, middle cervical and stellate ganglia to T10 ganglia were removed and processed for HRP identification. Following injections of HRP into the apex of the heart, the sinoatrial (SA) nodal region and the ventral wall of the right ventricle, we observed that HRP-labeled sympathetic neurons were localized predominantly in the right stellate ganglia, and to a lesser extent, in the right superior and middle cervical ganglia, and left stellate ganglia. Fewer labeled cells were found in the right T4-T6. T8 and T9. After HRP injection into the dorsal wall of the left ventricle, HRP-labeled cells were present mainly in the left stellate ganglia.     

Localization of efferent sympathetic neurons and afferent neurons in dorsal root ganglia innervating the heart

The retrograde transport of horseradish peroxidase (HRP) has been used to study the localization and the number of neurons innervating the heart in the right stellate ganglion and accessory cervical ganglion, spinal cord and dorsal root ganglia of the cat. HRP was applied to the central cuts of anastomose of the stellate ganglion with the vagal nerve, of the vagosympathetic trunk caudal to anastomose and of the inferior cardiac nerve. HRP-labelled neurons were detected in the stellate ganglion in the regions which give off nerves, whereas in the accessory cervical ganglion labelled neurons were distributed throughout the whole ganglion. HRP-stained cells were found in the anastomose. In the spinal cord labelled neurons were detected in the lateral horn of T1-T5 segments. In the dorsal root ganglion the greatest number of neurons was observed in T2-T4 segments.     

Anatomical demonstration of vagal input to nicotinamide acetamide dinucleotide phosphate diaphorase-positive (nitrergic) neurons in rat fundic stomach

Recent pharmacological evidence suggests that the nonadrenergic, noncholinergic (NANC) vagal inhibitory input responsible for receptive relaxation of the fundic stomach is mediated by nitric oxide-synthesizing enteric neurons. To demonstrate anatomically such direct vagal inputs to neurochemically identified enteric neurons, we utilized the nicotinamide acetamide dinucleotide phosphate (NADPH)-diaphorase histochemical reaction in conjunction with selective anterograde labeling of vagal efferents or afferents. Approximately 30% of all myenteric neurons of the fundic myenteric plexus stained positive for NADPH diaphorase, and the principal recipient of axonal projections from NADPH diaphorase-positive neurons was the circular muscle layer. In a group of animals showing the most complete labeling of vagal efferent preganglionics with the carbocyanine dye DiA, quantitative analysis of the half of the ventral fundic wall closer to the greater curvature revealed that 46.8% ± 4.4% of all myenteric neurons received some degree of vagal contacts and that 30.5% ± 6.6% of such vagally contacted neurons were also NADPH diaphorase positive. In another group of rats with the most successful selective labeling of vagal afferents through DiI injections into the left nodose ganglion, analysis of select ganglia throughout the ventral fundic wall revealed that, of a total of 454 neurons with vagal afferent contacts, 34.8% ± 2.8% were NADPH diaphorase positive. These findings support the view that, in the fundic stomach, some vagal preganglionic efferents terminate on nitric oxide-synthesizing neurons that, in turn, project to and relax the external smooth muscle layers. Furthermore, vagal afferent endings also contact NADPH diaphorase-positive neurons, suggesting the possibility of local axon reflexes originating from smooth muscular in-series tension receptors and terminating on nitrergic neurons of the myenteric plexus. © 1995 Wiley-Liss, Inc.     

GABA-ergic neurons in the crayfish nervous system: an immunocytochemical census of the segmental ganglia and stomatogastric system

We used an antiserum directed against gamma-aminobutyric acid (GABA) fixed with glutaraldehyde (Hoskins et al., Cell Tissue Res. 244:243-252, '86) to label neurons with GABA-like immunoreactivity (GLI) in wholemounts of the stomatogastric ganglion and each segmental ganglion of crayfish, except the brain. Each abdominal ganglion had an average of 63 labeled neurons, or 10% of all their neurons. Each peripheral nerve of each abdominal ganglion except the last contained labeled axons. Within each segment, the first peripheral nerve, N1, had five axons; the second peripheral nerve, N2, had at most four; and the third peripheral nerve, N3, had two. In the last ganglion, N2 had one labeled axon, N3 had two and N6 had two; the other nerves contained no labeled axons. A tabulation of the identified inhibitory neurons in the abdominal ganglia revealed that 40% of these GABA-ergic neurons have been identified. The subesophageal ganglion had many labeled neurons in clusters that formed a repeating pattern; it also had labeled neurons near its dorsal midline. The thoracic ganglia contained more labeled neurons than did the abdominals, but their patterns of labeling were similar. The commissural ganglia contained three clusters of labeled neurons and sent labeled axons to the esophageal ganglion. The esophageal ganglion contained four labeled neurons and many labeled axons. The stomatogastric ganglion contained labeled axon terminals but not labeled neurons.