Location of efferent sympathetic neurons and afferent neurons in spinal ganglia innervating the heart

Location and numbers of neurons associated with sympathetic innervation of the heart within the right stellate and accessory cervical ganglia, the spinal cord, and spinal ganglia were investigated using horseradish peroxidase retrograde axonal transport techniques in cats. The enzyme was applied to central sections of the anastomosis of the stellate ganglion with the vagus nerve, the inferior cardiac nerve, and the vagosympathetic trunk caudal to the anastomosis. Labeled neurons within the stellate ganglion were located close to the point of departure of the nerves and more thinly distributed in the accessory cervical ganglion. A group of labeled cells was found in the anastomosis itself. Preganglionic neurons associated with sympathetic innervation of the heat were detected at segmental levels T₁–T₅ in the spinal cord. Labeled neurons were diffusely located in the spinal ganglia, concentrated mainly at levels T₂–T₄.Medical Institute, Ministry of Public Health of the RSFSR, Yaroslavl'. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 106–111, January–February, 1989.     

Horseradish peroxidase localization of sympathetic postganglionic and parasympathetic preganglionic neurons innervating the monkey heart

The localization of the sympathetic postganglionic and parasympathetic preganglionic neurons innervating the monkey heart were investigated through retrograde axonal transport with horseradish peroxidase (HRP). HRP (4 mg or 30 mg) was injected into the subepicardial and myocardial layers in four different cardiac regions. The animals were euthanized 84-96 hours later and fixed by paraformaldehyde perfusion via the left ventricle. The brain stem and the paravertebral sympathetic ganglia from the superior cervical, middle cervical, and stellate ganglia down to the T9 ganglia were removed and processed for HRP identification. Following injection of HRP into the apex of the heart, the sinoatrial nodal region, or the right ventricle, HRP-labeled sympathetic neurons were found exclusively in the right superior cervical ganglion (64.8%) or in the left superior cervical ganglion (35%). Fewer labeled cells were found in the right stellate ganglia. After HRP injection into the left ventricle, labeled sympathetic cells were found chiefly in the left superior cervical ganglion (51%) or in the right superior cervical ganglion (38.6%); a few labeled cells were seen in the stellate ganglion bilaterally and in the left middle cervical ganglion. Also, in response to administration of HRP into the anterior part of the apex, anterior middle part of the right ventricle, posterior upper part of the left ventricle, or sinoatrial nodal region, HRP-labeled parasympathetic neurons were found in the nucleus ambiguus on both the right (74.8%) and left (25.2%) sides. No HRP-labeled cells were found in the dorsal motor nucleus of the vagus on either side.     

Catecholamine-containing neurons in Schistosoma mansoni

Summary Falck's method for the demonstration of monoamines was applied to the flatworm Schistosoma mansoni. Whole mount preparations and sections of frozen dried specimens showed that a primary catecholamine is present in four pairs of large nerve cells and in two longitudinal fiber tracts showing varicosities. Smaller neurons were found along these tracts. They are united by commissures and give off many branches which end in small dilatations.Contribution number 14 from the Schistosomiasis Research Unit, Institute of Biological Sciences, Federal University of Minas Gerais, Brazil. This study has been supported by research grants from the U.S. Army (DAAC 19-70-G-0023 and DAHG-70-G-0028), CAPES and Conselho Nacional de Pesquisas, Brazil. For technical assistance we thank Mr. Rubens Miranda, technician supported by the Conselho Nacional de Pesquisas of Brazil.     

Neuropeptide Y,enkephalin and noradrenaline coexist in sympathetic neurons innervating the bovine spleen

Summary The subcellular distribution of noradrenaline (NA), neuropeptide Y (NPY), Met and Leu-enkephalin (ENK), substance P (SP), somatostatin (SOM), and vasoactive intestinal polypeptide (VIP) was investigated in homogenates of bovine splenic nerve. The distribution of noradrenergic peptide-containing nerves in the bovine celiac ganglion, splenic nerve and terminal areas in spleen was studied by indirect immunofluorescence histochemistry using antisera to tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), NPY, enkephalin peptides, SP, SOM, VIP and peptide HI (PHI).After density gradient centrifugation, high levels of NPY and ENK-like immunoreactivity (LI) were found in high-density gradient fractions, coinciding with the main NA peak. SP, SOM and VIP were found in fractions with a lower density, VIP being also enriched in a heavy fraction; the latter three peptides were present in low concentrations.Immunohistochemistry revealed that staining for NPYLI and ENK-LI partly overlapped that for TH and DBH in celiac ganglia, splenic nerve axons and terminal areas of spleen. Almost all principal ganglion cells were TH- and DBH-immunoreactive. Many were also NPY-immunoreactive, whereas a smaller number were ENK-positive. In the celiac ganglion patches of dense SP-positive networks and some VIP/PHI- and ENK-immunoreactive fibers were seen around cell bodies.The results indicate that NPY and ENK are stored with NA in large dense-cored vesicles in unmyelinated axons of bovine splenic nerve. SP, SOM and VIP appear in different organelles in axon populations separate from sympathetic noradrenergic nerves.     

Double labelling immunohistochemical characterization of autonomic sympathetic neurons innervating the sow retractor clitoridis muscle

Retrograde neuronal tracing and immunohistochemical methods were used to define the neurochemical content of sympathetic neurons projecting to the sow retractor clitoridis muscle (RCM). Differently from the other smooth muscles of genital organs, the RCM is an isolated muscle that is tonically contracted in the rest phase and relaxed in the active phase. This peculiarity makes it an interesting experimental model. The fluorescent tracer fast blue was injected into the RCM of three 50 kg subjects. After a one-week survival period, the ipsilateral paravertebral ganglion S1, that in a preliminary study showed the greatest number of cells projecting to the muscle, was collected from each animal. The co-existence of tyrosine hydroxylase with choline acetyltransferase, neuronal nitric oxide synthase, calcitonin gene-related peptide, leu-enkephalin, neuropeptide Y, substance P and vasoactive intestinal polypeptide was studied under a fluorescent microscope on cryostat sections. Tyrosine hydroxylase was present in about 58% of the neurons projecting to the muscle and was found to be co-localized with each of the other tested substances. Within fast blue-labelled cells negative to the adrenergic marker, small populations of neurons singularly containing each of the other enzymatic markers or peptides were also observed. The present study documents the complexity of the neurochemical interactions that regulate the activity of the smooth myocytes of the RCM and their vascular components.     

Catecholamine-containing neurons in the peripheral nervous system of Aplysia

Numerous green-fluorescent neurons have been revealed by means of the glyoxylic acid histochemical method in cryostat sections of several organs of two Adriatic aplysiid gastropods, Aplysia depilans and A. fasciata. Catecholamine-containing perikarya and their processes have been found to be especially abundant in the vaginal portion of the large hermaphrodite duct, in the penis and its sheath, and in the gill. In the reproductive organs, two subpopulations of catecholamine-containing neurons could be distinguished according to their size and location. Axons of larger neurons formed bundles which seemed to project at the CNS.     

Angiotensinergic neurons in sympathetic coeliac ganglia innervating rat and human mesenteric resistance blood vessels

In contrast to the current belief that angiotensin II (Ang II) interacts with the sympathetic nervous system only as a circulating hormone, we document here the existence of endogenous Ang II in the neurons of rat and human sympathetic coeliac ganglia and their angiotensinergic innervation with mesenteric resistance blood vessels. Angiotensinogen - and angiotensin converting enzyme-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia, while renin mRNA was untraceable. Cathepsin D, a protease responsible for cleavage beneath other substrates also angiotensinogen to angiotensin I, was successfully detected in rat coeliac ganglia indicating the possibility of existence of alternative pathways. Angiotensinogen mRNA was also detected by in situ hybridization in the cytoplasm of neurons of rat coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia as well as with mesenteric resistance blood vessels. By using confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally with the mesenteric resistance blood vessels.     

Structure and localization of sympathetic neurons in the cat spinal cord

By the method of retrograde axonal transport of horseradish peroxidase (HP) structure and localization of sympathetic neurons sending axons to the cranial cervical ganglion (CCG) have been revealed ipsilaterally in the ventral horns and in 4 nuclei of the spinal cord: nucl ILp, nucl. ILf. nucl. IC, nucl. ICpe. Orientation of the neurons, their number, structure of the nuclei formed by them, degree of the CCG efferentation by the preganglionic fibres, which run from various nuclei, are different. In nucl. ILf two types of neurons have been revealed-triangle and spindle-shaped, they always orienting by their long axis in mediolateral direction. The greatest amount of HP-positive neurons are found in nucl. ILp. They form a well distinquished compact nucleus in the lateral horns. HP-labelled neurons in nucl. ILp are found at the level of segments T1-T8 with their maximal amount at the level of segments T1-T3. HP-positive neurons are detected in nucl. ILf beginning from the segment C8 up to the middle of T4, in nucl. IC-from the segment C8 up to T6, in nucl. ICpe-from the segment C8 up to T5, in the ventral horns-from the segment T1 up to T5. In rostocaudal direction from the segment C8 up to T8 the number of HP-positive neurons is decreasing, but the part of nucl. ILp neurons in the CCG efferentation, comparing to the neurons in other sympathetic structures of the spinal cord, is increasing.     

Mesodermal and neuronal retinoids regulate the induction and maintenance of limb innervating spinal motor neurons

During embryonic development, the generation, diversification and maintenance of spinal motor neurons depend upon extrinsic signals that are tightly regulated. Retinoic acid (RA) is necessary for specifying the fates of forelimb-innervating motor neurons of the Lateral Motor Column (LMC), and the specification of LMC neurons into medial and lateral subtypes. Previous studies implicate motor neurons as the relevant source of RA for specifying lateral LMC fates at forelimb levels. However, at the time of LMC diversification, a significant amount of retinoids in the spinal cord originates from the adjacent paraxial mesoderm. Here we employ mouse genetics to show that RA derived from the paraxial mesoderm is required for lateral LMC induction at forelimb and hindlimb levels, demonstrating that mesodermally synthesized RA functions as a second source of signals to specify lateral LMC identity. Furthermore, reduced RA levels in postmitotic motor neurons result in a decrease of medial and lateral LMC neurons, and abnormal axonal projections in the limb; invoking additional roles for neuronally synthesized RA in motor neuron maintenance and survival. These findings suggest that during embryogenesis, mesodermal and neuronal retinoids act coordinately to establish and maintain appropriate cohorts of spinal motor neurons that innervate target muscles in the limb.     

Oxytocin modulation of intrathoracic sympathetic ganglionic neurons regulating the canine heart

Substance P and vasoactive intestinal peptide are known to activate intrathoracic sympathetic neurons which regulate the heart. In the present series of experiments, when 1 I.U. of oxytocin in 0.1 cc of normal saline was administered into the cranial poles of stellate or the middle of middle cervical ganglia cardiac rate and force were augmented. The locations of ganglionic loci which, when injected, resulted in cardiac changes varied between animals. Twenty active sites were identified in the 12 dogs investigated. When the vehicle (0.1 cc of normal saline) was injected into these active sites minimal cardiac responses were induced in one instance. When 1 or 2 I.U. of oxytocin was administered into the superior vena cave of seven animals slight systemic hypotension occurred in two of these animals. Cardiac responses were induced when oxytocin was reinjected into active intrathoracic ganglionic sites after whole body administration of hexamethonium (10 mg/kg IV), but not after local administration of timolol into the ganglia. Thus, it appears that oxytocin can activate intrathoracic ganglionic neurons involved in efferent sympathetic cardiac regulation. That such responses persist in the presence of nicotinic blockade, but not following beta-adrenergic blockade of ganglionic neurons, indicates that oxytocin modifies beta-adrenergic and not nicotinic receptors on neurons in these ganglia.     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77–86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.     

Properties of axons of sympathetic preganglionic neurons in the cat lumbar spinal cord

Responses arising in ventral root filaments and antidromic discharges of single sympathetic preganglionic neurons in the lateral horn of gray matter in segment L₂ of the cat spinal cord were recorded during stimulation of the white rami communicantes in the same segment. Conduction velocities, thresholds, and refractory periods were determined for individual groups of sympathetic preganglionic fibers. Excitation was conducted more slowly along the intramedullary part of the axons of some sympathetic neurons than along the extramedullary part. In a third group of neurons studied the second antidromic discharge appeared in response to paired stimulation if the interstimulus interval was appreciably longer than their refractory period. It is postulated that axons of sympathetic preganglionic neurons in the lumber spinal cord have a thin intramedullary part and are supplied with recurrent collaterals.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 6, No. 2, pp. 143–151, March–April, 1974.     

Recurrent inhibition of sympathetic preganglionic neurons of the lateral horns of the spinal cord

Experiments on anesthetized and immobilized cats showed that repeated antidromic discharges can be evoked in 32.5% of sympathetic preganglionic neurons of the lateral horns in segments T3, T8–9, and L2 of the spinal cord, with intervals of 16 msec or more between them, which is much greater than the refractory period of these neurons. This feature was shown not to be connected with the properties of axons of that group of neurons and, in particular, with their after-subnormality. After orthodromic discharges in neurons of this group, for a much longer period of time than could be accounted for by possible collision, no antidromic discharges likewise were evoked. As a result of antidromic activation of some of these neurons in one segment, definite inhibition of the orthodromic response of other neurons of the same segment appeared, etiher by a reflex mechanism or through stimulation of descending pathways. The results point definitely to the existence of a mechanism of recurrent inhibition in some sympathetic preganglionic neurons of the lateral horns.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 382–389, July–August, 1977.     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord.

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77-86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.     

Peptidergic modulation of efferent sympathetic neurons in intrathoracic ganglia regulating the canine heart

When either substance P or vasoactive intestinal peptide was injected into an acutely decentralized intrathoracic sympathetic ganglion, short-lasting augmentation of cardiac chronotropism and inotropism was induced. These augmentations were induced before the fall in systemic arterial pressure occurred which was a consequence of these peptides leaking into the systemic circulation in enough quantity to alter peripheral vascular resistance directly. When similar volumes of normal saline were injected into an intrathoracic ganglion, no significant cardiac changes were induced. When substance P or vasoactive intestinal peptide was administered into an intrathoracic ganglion, similar cardiac augmentations were induced either before or after the intravenous administration of hexamethonium. In contrast, when these peptides were injected into an intrathoracic ganglion in which the beta-adrenergic blocking agent timolol (0.1 mg/0.1 ml of normal saline) had been administered no cardiac augmentation occurred. These data imply that in the presence of beta-adrenergic blockade intraganglionic administration of substance P or vasoactive intestinal peptide does not modify enough intrathoracic neurons to alter cardiac chronotropism and inotropism detectably. When neuropeptide Y was injected into an intrathoracic ganglion, no cardiac changes occurred. However, when cardiac augmentations were induced by sympathetic preganglionic axon stimulation these were enhanced following the intraganglionic administration of neuropeptide Y. As this effect occurred after timolol was administered into the ipsilateral ganglia, but not after intravenous administration of hexamethonium, it is proposed that the effects of neuropeptide Y are dependent upon functioning intrathoracic ganglionic nicotinic cholinergic synaptic mechanisms. Intravenous administration of either morphine or [D-ala2,D-leu5]enkephalin acetate did not alter the capacity of the preganglionic sympathetic axons to augment the heart when stimulated. Following the intravenous administration of naloxone, the positive inotropic cardiac responses induced by efferent preganglionic sympathetic axonal stimulation were enhanced minimally in control states and significantly following hexamethonium administration. Thus, it appears that enkephalins are involved in the modulation of intrathoracic ganglion neurons regulating the heart, perhaps via modification of beta-adrenergic receptors. Taken together these data indicate that substance P, vasoactive intestinal peptide, neuropeptide Y, or enkephalins modify intrathoracic ganglionic neurons which are involved in efferent sympathetic cardiac regulation.     

Localization of neurons innervating the columellar muscles of the snail

The topographic arrangement of large and small neurons participating in the mechanism of the defensive reflex was studied in the circumesophageal nerve ring ofHelix pomatia by a modified retrograde cobalt ion transport method. Comparison of the results with those of previous electrophysiological investigations of the mechanism of the defensive relfex leads to the conclusion that this reflex is effected by a system of neurons consisting of nine large and 60–80 small nerve cells.Research Institute of Neurocybernetics, State University, Rostov-on-Don. Translated from Neirofiziologiya, Vol. 12, No. 6, pp. 637–641, November–December, 1980.     

A study of single sympathetic preganglionic neurons in the lateral horns of the lumbar spinal cord

Antidromic discharges and spontaneous activity of single sympathetic preganglionic neurons in the lateral horns of the second lumbar segment of the spinal cord were recorded extracellularly in cats anesthetized with a mixture of chloralose and pentobarbital. A new technique used to identify antidromic discharges of sympathetic preganglionic neurons is described and the characteristics of discharges and sites where they were recorded are investigated. Changes in the frequency of spontaneous discharges of most neurons were shown to be connected with different types of fluctuation of the arterial pressure. Absence of functional specialization of the sympathetic preganglionic neurons is postulated.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol.6, No.3, pp.295–303, May–June, 1974.     

Calcitonin gene-related peptide neurons innervating the canine digestive system.

The pattern of nerve cells and fibers containing calcitonin gene-related peptide immunoreactivity (CGRP-IR) was investigated in the canine digestive tract by means of immunohistochemistry. CGRP-IR nerve fibers innervate all the layers of the gut, including the vasculature, with different densities depending on the region. CGRP-IR processes are sparse in the esophagus and stomach, where they are mostly confined to the enteric plexuses and vasculature. CGRP-IR fibers are quite abundant in the small and large intestine, where they form dense arborizations in the mucosa, and are numerous in the muscularis mucosae, deep muscular plexus and circular muscle. The myenteric and submucous plexuses of the intestine contain dense networks of CGRP-IR fibers and numerous CGRP-IR ganglion cells. On the other hand, in the enteric ganglia of the esophagus and stomach, in the intrapancreatic ganglia and in the ganglionated plexus of the gallbladder, CGRP-IR is restricted to non-varicose processes. A moderate density of CGRP-IR fibers supplies the endocrine and exocrine pancreas, and the fibromuscular layer and lamina propria of the gallbladder. The density of CGRP innervation in different regions can be summarized as follows: intestine > pancreas and gallbladder > or = antrum > cardia > gastric corpus and distal esophagus. CGRP- and tachykinin (TK)-IRs are colocalized in a substantial population of fibers, particularly those distributed to the mucosa, muscularis mucosae and vasculature, whereas there was no evidence of colocalization in intrinsic ganglion cells. The present results suggest that (1) the CGRP innervation of the dog digestive system includes an intrinsic and an extrinsic component, and (2) CGRP- and TK-IRs are co-expressed in extrinsic nerve fibers. These findings extend previous observations in rats and guinea pigs and provide insights into the sites of action of CGRP in the digestive system of the dog, which has served as a model for CGRP functional studies.