Light microscopic and ultrastructural localization of GABA-like immunoreactive input to retrogradely labeled sympathetic preganglionic neurons

The organization of gamma-aminobutyric acid-like immunoreactive (GABA-LIR) processes was studied within the sympathetic preganglionic neuropil of male Sprague-Dawley rats and pigeons (Columba livia). Sympathetic preganglionic neurons were retrogradely labeled following horseradish peroxidase (HRP) injections into either the adrenal medulla or superior cervical ganglion in rats or into the avian homologue of the mammalian stellate ganglion (paravertebral ganglion 14) in pigeons. GABA-LIR staining was visualized using peroxidase-antiperoxidase (PAP), avidin-biotin complex (ABC), or post-embedding immunogold methods. The pigeon preganglionic neuropil contained a dense network of GABA-LIR processes with punctate swellings that encircled sympathetic preganglionic perikarya within the principal preganglionic cell column (column of Terni) and the nucleus intercalatus spinalis. GABA-LIR spinal neurons were intermingled among HRP-labeled sympathetic preganglionic neurons within the column of Terni and throughout the zona intermedia. In the rat the density of the GABA-LIR processes within the four thoracic sympathetic preganglionic nuclei was less than that observed in the pigeon. Nevertheless, GABA-LIR profiles distinctively dotted preganglionic perikarya within the nuclei intermediolateralis pars principalis and pars funicularis, nucleus intercalatus spinalis and the central autonomic nucleus. GABA-LIR neurons were rarely observed within the nucleus intermediolateralis pars principalis, but were numerous in the zona intermedia and area X. No GABA-LIR spinal neurons in either vertebrate were retrogradely labeled with HRP. The ultrastructural arrangements of GABA-LIR processes within the sympathetic preganglionic neuropils of pigeons and rats were similar. GABA-LIR boutons formed symmetrical synaptic contacts and contained small round electron-lucent vesicles (50 nm) and one to several larger dense-core vesicles (80 nm). GABA-LIR terminals contacted HRP-labeled sympathetic preganglionic perikarya in all spinal nuclear regions in both vertebrates. More frequently, GABA-LIR boutons synapsed on dendrites. Occasionally, axo-axonic configurations were observed; each time only one of the axonal elements was GABA-LIR. Numerous unmyelinated and some thinly myelinated GABA-LIR axons coursed through the sympathetic preganglionic neuropils of both vertebrates. Synapses between GABA-LIR processes were present within the sympathetic preganglionic neuropil of both vertebrates. GABA-LIR dendrites were contacted by unlabeled terminals (predominantly small spherical vesicles with asymmetric synaptic specializations) and GABA-LIR terminals on GABA-LIR dendrites were similar in appearance to those synapsing on sympathetic preganglionic cell bodies and dendrites.     

Ultrastructural evidence of synaptic contacts between substance P-, enkephalin-, and serotonin-immunoreactive terminals and retrogradely labeled sympathetic preganglionic neurons in the rat: a study using a double-peroxidase procedure.

A double-peroxidase procedure was used to study the ultrastructural relationships between terminals and fibers containing three putative neurotransmitters and retrogradely identified sympathetic preganglionic neurons (SPNs) located in the intermediolateral cell column (IML) of the rat. SPNs with axons in the cervical sympathetic trunk were retrogradely labeled with horseradish peroxidase. In addition, terminals and fibers containing substance P, enkephalin, and serotonin were detected using immunohistochemistry. Sections containing both retrogradely labeled SPNs and immunoreactive processes were processed for electron microscopy. Ultrastructural examination revealed synaptic contacts between terminals containing each of these three neurotransmitters and retrogradely labeled dendrites from SPNs. Also, immunoreactive terminals were apposed to retrogradely labeled cell bodies. Therefore, these transmitters may alter sympathetic function by their direct action on SPNs.     

Ultrastructure of HRP-identified sympathetic preganglionic neurons in cats

Electron microscopic studies were conducted on cat sympathetic preganglionic neurons which were labeled using retrograde transport of horseradish peroxidase from the injection site in the stellate ganglion to somas in the thoracic spinal cord. Based on size, the identified somas were divided into two groups: large multipolar somas (25–35 μm), usually having three dendrites with a mean diameter of 8 μm, and small fusiform somas (12–24 μm), with two primary dendrites with a mean diameter of about 4 μm. The ultrastructures of the perikarya and dendrites were similar to other spinal neurons. Three types of terminals were identified: 1) round asymmetric terminals, 2) pleomorphic symmetric terminals, and 3) round symmetric terminals. On the soma, pleomorphic terminals were more numerous than round asymmetric terminals. On the dendrites, the density of round asymmetric terminals was similar to plemorphic symmetric terminals. Round symmetric terminals represent only a small percent on either the soma or dendrites. Because of the greater total density of terminals on the dendrites than soma, pleomorphic symmetric and round asymmetric terminals are primarily located on the dendrites. Very few dense-core vesicles characteristic of monoamine terminals were observed, perhaps due to the fixative used.     

Phenylethanolamine N-methyltransferase-containing terminals synapse directly on sympathetic preganglionic neurons in the rat

The ultrastructural morphology as well as neuronal and glial associations of phenylethanolamine N-methyltransferase (PNMT)-containing terminals in the intermediolateral cell column (IML) of the thoracic spinal cord were examined in the rat utilizing the peroxidase-antiperoxidase (PAP) method. The PNMT-immunoreactive terminals were 0.5-1.4 micron in diameter and contained a few mitochondria, a large population of small clear vesicles and from 1 to 6 large dense-core vesicles. The terminals formed synapses primarily with dendrites. The type of axodendritic association (i.e. symmetric or asymmetric) varied with the size of the dendrite, such that the majority of synapses on large dendrites were symmetric and those on smaller dendrites and dendritic spines were asymmetric. Moreover, most of the synaptic associations of PNMT-containing terminals were with the smaller dendritic processes. Many of the PNMT-labeled terminals, as well as their postsynaptic targets, were closely invested with, or apposed to fibrous astrocytic processes. In a subsequent set of experiments, we combined immunoautoradiographic labeling for PNMT with horseradish peroxidase (HRP) retrograde identification of sympathetic preganglionic neurons (SPNs) in the IML to determine whether or not SPNs receive direct synaptic input from the adrenergic terminals. In these sections, PNMT-containing terminals directly synapsed on the HRP-containing (i.e. retrogradely labeled SPNs) perikarya and dendrites. The axosomatic synapses observed between PNMT-labeled terminals and SPN perikarya were exclusively symmetric; whereas the type of axodendritic association varied depending upon the size of the dendrite such that the majority were asymmetric. The findings provide ultrastructural evidence that in the rat IML, adrenergic (i.e. PNMT-containing) terminals (1) may be either excitatory (asymmetric) or inhibitory (symmetric) depending on their site of termination and (2) can influence sympathetic nerve discharge through a direct effect on the SPN cell membrane.     

Phenylethanolamine N-methyltransferase-containing terminals synapse directly on sympathetic preganglionic neurons in the rat

The ultrastructural morphology as well as neuronal and glial associations of phenylethanolamine N-methyltransferase (PNMT)-containing terminals in the intermediolateral cell column (IML) of the thoracic spinal cord were examined in the rat utilizing the peroxidase-antiperoxidase (PAP) method. The PNMT-immunoreactive terminals were 0.5–1.4 μm in diameter and contained a few mitochondria, a large population of small clear vesicles and from 1 to 6 large dense-core vesicles. The terminals formed synapses primarily with dendrites. The type of axodendritic association (i.e. symmetric or asymmetric) varied with the size of the dendrite, such that the majority of synapses on large dendrites were symmetric and those on smaller dendrites and dendritic spines were asymmetric. Moreover, most of the synaptic associations of PNMT-containing terminals were with the smaller dendritic processes. Many of the PNMT-labeled terminals, as well as their postsynaptic targets, were closely invested with, or apposed to fibrous astrocytic processes. In a subsequent set of experiments, we combined immunoautoradiographic labeling for PNMT with horseradish peroxidase (HRP) retrograde identification of sympathetic preganglionic neurons (SPNs) in the IML to determine whether or not SPNs receive direct synaptic input from the adrenergic terminals. In these sections, PNMT-containing terminals directly synapsed on the HRP-containing (i.e. retrogradely labeled SPNs) perikarya and dendrites. The axosomatic synapses observed between PNMT-labeled terminals and SPN perikarya were exclusively symmetric: whereas the type of axodendritic association varied depending upon the size of the dendrite such that the majority were asymmetric. The findings provide ultrastructurral evidence that in the rat IML, adrenergic (i.e. PNMT-containing) terminals (1) may be either excitatory (asymmetric) or inhibitory (symmetric) depending on their site of termination and (2) can influence sympathetic nerve discharge through a direct effect on the SPN cell membrane.     

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.     

The intermediolateral cell column of the thoracic spinal cord is comprised of target-specific subnuclei: evidence from retrograde transport studies and immunohistochemistry

In this study we examined the hypothesis that the intermediolateral cell column (IML) of the thoracic spinal cord, the nucleus from which preganglionic sympathetic neurons originate, provides an anatomical substrate through which selective regulation of sympathetic nervous system targets is accomplished. Preganglionic sympathetic neurons of rats were retrogradely labeled by the simultaneous exposure of the cervical sympathetic trunk (CST) and the adrenal medulla to Fluoro-Gold and True blue, contrasting fluorescent dyes. Retrograde labeling from these sites revealed 2 populations of sympathetic preganglionic neurons in IML whose distribution overlapped between segments T1 and T4. In regions where these 2 groups of retrogradely labeled neurons overlapped, sympathoadrenal preganglionic (SAP) neurons occupied the most lateral aspect of the nucleus. It was also determined whether individual retrogradely labeled neurons within these two groups sent axon collaterals to both the CST and adrenal medulla. Diamidino yellow, a fluorescent retrograde tracer dye that labels only nuclei, was substituted for Fluoro-Gold and used in combination with True blue to simultaneously label preganglionic sympathetic neurons projecting to either the CST or adrenal medulla. No double-labeled cell bodies were observed in spinal cords of rats treated in this manner. Thus it appeared that the efferent projections of these 2 cell populations in IML were target-specific. Immunohistochemical analysis of the relationship between nerve fibers in the IML and preganglionic sympathetic neurons was also undertaken in an attempt to classify further these 2 populations of sympathetic preganglionic neurons. Equal proportions of identified CST and SAP neurons appeared to be apposed by varicosities immunoreactive for either somatostatin or serotonin. On the other hand, when the comparison was based on whether oxytocin-immunoreactive varicosities appeared to appose these 2 populations of retrogradely labeled sympathetic neurons, a highly significant difference was revealed. That is, oxytocin-immunoreactive fibers and terminals appeared to avoid SAP neurons. Thus these data support the hypothesis that an anatomical substrate exists in spinal cord IML whereby selective regulation of sympathetic nervous system targets may be mediated. Moreover, the lack of oxytocin-immunoreactive varicosities apposing SAP neurons in IML suggests that if the paraventricular nucleus innervates SAP neurons in IML, it does so via a population of neurons that do not use oxytocin as a neurotransmitter.     

The fine structural organization of tyrosine hydroxylase immunoreactive neurons in rat arcuate nucleus

The fine structure of the tyrosine hydroxylase (TH) immunoreactive neurons of the hypothalamic arcuate nucleus was examined by means of immunocytochemistry [peroxidase-antiperoxidase (PAP) method], utilizing an antibody against TH. Immunolabeled axon terminals were observed infrequently and were located predominantly in the lateral region, whereas numerous labeled perikarya and dendrites were found throughout the nucleus. The labeled terminals, containing primarily clear and occasionally dense core vesicles, were never observed in synaptic contact. On the other hand, unlabeled axon terminals were frequently seen synapsing on labeled dendrites. In addition, the labeled dendrites were often seen in direct apposition to other neuronal elements such as both labeled and unlabeled perikarya. In contrast, unlabeled dendrites were never seen apposed to labeled perikarya. Labeled dendrites also occurred in direct contact with one another and with unlabeled dendrites. Moreover, numerous labeled dendrites were encountered along tanycytic processes. Dendrites engaged in tanycytic appositions were occasionally partially encompassed by thin sheaths emanating from the tanycytic process. The extensive contact made by the labeled dendritic profiles on both labeled perikarya and dendrites suggests that tubero-infundibular dopaminergic (TIDA) cells may communicate with each other by means of dendritic release of dopamine. The presence of appositions between labeled dendrites and both unlabeled perikarya and dendrites suggests that the TIDA system also influences other neuronal populations through its dendrites. Finally, the dendrotanycytic relationship suggests that the TIDA system may play some role in the regulation of tanycytic function.     

Ultrastructural localization and afferent sources of corticotropin-releasing factor in the rat rostral ventrolateral medulla: Implications for central cardiovascular regulation

We investigated the ultrastructural localization, afferent sources, and arterial pressure effects of corticotropin-releasing factor (CRF) in the nucleus reticularis rostroventrolateralis (RVL), a region of the ventrolateral medulla containing C1 adrenergic neurons and sympatho-excitatory reticulospinal afferents to sympathetic preganglionic neurons. A polyclonal antibody, to CRF was localized in acrolein-fixed sections through the rat RVL by the peroxidase–antiperoxidase (PAP) method. Light microscopy showed that 1–7 perikarya/30 μm section and numerous varicose processes contained CRF-like immunoreactivity (CRF-LI). By electron microscopy, CRF-LI was most intensely localized to large (80–100 nm) dense-core vesicles within numerous terminals and a few perikarya and large dendrites. Approximately half of the terminals containing CRF-LI were in direct contact with unlabeled perikarya or dendrites; the remainder were in apposition to either unlabeled terminals or astrocytes. Most synaptic specializations were asymmetric synapses on small, unlabeled dendrites. To examine potential extrinsic sources of CRF-containing terminals in the C1 area of the RVL, PAP immunocytochemical localization of CRF was combined with retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). In all cases examined, a number of dually labeled neurons were found in the paraventricular nucleus (PVN) of the hypothalamus and a few dually labeled neurons were observed in the nuclei of the solitary tract; these labeled neurons were ipsilateral to the unilateral injection of WGA-HRP into the C1 area. Fewer dually labeled perikarya were detected in the lateral hypothalamic area and the lateral parabrachial nuclei, ipsilateral to the WGA-HRP injection. Additional physiological studies showed that bilateral microinjections of CRF into the C1 area of the RVL of urethane-anesthetized rats elicited a dose-related increase in arterial pressure. The results suggest that within the C1 area of the RVL, CRF released from terminals, arising predominantly from the PVN of the hypothalamus and probably from local neurons as well, may excite sympathoexcitatory reticulospinal neurons. © 1993 Wiley-Liss, Inc.     

Relationship of Met-enkephalin-like immunoreactivity to vagal afferents and motor dendrites in the nucleus of the solitary tract: a light and electron microscopic dual labeling study

Methionine (Met⁵)-enkephalin has been implicated in autonomic functions involving vagal reflexes within the nucleus of the solitary tract (NTS). We examined the light and electron microscopic relationships between neurons containing methionine (Met⁵)-enkephalin-like immunoreactivity (MELI) and vagal afferents and motor dendrites in the rat NTS. A polyclonal antibody raised against Met⁵-enkephalin and showing maximal cross-reactivity with this peptide was localized by immunoautoradiography. In the same sections, vagal afferents and motor neurons were identified by histochemical detection of anterogradely and retrogradely transported horseradish peroxidase (HRP). By light microscopy, the MELI was detected in perikarya distributed principally in the dorsomedial, intermediate and parasolitary subdivisions of the NTS. These subnuclei as well as medial and commissural divisions of the NTS also showed: (1) aggregates of silver grains thought to overlie terminals containing MELI, and (2) anterogradely transported HRP in varicose processes. Electron microscopic analysis of the dorsomedial NTS at the level of the area postrema established that MELI was detectable in perikarya, dendrites, and axon terminals. Most of the MELI was associated with large dense core vesicles (dcvs). These opioid terminals formed primarily symmetric synapses on proximal and asymmetric synapses on distal dendrites. Analysis of the dendritic targets of terminals containing MELI revealed that 13/222 were in synaptic contact with dendrites also containing MELI. The remainder of the terminals containing MELI either lacked recognized junctions or formed synapses with unlabeled dendrites. In comparison to the terminals containing MELI in the same series of sections, anterogradely labeled vagal terminals extensively formed asymmetric junctions with distal dendrites and spines. Of the observed anterogradely labeled terminals 6/84 formed synapses with dendrites containing MELI and 3/84 with dendrites containing retrogradely transported HRP. The remainder of the junctions were with dendrites lacking detectable immunoautoradiographic or HRP-labeling. The majority of the recognized synapses on labeled dendrites were at more proximal sites possibly reflecting more limited detection of both MELI and retrogradely transported HRP in smaller dendrites. However, the presence of even a few junctions at proximal sites on dendrites where synaptic transmission is known to be more effective suggests a potentially strong modulation of both opioid and vagal motor neurons by visceral afferents in the NTS. In addition to forming synapses on dendrites, both vagal afferents and terminals containing MELI showed frequent synaptic associations with unlabeled terminals, but not with each other. This finding suggests that the previously demonstrated opiate binding sites on vagal afferents is most likely attributed to other endogenous opiates.     

Development and migration of avian sympathetic preganglionic neurons.

Modern neuronanatomical techniques were used to investigate the development of the avian sympathetic preganglionic cell column in the spinal cord of the chick embryo. [3H]thymidine autoradiography indicated that the majority of these preganglionic, or "Terni column" neurons are generated between stages 18 and 24 (days 2-4). This coincides with the genesis of the somatic motoneurons in the thoracic levels of the cord, and therefore differences in the time of origin cannot explain the divergent fates of these two neuronal populations. Data obtained from short-survival autoradiographic experiments indicated that many early born cells remain close to the ventral region of the ventricular epithelium until day 5 of incubation. Ventral root injections used to label retrogradely neurons projecting an axon into the ventral root (Terni cells and somatic motoneurons) have labeled neurons next to the ventricular epithelium at the same early stages. Thus, it seems likely that some Terni cells, if not all, maintain medial positions and do not migrate laterally to join a common motor column before initiating a dorsal migration. Analysis of a closely staged series of embryos, whose Terni column neurons were retrogradely labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP), revealed that between days 5 and 8 of incubation, Terni column neurons migrated dorsally to attain their adult position adjacent to the central canal. These changes in position were reflected in the changing morphology of the Terni column neurons, visualized by the Golgi-like HRP labeling. The positions of the migrating Terni cells differed from those of commissural cells, indicating that these fibers are not the substrate for the dorsal migration. The dorsal migration of Terni column cells was not disrupted by the surgical removal of the sympathetic ganglia, the synaptic targets of these neurons, nor by disruption of spinal afferents. Taken together, these results suggest that the migratory behavior of Terni cells in distinctive when compared to that of somatic motoneurons, and that local and/or intrinsic cues within the spinal cord guide the dorsal migration of Terni column cells.     

Projections of neurons in the ventromedial medulla to pontine catecholamine cell groups involved in the modulation of nociception

Stimulation of neurons in the nucleus raphe magnus (RMg) or the adjacent gigantocellular nucleus pars alpha (Gi) and paragigantocellular nucleus (PGi) produces antinociception which is partially mediated by bulbospinal noradrenergic neurons. Since no norepinephrine-containing neurons are located in either the RMg or the Gi/PGi, it is likely that neurons located in these nuclei have axonal connections with the spinally-projecting catecholamine neurons located in the A5, A6 (locus coeruleus), or A7 catecholamine cell groups. To provide evidence for such connections, the anterograde tracer, Phaseolus vulgaris-leucoagglutinin (PHA-L), was injected into the RMg or Gi/PGi and labeled axons were identified near catecholamine-containing neurons labeled with dopamine-β-hydroxylase-immunoreactivity (DβH-ir). A dense field of PHA-L-positive terminals was seen within the A7 cell group which was mainly ipsilateral to PHA-L injections made into either the RMg or the Gi/PGi. Many PHA-L-positive terminals were closely apposed to DβH-ir A7 perikarya or proximal dendrites. A modest number of terminals was seen within the A5 and LC cell groups. In the second experiment, a unilateral injection of the retrograde tracer, Fluoro-Gold, was made into the A7 cell group and brainstem sections were processed for serotonin (5-HT) immunocytochemistry. Many neurons retrogradely labeled with Fluoro-Gold were seen in the RMg, but a much larger number were found in the Gi/PGi. Less than 5% of these Fluoro-Gold-labeled cells contained 5-HT-immunoreactivity. The results of these experiments indicate that the RMg and Gi/PGi have a substantial population of non-serotonergic neurons which project to the A7 noradrenergic cell group.     

Anterograde and retrograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) from the globus pallidus to the striatum of the rat

Iontophoretic administration of PHA-L into the globus pallidus of rats resulted in the labeling of neuronal perikarya in the striatum as well as axons and terminals in the striatum, entopeduncular nucleus, subthalamus and substantia nigra. The labeled striatal perikarya were densely stained in Golgi fashion with virtually complete filling of the dendrites and spines. It is concluded that the striatal cells were filled by the retrograde transport of PHA-L and represent either striatopallidal cells, or striatonigral cells whose axons were interrupted as they passed through the injection site. The anterogradely labeled axon terminals in the striatum were observed in close apposition to the dendrites of the retrogradely labeled neurons suggesting the existence of synaptic contacts between the two groups of cells. This study demonstrates that PHA-L can be transported retrogradely as well as anterogradely following iontophoretic injections.     

Light and electron microscopic study of tyrosine hydroxylase-immunoreactive neurons within the developing rat arcuate nucleus

The topography, fine structure, and patterns of connections of tyrosine hydroxylase (TH)-immunoreactive tubero-infundibular dopaminergic (TIDA) neurons were examined by light and electron microscopic immunocytochemistry in the arcuate nucleus of 2-, 15- and 30-day-old female Wistar rats. In 2-day-old animals, TH-immunoreactive perikarya were mainly located in the ventrolateral portion of the arcuate nucleus. In 15-day-old rats numerous TH-positive cell bodies were still present ventrolaterally, but a cluster of labeled cells was also apparent in the mediodorsal segment of the nucleus. In the 30-day-old rats, most TH-immunoreactive neurons were concentrated mediodorsally, as seen in the adult. At the ultrastructural level, TH-immunoreactive somata exhibited, in all age groups, a large nucleus surrounded by a thin rim of cytoplasm containing mitochondria, Golgi apparatus, endoplasmic reticulum, multivesicular bodies and lysosomes. These labeled somata were synaptically contacted by unlabeled axon terminals and often laid adjacent to either labeled or unlabeled dendrites. Similarly, in all age groups, labeled dendrites were synaptically contacted by unlabeled axon terminals and were often directly apposed to either labeled or unlabeled perikarya and dendrites, or to tanycytic processes. These results indicate that TIDA neurons establish extensive connections early in development, and that their pattern of intercellular relationships remains qualitatively unchanged from 2 days to adulthood. It is suggested that TIDA neurons may be already functional at birth, and could therefore, influence the maturation of other arcuate neuronal populations.     

Ultrastructural localization of the binding fragment of tetanus toxin in putative gamma-aminobutyric acidergic terminals in the intermediolateral cell column: a potential basis for sympathetic dysfunction in generalized tetanus

Tetanus toxin (TeTx) causes sympathetic hyperactivity, a major cause of mortality in generalized tetanus, apparently by obstructing the inhibition of sympathetic preganglionic neurons (SPNs). Neuroanatomic tracing and immunohistochemistry were used to investigate whether axon terminals in the intermediolateral cell column (IML) that synapse on SPNs and use the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) may be infected transsynaptically with TeTx. The binding fragment of TeTx (TTC; an atoxic surrogate of TeTx) and the cholera toxin B subunit (CTB; a retrograde tracer) were injected into the rat superior cervical ganglion and, over 16-48 hours, were transported to the ipsilateral IML in the caudal half of the last cervical and first three thoracic spinal cord segments. With light microscopy, diffuse CTB immunolabeling extended throughout SPN perikarya and dendrites. Punctate TTC and GABA immunolabeling were accumulated densely in the neuropil between and surrounding SPN processes. With electron microscopy, 54% of the axon terminals in the IML (n = 1,337 terminals) were TTC immunolabeled (TTC(+)), and 25% contained putative neurotransmitter levels of GABA immunolabeling (GABA(+)). On average, GABA(+) terminals had a 76% chance of also being TTC(+) and a 62% greater chance of being TTC(+) than GABA(-) terminals (P < 0.000001). Axon terminals were just as likely to be TTC(+) and/or GABA(+) regardless of whether the dendrites they synapsed on were large (>1 microM) or small in cross-sectional area or were labeled retrogradely. Sympathetic hyperactivity in tetanus may involve 1) retrograde and transsynaptic transport of TeTx by SPNs and 2) at least in part, an infection of GABAergic terminals in the IML.     

Central pupillary light reflex circuits in the cat: II. Morphology,ultrastructure, and inputs of preganglionic motoneurons

Preganglionic motoneurons supplying the ciliary ganglion control lens accommodation and pupil diameter. In cats, these motoneurons make up the preganglionic Edinger‐Westphal population, which lies rostral, dorsal, and ventral to the oculomotor nucleus. A recent cat study suggested that caudal motoneurons control the lens and rostral motoneurons control the pupil. This led us to examine the morphology, ultrastructure, and pretectal inputs of these populations. Preganglionic motoneurons retrogradely labeled by introducing tracer into the cat ciliary ganglion generally fell into two morphologic categories. Fusiform neurons were located rostrally, in the anteromedian nucleus and between the oculomotor nuclei. Multipolar neurons were found caudally, dorsal and ventral to the oculomotor nucleus. The dendrites of preganglionic motoneurons within the anteromedian nucleus crossed the midline, providing a possible basis for consensual responses. Ultrastructurally, several different classes of synaptic profiles contact preganglionic motoneurons, suggesting that their activity may be modified by a variety of inputs. Furthermore, there were differences in the synaptic populations contacting the rostral vs. caudal populations, supporting the contention that these populations display functional differences. Anterogradely labeled pretectal terminals were observed in close association with labeled preganglionic motoneurons, particularly in the rostral population. Ultrastructural analysis revealed that these terminals, packed with clear, spherical vesicles, made asymmetric synaptic contacts onto motoneurons in the rostral population, indicating that these cells serve the pupillary light reflex. Thus, the preganglionic motoneurons found in the cat display morphologic, ultrastructural, and connectional differences suggesting that this rostral preganglionic population is specialized for pupil control, whereas more caudal elements control the lens. J. Comp. Neurol. 522:3978–4002, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Glutamate-immunoreactive synapses on retrogradely-labelled sympathetic preganglionic neurons in rat thoracic spinal cord

Retrograde tracing with cholera toxin B subunit (CTB) combined with post-embedding immunogold labelling was used to demonstrate the presence of glutamate-immunoreactive synapses on sympathetic preganglionic neurons that project to the adrenal medulla or to the superior cervical ganglion in rat thoracic spinal cord. At the electron microscope level, glutamate-immunoreactive synapses were found on retrogradely labelled nerve cell bodies and on dendrites of all sizes. Two-thirds of the vesicle-containing axon profiles that were directly apposed to, or synapsed on, CTB-immunoreactive sympathoadrenal neurons were glutamate positive. The proportion of glutamate-immunoreactive contacts and synapses on sympathoadrenal neurons decreased to zero when the anti-glutamate antiserum was absorbed with increasing concentrations of glutamate from 0.1 mM to 10 mM. Double immunogold labelling for glutamate and gamma-aminobutyric acid (GABA) showed that glutamate-immunoreactive profiles did not contain GABA and that GABA-immunoreactive profiles did not contain glutamate. These results suggest that glutamate is the major excitatory neurotransmitter to sympathoadrenal neurons and possibly to other sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord.     

The intermediolateral nucleus: an ‘open’ or ‘closed’ nucleus?

The sympathetic preganglionic neurons located in the intermediolateral nucleus (IML) that project to the superior cervical ganglion of the rabbit were observed to have two major dendritic orientations after retrograde labeling with horseradish peroxidase. One projection extends longitudinally within IML. The second projection courses medially and presents a triangular shape in horizontal sections. The labeled processes that project medially arise from cells in IML and project through the intercalated nucleus towards the central autonomic area and follow the contour of the central canal. Medially oriented dendrites intruding into other areas of the intermediate grey matter show that IML is an ‘open’ rather than a ‘closed’ nucleus as has been recently suggested. The location and distribution of the sympathetic preganglionic neurons projecting to the superior cervical ganglion in the rabbit are compared with those reported for other species.