Ultrastructure and synaptology of the nucleus ambiguus in the rat: The semicompact and loose formations

The fine structure of the pharyngomotor semicompact and laryngomotor loose format formations of the rat nucleus ambiguus was studied in single and serial sections by means of light and electron microscopy. Motoneurons and their dendrites were identified after retrograde labelling by injections of neuroanatomical tracers into pharyngeal and laryngeal muscles or nerves. Pharyngeal motoneurons measured 39 × 29 μm and had 2–25 axosomatic synapses per somatic profile, representing an estimated average of 1S2 synapses per soma. Laryngeal motoneurons measured 42 × 30 μm with 6–33 synapses per profile, or an average of 339 synapses per soma. In both subdivisions, axon terminals that contained round vesicles and formed asymmetric junctions and terminals that contained pleomorphic vesicles and formed symmetric junctions were distributed in approximately equal proportions on somata and dendrites, forming over 90% of the synapse population. A small percentage (2–8%) of synapses had a subsurface cistern situated below the axon terminal (C type). Small, atypical motoneurons measuring 15 × 5 μm with an invaginated nucleus were also present in both subdivisions. The ultrastructure and synaptology of pharyngeal and laryngeal motoneurons are characterized by similarities to those of spinal motoneurons and by their relatively large numbers of axosomatic synapses in comparison to esophageal motoneurons of the compact formation of the nucleus ambiguus. © 1996 Wiley-Liss, Inc.     

Axonal projections and synapses from the supratrigeminal region to hypoglossal motoneurons in the rat

Neural circuits from the supratrigeminal region (Vsup) to the hypoglossal motor nucleus were studied in rats using anterograde and retrograde neuroanatomical tracing methodologies. Iontophoretic injection of 10% biotinylated dextran amine (BDA) unilaterally into the Vsup anterogradely labeled axons and axon terminals bilaterally in the hypoglossal nucleus (XII) as well as other regions of the brainstem. In the ipsilateral XII, the highest density of BDA labeling was found in the dorsal compartment and the ventromedial subcompartment of the ventral compartment, where BDA labeling formed a dense, patchy distribution. Microinjection of 20% horseradish peroxidase (HRP) ipsilaterally or bilaterally into the tongue resulted in retrograde labeling of XII motoneurons confined to the dorsal and ventral compartments of the hypoglossal motor nucleus. Under light microscopical examination, BDA-labeled terminals were observed closely apposing the somata and primary dendrites of HRP-labeled hypoglossal motoneurons. Two hundred and sixty-five of these BDA-labeled terminals were examined at the ultrastructural level. One hundred and twelve BDA-labeled axon terminals were observed synapsing with either the somata (39%, 44/112) or the large or medium-size dendrites (61%, 68/112) of retrogradely labeled hypoglossal motoneurons. Axon terminals containing spherical vesicles (S-type) formed asymmetric synapses with HRP-labeled hypoglossal motoneuron dendrites. In contrast to this, F_F-type axon terminals, containing flattened vesicles, formed symmetric synapses with both the somata and dendrites of HRP-labeled hypoglossal motoneurons with a preponderance of the contacts on their somata. Axon terminals containing pleomorphic vesicles (F_P-type) were noted forming both symmetric and asymmetric synapses with HRP-labeled hypoglossal motoneuron somata and dendrites. The present study provides anatomical evidence of neuronal projections and synaptic connections from the supratrigeminal region to hypoglossal motoneurons. These data suggest that the supratrigeminal region, as one of the premotor neuronal pools of the hypoglossal nucleus, may coordinate and modulate the activity of tongue muscles during oral motor behaviors.     

Morphology and frequency of axon terminals on the somata, proximal dendrites, and distal dendrites of dorsal neck motoneurons in the cat.

The purpose of the present study was to compare the frequency of different classes of axon terminals on selected regions of the somatodendritic surface of dorsal neck motoneurons. Single motoneurons supplying neck extensor muscles were antidromically identified and intracellularly stained with horseradish peroxidase. By using light microscopic reconstructions as a guide, axon terminals on the somata, proximal dendrites (within 250 microns of the soma), and distal dendrites (more than 540 microns from the soma) were examined at the electron microscopic level. Axon terminals were divided into several classes based on the shape, density, and distribution of their synaptic vesicles. The proportion of axon terminals belonging to each axon terminal class was similar on the somata and proximal dendrites. However, there were major shifts in the relative frequency of most classes of axon terminals on the distal dendrites. The most common classes of axon terminals on the somata and proximal dendrites contained clumps of either spherical or pleomorphic vesicles. These types of axon terminals accounted for more than 60% of the axon terminals on these regions. In contrast, only 11% of the axon terminals found on distal dendrites belonged to these types of axon terminals. The most commonly encountered axon terminal on distal dendrites contained a dense collection of uniformly distributed spherical vesicles. These types of axon terminals accounted for 40% of all terminals on the distal dendrites, but only 5-7% of the axon terminals on the somata and proximal dendrites. Total synaptic density on each of the three regions examined was similar. However, the percentage of membrane in contract with axon terminals was approximately four times smaller on distal dendrites than somata or proximal dendrites. Axon terminals (regardless of type) were usually larger on somata and proximal dendrites than distal dendrites. These results indicate that there are major differences in the types and arrangement of axon terminals on the proximal and distal regions of dorsal neck motoneurons and suggest that afferents from different sources may preferentially contact proximal or distal regions of the dendritic trees of these cells.     

A Golgi and ultrastructural study of the monkey globus pallidus

Golgi preparations reveal that the most frequent type of pallidal neuron (principal cell), which has been recognized in all previous reports, is large (20–50 (Am)), fusiform, with dendrites up to 700 μm long. Large neurons of globular shape are less frequently impregnated. The morphology of dendrites varies considerably within the same neuron. Some exhibit numerous spines and protrusions and are seen to terminate in elaborate arborizations. A small interneuron (12 (μm)), with relatively short dendrites, up to 150 μm, and a short sparsely branching axon is observed less frequently. At least two types of afferent axons are present. A small-diameter fiber from the neostriatum enters the pallidum in bundles and gives rise to numerous thin branching processes with varicosit es about 1 μm in size. The axon collaterals are oriented orthogonal to the main axon and parallel to the dendrites of principal cells. A large-caliber fiber with clusters of 2–3-μm swellings can also be seen in close proximity to large pallidal dendrites. Ultrastructurally, principal cell dendrites (trunks, spines, and protrusions) are totally covered by synapsing axon terminals. In contrast, some small dentrites, presumed to belong to interneurons, form very few synapses. At least six categories of profiles containing vesicles are observed. One group has cytologic features of dendrites and participates in serial and triadic synapses with other profiles in the pallidal neuropil. Results suggest that the synaptic organization of the globus pallidus may be viewed as a repetitive, geometric arrangement of striatal and other afferent axons ensheathing and synapsing with the dendrites of principal cells. This pattern is interrupted by the presence of presynaptic dendrites, probably belonging to interneurons, which participate in complex synaptic arrangements.     

Distribution of glycinergic terminals on lumbar motoneurons of the adult cat: an ultrastructural study

The distribution of glycine-like immunoreactivity on cat lumbar motoneurons was examined in electron microscopy, using pre-embedding immunocytochemistry. In the dorsolateral portion of the ventral horn, numerous labeled axon terminals were presynaptic to somatic and dendritic profiles of alpha-motoneurons. Most of the glycinergic boutons contained pleomorphic vesicles and showed symmetrical contacts. On the somatic and proximal dendritic compartments, glycinergic terminals accounted for, respectively, 24.6 and 26.6% of the total number of terminals. There were very few glycinergic terminals on gamma-motoneurons. Immunoreactive axons, dendrites and cell bodies were also observed near the motoneurons. These results support the view that glycine plays a major role in the inhibition of alpha-motoneurons and suggest that inhibitory mechanisms occur on the soma as well as on dendrites.     

Immunocytochemical localization of gamma-aminobutyric acid in the hypoglossal nucleus of the macaque monkey, Macaca fuscata: a light and electron microscopic study

The hypoglossal nucleus of the macaque monkey Macaca fuscata was investigated with light and electron microscopic immunocytochemistry with an antibody directed against gamma-aminobutyric acid (GABA). At the light microscopic level, GABA immunoreactivity was present in small neurons, punctate structures, and thin, fiberlike structures. These GABA-positive elements were distributed throughout the hypoglossal nucleus at rostrocaudal levels. There was no immunoreactivity in the hypoglossal motoneurons. The GABA-positive small neurons were fusiform or ovoid (15 X 9 micron) and extended a few proximal dendrites from both poles. At the ultrastructural level, these small neurons were characterized by a markedly invaginated nucleus and a scanty cytoplasm in which cisternae of rough endoplasmic reticulum were not organized into extensive lamellar arrays as seen in the motorneurons. The GABA-positive punctate structures at the light microscopic level were identified as vesicle-containing axon boutons at the electron microscopic level. These GABA-positive axon terminals made synaptic contacts mainly with the dendrites of the motoneurons and infrequently with the somata. The majority of them made symmetric synapses and they contained pleomorphic synaptic vesicles. However, a small number of GABA-positive terminals (7%) formed asymmetric synapses with the dendrites of motoneurons, and these contacts exhibited postsynaptic dense bars or Taxi bodies lying beneath the postsynaptic membranes. There were no GABA-positive boutons that contacted the cell bodies of the small neurons. Although GABA-positive myelinated and unmyelinated axons were seen as thin, fiberlike structures, these myelinated and unmyelinated axons rarely gave rise to boutons on the motoneurons. The present study suggests that GABAergic inhibition in the monkey hypoglossal nucleus occurs mainly on the dendrites of the motoneurons and to some extent on the somata.     

Ultrastructural identification of synaptic terminals from the axon of type 3 interneurons in the cat lateral geniculate nucleus

Synaptic terminals from the axons of type 3 neurons in the A-laminae of the cat LGN impregnated with the Golgi gold-toning procedure were examined at light and electron microscopic levels. The axons were identified by their somatic origin, thin diameter, and, in one of these cells, by dense undercoating beneath the axolemma, which is a known characteristic of the axon initial segment. The axon of one of the analyzed cells was profusely branched and extended throughout most of lamina A within the dendritic domains of the cell, and both types of processes were oriented along projection lines in LGN. This suggests that the dendrites and axons of type 3 cells receive inputs and exert effects, of probably inhibitory nature, within restricted retinotopic regions of LGN. The vast majority of the axon terminals of these cells were distributed in series along axonal branches. In one of the type 3 cells, however, a dense cluster of terminals arising from a secondary axonal branch was observed. Ultrastructurally, the analyzed synaptic terminals of the type 3 cells contained flattened or pleomorphic synaptic vesicles, dark mitochondria, and established synapses that appeared to be of symmetrical type when the membranes were perpendicularly cut. On the basis of these characteristics these terminals are classified as F boutons, following Guillery's (Z. Zellforsch. 96:1-38, '69), nomenclature. The postsynaptic elements to the axon terminals were dendrites of small to medium size, which received "en passant" synaptic contacts in extraglomerular regions of the geniculate neuropil by the terminals distributed in series. The axon terminals located in clusters, however, made synapses with dendrites in glomerular regions of the neuropil, where they were not seen postsynaptic to retinal or other types of terminals. This is in contrast to the postsynaptic nature of F2 boutons in the same glomeruli, which have been identified as dendritic appendages of the GABA positive type 3 neurons in the cat LGN (Montero: J. Comp. Neurol. 254:228-245, '86). On the other hand, the axonal F terminals differ from F1 boutons in terms of synaptic relations and ultrastructure, since the latter have been shown to be presynaptic to F2s and somata and to contain crowded populations of flat synaptic vesicles which give them a characteristic dark appearance. Terminals equivalent to F1 boutons have been shown to originate from perigeniculate cells in the rat LGN. From these observations it is suggested that the geniculate GABAergic interneurons support two morphologically and functionally different type of inhibitory terminals synapsing the dendrites of relay cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The synaptology of parvalbumin-immunoreactive neurons in the primate prefrontal cortex.

Electron microscopy and immunocytochemistry with a monoclonal antibody against parvalbumin (PV) were combined to analyze the distribution and morphology of PV-immunoreactive (PV-IR) neurons and the synaptology of PV-IR processes in the principal sulcus of the macaque prefrontal cortex. Parvalbumin-IR neurons are present in layers II-VI of the macaque principal sulcus (Walker's area 46) and are concentrated in a band centered around layer IV. PV-IR cells are exclusively non-pyramidal in shape and are morphologically heterogeneous with soma sizes ranging from less than 10 microns to greater than 20 microns. Well-labeled neurons that could be classified on the basis of soma size and dendritic configuration resembled large basket and chandelier cells. A novel finding is that supragranular PV-IR neurons exhibit dendritic patterns with predominantly vertical orientations, whereas infragranular cells exhibit mostly horizontal or oblique dendritic orientations. PV-IR cells within layer IV exhibit a mixture of dendritic arrangements. Vertical rows of PV-IR puncta, 15-30 microns in length, resembling the "cartridges" of chandelier cell axons were most dense in layers II, superficial III, and the granular layer IV but were not observed in the infragranular layers. Cartridges were often present beneath unlabeled, presumed pyramidal cells. PV-IR puncta also formed pericellular nests around pyramidal cell somata and proximal dendrites, suggestive of basket cell innervation. PV-IR axons were occasionally observed in the white matter underlying the principal sulcus. Electron microscopic analysis revealed that PV-IR somata and dendrites are densely innervated by nonimmunoreactive terminals forming asymmetric (Gray type I) synapses as well as by fewer terminals forming symmetric (Gray type II) synapses. The majority of terminals forming symmetric synapses with PV-IR post-synaptic structures were not immunolabeled; however, some of these boutons did contain PV-immunoreactivity. PV-IR boutons exclusively form symmetric synapses and heavily innervate layer II/III pyramidal cells. PV-IR axon cartridges formed numerous axo-axonic synapses with the axon initial segments of pyramidal cells 15-20 microns beneath the axon hillock and also terminated on large axonal spines of the initial segment. Furthermore, we failed to observe a mixture of PV-immunoreactive and non-immunoreactive boutons composing a single axon cartridge. Pyramidal cell somata and proximal dendrites were also heavily innervated by PV-IR boutons forming symmetric synapses, again, consistent with basket cell innervation. In addition, PV-IR axon terminals frequently formed symmetric synapses with dendritic shafts and spines of unidentified neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ultrastructure of the subnucleus gelatinosus of the nucleus of the tractus solitarius in the cat

The dorsomedial region of the nucleus of the tractus solitarius termed the subnucleus gelatinosus (SNG) was studied at the light and electron microscopic level in the cat. In cresyl violet and luxol fast blue stained sections the SNG contained small neuronal somata that were scattered throughout a pale-staining neuropil containing few myelinated fibers. These neurons were difficult to impregnate with Golgi staining techniques, but in successful impregnations the somata were observed to be 10--19 micrometers in diameter and bore few sparsely branching primary dendrites. Spines were present on the dendrites of some neurons and were more numerous on distal portions of the dendritic tree. Ultrastructural examination of the SNG revealed that the neuronal complement consisted of round, oval, or spindle shaped neurons with little or no organized Nissl substance. Rare myelin-like ensheathments of neuronal perikarya were also observed. Bundles of fine unmyelinated axons that coursed mainly longitudinally were a prominent feature of the area. The most common type of axon terminal observed contained mainly round clear vesicles, approximately 31 nm in diameter, and made asymmetrical synaptic contact with a dendritic profile. Pleomorphic vesicle-containing terminals involved in symmetrical synaptic contact were also commonly seen. Axodendritic and axosomatic synapses were associated with terminals containing either round clear vesicles or pleomorphic vesicles. Less commonly, dendrodendritic and dendrosomatic synapses were seen, the presynaptic elements of which contained pleomorphic vesicles. Following removal of a nodose ganglion, degenerating terminals of vagal afferent fibers were observed throughout the neuropil. Such terminals contained round, clear vesicles with an occasional large, dense-cored vesicle, and made axodendritic and axosomatic synaptic contacts.     

Dual ultrastructural immunocytochemical labeling of μ and δ opioid receptors in the superficial layers of the rat cervical spinal cord

The δ opioid receptor (DOR) and μ opioid receptor (MOR) are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. To determine the cellular basis for these functional associations, we examined the electron microscopic immunocytochemical localization of DOR and MOR in single sections through the superficial layers of the dorsal horn in the adult rat spinal cord (C2–C4). From a total of 270 DOR-labeled profiles, 49% were soma and dendrites, 46% were axon terminals and small unmyelinated axons, and 5% were glial processes. 6% of the DOR-labeled soma and dendrites, and <1% of the glial processes also showed MOR-like immunoreactivity (MOR-LI). Of 339 MOR-labeled profiles, 87% were axon terminals and small unmyelinated axons, 12% were soma and dendrites, and 2% were glial processes. 21% of the MOR-labeled soma and dendrites, but none of the axon terminals also contain DOR-LI. The subcellular distributions of MOR and DOR were distinct in axon terminals. In axon terminals, both DOR-LI and MOR-LI were detected along the plasmalemma, but only DOR-LI was associated with large dense core vesicles. DOR-labeled terminals formed synapses with dendrites containing MOR and conversely, MOR-labeled terminals formed synapses with DOR-labeled dendrites. These results suggest that the synergistic actions of selective MOR- and DOR-agonists may be attributed to dual modulation of the same or synaptically linked neurons in the superficial layers of the spinal cord.     

The fine structure of the rat subthalamic nucleus: an electron microscopic study

The normal ultrastructure of the rat subthalamic nucleus (STH) was studied. The STH consisted of tightly packed neurons distributed within a neuropil filled with large numbers of blood vessels and thinly myelinated fibers. The somata of STH neurons (diameters, D, between 10 and 25 micron) contained abundant organelles but had only a small amount of both smooth and rough endoplasmic reticulum. The nuclei had deeply invaginated nuclear envelopes and pale nucleoplasm with little heterochromatin. STH neurons often were tightly apposed without any intervening glial membranes. Similar appositions were also found between somata and dendrites, dendrites and dendrites, and dendrites and initial axon segments. Although puncta adhaerentia were often observed, no gap junctions were found on any of these membrane appositions. In the neuropil, the dendrites were mostly smooth and thin (D between 0.5 and 1 micron) with an occasional stubby spine or thin dendritic appendage. At least two types of axon terminals were identified. Type 1 terminals (D up to 1 micron) contained medium-sized round vesicles (D about 45 nm) and formed asymmetrical synapses. Type 2 terminals were often large (D up to 5 micron) and contained both round and slightly flattened vesicles (D up to 50 nm). The type 2 terminals frequently formed adherens junctions with their postsynaptic targets in addition to forming relatively symmetrical synaptic junctions. The remaining axon terminals included a small number of terminals with various morphological characteristics and possibly some tangentially sectioned type 1 and type 2 terminals. Therefore they have not been classified as individual types in this study. A quantitative analysis indicated that the type 1 terminals formed synapses mainly with thin dendrites whereas the type 2 terminals formed synapses mainly with somata and larger dendrites.     

Fine structure and synaptic connections of the common spiny neuron of the rat neostriatum: A study employing intracellular injection of horseradish peroxidase

Medium-sized spiny neurons of the rat neostriatum, identified by intracellular injection of horseradish peroxidase, were examined at both light and electron microscopic levels. These neurons were characterized by their heavy investment of dendritic spines, beginning about 20 μm from the soma and continuing to the tips of the dendrites. Their axons arose from the soma or from a large dendritic trunk very near the soma, and tapered rapidly to form a main axonal branch from which arose several smaller initial collaterals. These arborized extensively throughout an area of about the same size as, and highly overlapping with, the dendritic field of the cell, while the main axon could be followed for distances of up to 1 mm in the direction of the globus pallidus. Three major synaptic types were seen in contact with spiny neurons. Boutons containing small round synaptic vesicles formed synapses exclusively with spiny regions of the dendrites, and most of these were axo-spinous. Small, very pleomorphic synaptic vesicles characterized a second bouton type of unknown origin, which made contacts with somata, initial segments, and dendrites, but not dendritic spines. Boutons containing large pleomorphic synaptic vesicles had the most widespread distribution, contacting all regions including dendritic spines. Spines receiving these contacts also were postsynaptic to boutons containing small round vesicles. Axon collaterals of spiny cells formed synapses with large pleomorphic vesicles and made synapses with somata, initial segments of axons, dendrites, and dendritic spines of striatal neurons, including other spiny cells.     

Neuronal associations in the rat suprachiasmatic nucleus demonstrated by immunoelectron microscopy.

The synaptic associations of neurons in the suprachiasmatic nucleus (SCN) of rats were examined by single immunolabeling for somatostatin (SRIH) and arginine vasopressin (AVP), and double immunolabeling for SRIH plus AVP and vasoactive intestinal polypeptide (VIP) plus AVP. Single immunolabeling showed that SRIH neurons, which displayed some somatic and dendritic spines, formed synaptic contacts with immunonegative and positive axon terminals. AVP neurons also formed synaptic contacts with both immunonegative and positive axon terminals. The immunonegative terminals contained small, spherical clear vesicles or flattened clear vesicles. A few immunopositive AVP fibers made synapses with immunonegative somatic or dendritic spines. Double immunolabeling showed synaptic associations between SRIH axons and AVP cell bodies or dendritic processes, and between AVP axons and the somata or dendrites of SRIH neurons. These findings suggest a reciprocal relation between the two types of neurons. Synaptic contacts between AVP neurons and VIP axon terminals were also demonstrated. Previously, we found synapses between SRIH axons and VIP neurons. Thus SRIH neurons appeared to regulate AVP and VIP neurons. On the basis of these findings, two possible oscillation systems of the SCN are proposed.     

Morphology of single axons of tectospinal neurons in the upper cervical spinal cord

Morphology of single axons of tectospinal (TS) neurons was investigated by intraaxonal injection of horseradish peroxidase (HRP) at the upper cervical spinal cord of the cat. TS axons were electrophysiologically identified by their direct responses to stimulation of the contralateral superior colliculus (SC). None of these axons responded to thoracic stimulation at Th2. Three-dimensional reconstructions of the axonal trajectories were made from 20 well-stained TS axons at C1-C3. Cell bodies of these axons were located in the intermediate or deep layers of the caudal two-thirds of the SC. Usually, TS axons had multiple axon collaterals, and up to seven collaterals were given off per stem axon [2.7 ± 1.6 (mean ± S.D.); n = 20]. Collaterals had simple structures and ramified a few times mainly in the transverse plane. The number of terminals for each collateral was small. These collaterals terminated in the lateral parts of laminae V–IX, mainly in laminae VI, VII, and VIII. There were usually gaps free from terminal arborizations between adjacent collaterals, because the rostrocaudal spread of each collateral (mean = 700 μm) was narrower than the intercollateral interval (mean = 2,500 μm). Seven of the 19 TS axons had terminals in the lateral parts of laminae V–VIII, with little projection to lamina IX, and the other 12 axons had terminals in lamina IX besides the projection to the lateral parts of laminae V–VIII. Axon terminals in lamina IX did not appear to make contacts with the somata or proximal dendrites of retrogradely labeled motoneurons, but contacts were found with the somata of counterstained interneurons in the lateral parts of laminae V–VIII. Three spinal interneurons (two in lamina VIII and one in lamina V at C1) that received monosynaptic excitation from the SC were stained, and their axonal trajectories were reconstructed. They had multiple axon collaterals at C1-C2 and mainly projected to laminae VIII and IX, with smaller projections to lamina VII. Many axon terminals of the interneurons were found in multiple neck motor nuclei, where some of them made contacts with retrogradely labeled motoneurons. The present finding provides evidence that the direct TS projection to the spinal cord may influence activities of multiple neck muscles, mainly via spinal interneurons, and may play an important role in control of head movement in parallel with the tectoreticulospinal system. © 1996 Wiley-Liss, Inc.     

Direct synaptic projections to esophageal motoneurons in the nucleus ambiguus from the nucleus of the solitary tract of the rat

Neurons of the nucleus of the solitary tract (NTS) serve as interneurons in swallowing. We investigated the synaptology of the terminals of these neurons and whether they project directly to the esophageal motoneurons in the compact formation of the nucleus ambiguus (AmC). Following wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) injection into the NTS, many anterogradely labeled axodendritic terminals were found in the neuropil of the AmC. The majority of labeled axodendritic terminals (89%) contained round vesicles and made asymmetric synaptic contacts (Gray's type I), but a few (11%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II). More than half of the labeled terminals contacted intermediate dendrites (1-2 μm diameter). There were no retrogradely labeled medium-sized motoneurons, but there were many retrogradely labeled small neurons having anterogradely labeled axosomatic terminals. A combined retrograde and anterograde transport technique was developed to verify the direct projection from the NTS to the esophageal motoneurons. After the esophageal motoneurons were retrogradely labeled by cholera toxin subunit B conjugated HRP, the injection of WGA-HRP into the NTS permitted ultrastructural recognition of anterogradely labeled axosomatic terminals contacting directly labeled esophageal motoneurons. Serial sections showed that less than 20% of the axosomatic terminals were labeled in the esophageal motoneurons. They were mostly Gray's type I, but a few were Gray's type II. In the small neurons, more than 30% of axosomatic terminals were labeled, which were exclusively Gray's type I. These results indicate that NTS neurons project directly not only to the esophageal motoneurons, but also to the small neurons which have bidirectional connections with the NTS. J. Comp. Neurol. 381:18-30, 1997. © 1997 Wiley-Liss, Inc.     

Principal neurons of the basilar pons as the source of a recurrent collateral system

The basilar pontine nuclei in the opossum are composed of two general categories of neurons, intrinsic cells and the principal or projection neurons. Observations from Golgi material indicate that principal neurons whose primary axons project to the cerebellar cortex may also give rise to recurrent branches distributing within the pontine gray. Such collaterals were observed to arise near the soma and at some distance from the cell body of the parent axon. The electron microscopic correlate of such a system was identified in the basilar pontine neuropil in animals subjected to lesions of the cerebellar cortex. These lesions destroyed mossy terminals and their parent axons and thus initiated a retrograde reaction in basilar pontine projection neurons which manifested itself in the form of morphologic alterations observed in somata, dendrites, and a class of axonal boutons. Similar altered axon terminals were not observed in control material and did not correspond to the terminals of cerebello-pontine axons described in previous work. It was therefore suggested that such boutons represented the terminals of the recurrent collateral system observed in Golgi material.     

Dopamine-acetylcholine interaction in the rat striatum: A dual-labeling immunocytochemical study

The relationship between cholinergic neurons and dopaminergic axons in the rat striatum was examined by a dual-labeling immunocytochemical method. Cholinergic neurons were identified by their immunoreactivity for choline acetyltransferase (ChAT), and dopaminergic axon terminals were identified by their positive immunoreactivity for tyrosine hydroxylase (TH). Electron microscopic analysis of dual-labeled sections revealed that while most TH-positive terminals formed synapses with unlabeled striatal neurons and dendrites, a number of TH-positive terminals formed close appositions, highly suggestive of synapses, with both large and small dendrites as well as somata of ChAT-positive neurons. Tight appositions were also found between TH-positive terminals and ChAT-positive terminals. Moreover, TH-positive terminals and ChAT-positive terminals were found to form synapses with common dendrites of unlabeled striatal neurons. These results indicated that 1) dopaminergic axon terminals could interact directly with striatal cholinergic interneurons via tight appositions with distances comparable to conventional synapses; and 2) there is a convergence of dopaminergic and cholinergic axon terminals on noncholinergic striatal neurons.     

Anatomic evidence suggestive of dendrodendritic synapses in the opossum basilar pons

The basilar pontine nuclei in the opossum are composed of two general categories of neurons, intrinsic cells and the principal or projection neurons. Observations from Golgi material indicate that principal neurons whose primary axons project to the cerebellar cortex may also give rise to recurrent branches distributing within the pontine gray. Such collaterals were observed to arise near the soma and at some distance from the cell body of the parent axon. The electron microscopic correlate of such a system was identified in the basilar pontine neuropil in animals subjected to lesions of the cerebellar cortex. These lesions destroyed mossy terminals and their parent axons and thus initiated a retrograde reaction in basilar pontine projection neurons which manifested itself in the form of morphologic alterations observed in somata, dendrites, and a class of axonal boutons. Similar altered axon terminals were not observed in control material and did not correspond to the terminals of cerebello-pontine axons described in previous work. It was therefore suggested that such boutons represented the terminals of the recurrent collateral system observed in Golgi material.     

Electron microscopic localization of corticotropin‐releasing factor (CRF) and CRF receptor in rat and mouse central nucleus of the amygdala

Corticotrophin‐releasing factor (CRF) is expressed in the central nucleus of the amygdala (CeA), where the CRF receptor (CRFr) plays an important role in anxiety‐ and stress‐related behaviors. To determine the subcellular sites of CRFr activation in this region, we examined the electron microscopic immunolabeling of antisera recognizing CRF or CRFr. The ultrastructural analysis was principally conducted in the lateral subdivision of the rat CeA, with comparisons being made in mice so as to optimally utilize mutant mice in control experiments. The CRFr labeling was seen in many small dendrites and dendritic spines as well as in a few somata, large dendrites, axons, and axon terminals or more rarely in glial processes. Approximately 35% of the CRFr‐labeled dendrites contained CRF immunoreactivity, which was distributed diffusely throughout the cytoplasm, or specifically affiliated with either endomembranes or large dense‐core vesicles. The CRF‐immunoreactive vesicles also were present in somata and axon terminals with or without CRFr labeling. The CRF immunoreactivity was usually absent from both terminals and dendrites joined by asymmetric, excitatory‐type synapses, where a postsynaptic location of the CRFr was commonly observed. Numerous terminals containing both CRF and CRFr were seen, however, within the neuropil and sometimes apposing the excitatory synapses. These results provide ultrastructural evidence for a primary involvement of CRF receptors in modulation of the postsynaptic excitability of CeA neurons, an effect that may be limited by the availability of CRF. The findings have important implications for understanding CRF mediation of rapid responses to stress. J. Comp. Neurol. 512:323–335, 2009. © 2008 Wiley‐Liss, Inc.     

Vesicle shape and amino acids in synaptic inputs to phrenic motoneurons: Do all inputs contain either glutamate or GABA?

Varicosities that made synapses or direct contacts with retrogradely labelled rat phrenic motoneurons were examined for their content of immunoreactivity for either glutamate or glutamate decarboxylase, the enzyme involved in synthesis of α-aminobutyric acid (GABA). Phrenic motoneurons were identified by retrograde tracing from the diaphragm with cholera toxin B subunit conjugated to horseradish peroxidase. Cell bodies and medium-sized to large dendrites were labelled. Preembedding immunocytochemistry identified glutamate decarboxylase-immunoreactive nerve fibres; glutamate-immunoreactive nerve terminals were identified using postembedding immunogold labelling of ultrathin sections. The presence of glutamate- or glutamate decarboxylase immunoreactivity in nerve terminals was correlated with the morphology of the synaptic vesicles. Two major classes of nerve terminals were identified. Nerve terminals with round (presumably spherical) synaptic vesicles (S terminals) comprised 55% of synapses and contacts on phrenic motoneuron somata and 58% of synapses and direct contacts with dendrites. Nerve terminals with flattened synaptic vesicles (F terminals) comprised 42% of synapses direct contacts with somata and 41% of synapses and direct contacts with dendrites. Analysis of immunogold-labelled sections showed that S terminals contained statistically higher levels of glutamate immunoreactivity than F terminals. At the light microscope level, many glutamate decarboxylase-immunoreactive nerve terminals surrounded retrogradely labelled motoneurons. Varicosities with glutamate decarboxylase immunoreactivity made 33% of all synapses and direct contacts on somata, and 33% of synapses and direct contacts with dendrites of the retrogradely labelled phrenic motoneurons. Flattened synaptic vesicles were present in those glutamate decarboxylase-immunoreactive nerve terminals in which synaptic vesicle morphology could be judged. An additional 10% of all nerve terminals were of the F type, but were not glutamate decarboxylase-immunoreactive. Three percent of terminals on somata and 1% of nerve terminals on dendrites could not be classified as S or F types. These findings suggest that more than 90% of all inputs to phrenic motoneuron cell bodies and proximal dendrites could contain either GABA or glutamate. Some of these glutamatergic and GABAergic nerve fibres undoubtedly represent the source of inspiratory drive to, or expiratory inhibition of, phrenic motoneurons. © 1996 Wiley-Liss, Inc.