Ultrastructure of the mammillotegmental projections to the ventral tegmental nucleus of Gudden in the rat.

This study examines the termination pattern of axons from the medial mammillary nucleus within the ventral tegmental nucleus of Gudden (TV) in rats by using anterograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) and visualized with tetramethylbenzidine. The neuropil of TV contains three classes of axodendritic terminals, that is, terminals containing round, flat, and pleomorphic synaptic vesicles. These types make up 55.6%, 26.1%, and 18.3%, respectively, of all normal axodendritic terminals. Injection of WGA-HRP into the medial mammillary nucleus permits ultrastructural recognition of anterogradely labeled terminals within the TV. More than 80% of the labeled terminals contain round synaptic vesicles and form asymmetric synaptic contacts, whereas about 16% contain flat synaptic vesicles with symmetric synaptic contacts. There are a few labeled terminals with pleomorphic vesicles and only a few axosomatic terminals. Almost all labeled terminals are small, having diameters of less than 1.5 microns. Compared with the distributions of normal and labeled terminals with round vesicles, there is an increase of the percentage of labeled terminals with round vesicles on the intermediate dendrites (1-2 microns diameter) and a decrease on the distal dendrites (less than 1 micron diameter). Anterogradely labeled axon terminals often contact retrogradely labeled dendrites. These results suggest that the medial mammillary neurons send mainly excitatory as well as a few inhibitory inputs to the dendrites of TV and have direct reciprocal contacts with the TV neurons.     

Topography and synaptology of mamillary body projections to the mesencephalon and pons in the rat.

The anterograde and retrograde transport of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) was used to study the anatomical organization of descending projections from the mamillary body (MB) to the mesencephalon and pons at light and electron microscopic levels. Injections of WGA-HRP into the medial mamillary nucleus resulted in dense anterograde and retrograde labeling in the ventral tegmental nucleus, while injections in the lateral mamillary nucleus resulted in dense anterograde labeling in the dorsal tegmental nucleus pars dorsalis and dense anterograde and retrograde labeling in the pars ventralis of the dorsal tegmental nucleus. Anterogradely labeled fibers in the mamillotegmental tract diverged from the principal mamillary tract in an extensive dorsocaudally oriented swath of axons which extended to the dorsal and ventral tegmental nuclei, and numerous axons turned sharply ventrally and rostrally to terminate topographically in the dorsomedial nucleus reticularis tegmenti pontis and rostromedial pontine nuclei. The anterograde labeling in these two precerebellar relay nuclei was distributed near the midline such that projections from the lateral mamillary nucleus terminated mainly dorsomedial to the terminal fields of projections from the medial mamillary nucleus. In the dorsal and ventral tegmental nuclei, labeled axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions primarily with small diameter dendrites and to a lesser extent with neuronal somata. A few labeled terminals contained pleomorphic vesicles and formed symmetric synaptic junctions with dendrites and neuronal somata. Labeled axon terminals were also frequently found in synaptic contact with retrogradely labeled dendrites and neuronal somata in the dorsal and ventral tegmental nuclei. These findings indicate that neurons in the dorsal and ventral tegmental nuclei are reciprocally connected with MB projection neurons. In the nucleus reticularis tegmenti pontis and medial pontine nuclei, labeled axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions primarily with small diameter dendrites. The present study demonstrates that projections from the medial and lateral nuclei of the MB are topographically organized in the mesencephalon and pons. The synaptic morphology of mamillotegmental projections suggests that they may have excitatory influences primarily on the distal dendrites of neurons in these brain regions.     

Mamillary body in the rat: topography and synaptology of projections from the subicular complex, prefrontal cortex, and midbrain tegmentum

The retrograde and anterograde transport of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) has been used to trace afferent connections of the rat mamillary body (MB) at the light and electron microscopic levels. Injections of WGA-HRP into different parts of the MB resulted in heavy retrograde labeling in the subicular complex, medial prefrontal cortex, and dorsal and ventral tegmental nuclei. Injections of WGA-HRP into each of these brain regions, respectively, resulted in anterograde labeling with specific distributions and characteristic synaptic organizations in the MB. Projections from the rostrodorsal and caudoventral subiculum terminated in a topographically organized laminar fashion in the medial mamillary nucleus bilaterally, whereas afferent projections from the presubiculum and parasubiculum terminated only in the lateral mamillary nucleus. Labeled axon terminals which originated from the subicular complex were characterized by round vesicles and formed asymmetric synaptic junctions with small-diameter dendrites and dendritic spines in the medial and lateral mamillary nuclei. Projections from the prefrontal cortex originated mainly in the infralimbic area and to a lesser degree in the prelimbic and anterior cingulate areas. Injections of tracer into these brain regions gave rise to dense labeling of axon terminals in the medial mamillary nucleus, pars medianus, and in the anterior dorsomedial portion of the pars medialis. The labeled terminals were characterized by round vesicles and formed asymmetric synaptic junctions with small-diameter dendrites and dendritic spines. Projections from the dorsal tegmental nucleus terminated in the ipsilateral lateral mamillary nucleus, whereas afferent projections from the anterior and posterior subnuclei of the ventral tegmental nucleus terminated topographically in the medial mamillary nucleus. The ventral tegmental nucleus, pars anterior projected to the midline region of the medial nucleus and the dorsolateral and ventromedial subdivisions of the pars posterior projected to medial and lateral parts of the medial nucleus, respectively. In contrast to the synaptic morphology of subicular complex and medial prefrontal cortex axon terminals in the MB, labeled axon terminals in the MB which originated from the midbrain tegmentum were characterized by pleomorphic vesicles and formed symmetric synaptic junctions with neuronal somata and proximal dendrites as well as distal dendrites and dendritic spines.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

The GABAergic neurons and axon terminals in the lateralis medialis-suprageniculate nuclear complex of the cat: GABA-immunocytochemical and WGA-HRP studies by light and electron microscopy

In order to get more detailed information on the neural circuit of the lateralis medialis-suprageniculate nuclear (LM-Sg) complex of the cat, the GABAergic innervation of this complex was studied by GABA immunohistochemical techniques. Small immunoreactive cells were found throughout the LM-Sg complex. On the basis of their ultrastructural features, these GABAergic cells were identified as Golgi type II interneurons. The neuropil of this nucleus displayed a conspicuous granular immunoreactivity. Ultrastructurally, the immunoreactive neural profiles in the neuropil were identified as the presynaptic dendrites of interneurons, myelinated axons, or axon terminals. The GABAergic dendritic profiles, containing pleomorphic synaptic vesicles, were involved in synaptic glomeruli. Additionally, GABAergic axon terminals containing pleomorphic synaptic vesicles formed symmetric axodendritic synaptic contacts mainly in the extraglomerular neuropil. They appeared to correspond to either axon terminals from the thalamic reticular nucleus (TRN) or the axon terminals of interneurons. The projections from the TRN to the LM-Sg complex were studied by using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Following injection of WGA-HRP into the LM-Sg complex, a number of retrogradely labeled cells were observed in the TRN. The connections between the TRN and the LM-Sg complex appeared to be topographically organized, the dorsal TRN being connected mainly with the dorsomedial portion of the LM-Sg complex, and the ventral TRN being connected chiefly with the ventrolateral portion of the LM-Sg complex. Following injection of the tracer into the TRN, ultrastructural examination of anterograde labeling in the LM-Sg complex revealed that labeled terminals contain pleomorphic vesicles and make symmetric synaptic contacts mainly with small to medium-sized dendrites. The labeled terminals were not involved in synaptic glomeruli. The present results provide anatomic support for the contention that the projection cells of the LM-Sg complex may be inhibited by both the TRN axons and interneurons, probably through the mediation of GABA.     

Ultrastructure and synaptic organization of axon terminals from brainstem structures to the mediodorsal thalamic nucleus of the rat.

The ultrastructural characteristics and synaptic organization of afferent terminals from the brainstem to the mediodorsal thalamic nucleus (MD) of the rat have been studied with the electron microscope, by means of anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Labeled fibers were seen predominantly in the lateral portion of MD after the injections of WGA-HRP into the substantia nigra pars reticulata (SNr), the superior colliculus (SC), and the dorsal tegmental region (DT). The boutons arising from the SC were relatively small (less than 1.5 microns in diameter), formed asymmetric synaptic contacts with small dendrites and dendritic spines, and contained round synaptic vesicles. The axon terminals from the DT were mostly large boutons (2-4.5 microns) with asymmetric synaptic specializations and round vesicles. These boutons and their postsynaptic targets formed synaptic glomeruli that were entirely or partially ensheathed by glial lamellae. The ultrastructural features are almost identical to those of boutons in the medial and central segments of MD that were previously shown to originate from the basal amygdaloid nucleus and the piriform cortex. The boutons from the SNr had a wide range in size, but the majority were medium-sized to large (1.5-4 microns). The nigral boutons established symmetric synaptic contacts with dendritic shafts and occasionally with somata, and contained pleomorphic vesicles. However, like the DT terminals, they participated in glomerular formations. The nigral terminals closely resemble previously described terminals in the medial part of MD from the ventral pallidum, except that the nigral terminals formed en passant and axosomatic synapses as well as axodendritic synapses. A combined immunohistochemistry and WGA-HRP tracing study revealed that the nigral inputs were immunoreactive for glutamic acid decarboxylase and the axon terminals from the DT were immunoreactive for choline acetyltransferase. In a separate study, the colliculothalamic fibers have been shown to take up and transport the transmitter specific tracer [3H]-D-aspartate, and are therefore putatively glutamatergic and/or aspartatergic. Taken together with this, the present results suggest that the collicular afferents are excitatory and glutamatergic and/or aspartatergic, that the inputs from the DT are also excitatory and cholinergic, while the nigral inputs are inhibitory and GABAergic.     

Direct synaptic contacts on the myenteric ganglia of the rat stomach from the dorsal motor nucleus of the vagus

The myenteric ganglia regulate not only gastric motility but also secretion, because a submucous plexus is sparsely developed in the rodent stomach. We have examined whether the neurons of the dorsal motor nucleus of the vagus (DMV) have direct synaptic contacts on the myenteric ganglia and the ultrastructure of the vagal efferent terminals by using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The myenteric ganglia of the rat were composed of four types of neurons, i.e., small, medium-sized, large, and elongated neurons. The average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. More than half of the axosomatic terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized, and large neurons. About 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. When WGA-HRP was injected into the DMV, many anterogradely labeled terminals were found around the myenteric neurons. The labeled terminals were large (3.16 +/- 0.10 microm) and contacted exclusively the somata. Most of them (about 90%) contained round vesicles and formed asymmetric synaptic contacts. Serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the DMV. The vagal axon terminals generally contacted the medium-sized or the elongated neurons, whereas a few labeled terminals contacted the small and the large neurons. The present results indicate that the DMV projects to all types of neurons and that their axon terminals contain mostly round synaptic vesicles and form asymmetric synaptic contacts.     

Synaptic organization of the rat parafascicular nucleus,with special reference to its afferents from the superior colliculus and the pedunculopontine tegmental nucleus

The synaptic organization of afferents to the parafascicular nucleus (Pf) of the thalamus was studied in rats. In the Pf, three types of axon terminals were identified: the first type was a small terminal with round synaptic vesicles forming an asymmetric synapse, the second type was a large terminal with round synaptic vesicles forming an asymmetric synapse, and the third type was a terminal with pleomorphic vesicles forming a symmetric synapse. They were named SR, LR and P boutons, respectively. In order to determine the origin of these axon terminals, biotinylated dextran amine (BDA) was injected into the main afferent sources of the Pf, the superior colliculus (SC) and the pedunculopontine tegmental nucleus (PPN). Axon terminals from the SC were both SR and LR boutons which made synaptic contacts with somata and dendrites. PPN afferents were SR boutons, which made synaptic contacts with somata and smaller dendrites. Double-labeled electron microscopic studies, in which a retrograde tracer (wheat germ agglutinin conjugated to horseradish peroxidase: WGA-HRP) was injected into the striatum and an anterograde tracer (BDA) into the SC revealed that SC afferent terminals made synapses directly with Pf neurons that projected to the striatum. Another experiment was performed to find out whether two different afferents converged onto a single Pf neuron. To address this question, two different tracers were injected into the SC and PPN in a rat. Electron microscopically, both afferent terminals from the SC and PPN made synaptic contacts with the same dendrite. Our results prove that a single neuron of the rat Pf received convergent projections from two different sources.     

Synaptic organization of projections from basal forebrain structures to the mediodorsal thalamic nucleus of the rat.

The synaptic organization of the mediodorsal thalamic nucleus (MD) in the rat was studied with the electron microscope, and correlated with the termination of afferent fibers labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Presynaptic axon terminals were classified into four categories in MD on the basis of the size, synaptic vesicle morphology, and synaptic membrane specializations: 1) small axon terminals with round synaptic vesicles (SR), which made asymmetrical synaptic contacts predominantly with small dendritic shafts; 2) large axon terminals with round vesicles (LR), which established asymmetrical synaptic junctions mainly with large dendritic shafts; 3) small to medium axon terminals with pleomorphic vesicles (SMP), which formed symmetrical synaptic contacts with somata and small-diameter dendrites; 4) large axon terminals with pleomorphic vesicles (LP), which made symmetrical synaptic contacts with large dendritic shafts. Synaptic glomeruli were also identified in MD that contained either LR or LP terminals as the central presynaptic components. No presynaptic dendrites were identified. In order to identify terminals arising from different sources, injections of WGA-HRP were made into cortical and subcortical structures known to project to MD, including the prefrontal cortex, piriform cortex, amygdala, ventral pallidum and thalamic reticular nucleus. Axons from the amygdala formed LR terminals, while those from the prefrontal and insular cortex ended exclusively in SR terminals. Fibers labeled from the piriform cortex formed both LR and SR endings. Based on their morphology, all of these are presumed to be excitatory. In contrast, the axons from the ventral pallidum ended as LP terminals, and those from the thalamic reticular nucleus formed SMP terminals. Both are presumed to be inhibitory. At least some terminals from these sources have also been identified as GABAergic, based on double labeling with anterogradely transported WGA-HRP and glutamic acid decarboxylase (GAD) immunocytochemistry.     

Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area.

Physiological and pharmacological studies indicate that descending projections from the prefrontal cortex modulate dopaminergic transmission in the nucleus accumbens septi and ventral tegmental area. We investigated the ultrastructural bases for these interactions in rat by examining the synaptic associations between prefrontal cortical terminals labeled with anterograde markers (lesion-induced degeneration or transport of Phaseolus vulgaris leucoagglutinin; PHA-L) and neuronal processes containing immunoreactivity for the catecholamine synthesizing enzyme, tyrosine hydroxylase. Prefrontal cortical terminals in the nucleus accumbens and ventral tegmental area contained clear, round vesicles and formed primarily asymmetric synapses on spines or small dendrites. In the ventral tegmental area, these terminals also formed asymmetric synapses on large dendrites and a few symmetric axodendritic synapses. In the nucleus accumbens septi, degenerating prefrontal cortical terminals synapsed on spiny dendrites which received convergent input from terminals containing peroxidase immunoreactivity for tyrosine hydroxylase, or from unlabeled terminals. In single sections, some tyrosine hydroxylase-labeled terminals formed thin and punctate symmetric synapses with dendritic shafts, or the heads and necks of spines. Close appositions, but not axo-axonic synapses, were frequently observed between degenerating prefrontal cortical afferents and tyrosine hydroxylase-labeled or unlabeled terminals. In the ventral tegmental area, prefrontal cortical terminals labeled with immunoperoxidase for PHA-L were in synaptic contact with dendrites containing immunogold reaction product for tyrosine hydroxylase, or with unlabeled dendrites. These results suggest that: (1) catecholaminergic (mainly dopaminergic) and prefrontal cortical terminals in the nucleus accumbens septi dually synapse on common spiny neurons; and (2) dopaminergic neurons in the ventral tegmental area receive monosynaptic input from prefrontal cortical afferents. This study provides the first ultrastructural basis for multiple sites of cellular interaction between prefrontal cortical efferents and mesolimbic dopaminergic neurons.     

Synaptology of the direct projections from the nucleus of the solitary tract to pharyngeal motoneurons in the nucleus ambiguus of the rat

During the pharyngeal phase of the swallowing reflex, the nucleus of the solitary tract (NTS) receives peripheral inputs from the pharynx by means of the glossopharyngeal ganglion and is the location of premotor neurons for the pharyngeal (PH) motoneurons. The semicompact formation of the nucleus ambiguus (AmS) is composed of small and medium-sized neurons that do not project to the pharynx, and large PH motoneurons. We investigated whether the neurons in the NTS projected directly to the PH motoneurons or to the other kinds of neurons in the AmS by using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) injections into the pharyngeal muscles of male Sprague-Dawley rats, many nerve terminals anterogradely labeled with WGA-HRP were found to contact PH motoneurons retrogradely labeled with CT-HRP. Most of the labeled axodendritic terminals (63%) contained pleomorphic vesicles with symmetric synaptic contacts (Gray's type II), and the remaining ones contained round vesicles with asymmetric synaptic contacts (Gray's type I). About 14% of the axosomatic terminals on PH motoneuron in a sectional plane were anterogradely labeled, and about 70% of the labeled axosomatic terminals were Gray's type II. Observations of serial ultrathin sections revealed that both the small and the medium-sized neurons received only a few labeled axosomatic terminals that were exclusively Gray's type I. These results indicate that the NTS neurons may send mainly inhibitory as well as a few excitatory inputs directly to the PH motoneurons in the AmS. J. Comp. Neurol. 393:391–401, 1998. © 1998 Wiley-Liss, Inc.     

Synaptic organization of septal projections in the rat medial habenula: a wheat germ agglutinin-horseradish peroxidase and immunohistochemical study.

The synaptic organization of septal inputs to the rat habenular complex of the dorsal diencephalon was examined employing the anterograde tracer wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The cellular distribution of substance P (SP) and choline acetyltransferase (ChAT) immunoreactivity was also studied at the light and electron microscopic level. Following placements of tracer within the entire septum, labeled axons were observed in the stria medullaris and in the medial and lateral subnuclei of the habenula. Following injections of tracer in the nuclei triangularis and septofimbrialis of the posterior septum, the medial subnucleus was heavily labeled, whereas the lateral subnucleus was devoid of peroxidase activity. The medial subnucleus possessed labeled myelinated axons and terminals that contained clear, spherical vesicles and formed asymmetric contacts with dendritic spines and shafts. Terminals possessing WGA-HRP activity also formed non-synaptic junctions with other labeled or unlabeled terminals. SP and ChAT immunoreactivity in normal and colchicine-treated animals was confined to dendrites and somata within the medial habenula. Terminals containing clear spherical vesicles formed asymmetric synaptic contacts with these immunoreactive somatic and dendritic profiles. Based on the combined anterograde tracing and immunohistochemical data, it is proposed that septal projections provide a direct innervation to habenular neurons that contain ChAT or SP activity. These septal inputs may play an important role in the facilitation of the ChAT- and SP-positive habenular neurons, both of which provide prominent afferent inputs to the interpeduncular nucleus. Thus, neurons of the habenula and interpeduncular nucleus are under the direct and indirect influence of septal neurons within the limbic forebrain circuit.     

Ultrastructure of the rostral ventral respiratory group neurons in the ventrolateral medulla of the rat

The neurons in the ventrolateral medulla that project to the spinal cord are called the rostral ventral respiratory group (rVRG) because they activate spinal respiratory motor neurons. We retrogradely labeled rVRG neurons with Fluoro-Gold (FG) injections into the fourth cervical spinal cord segment to determine their distribution. The rostral half of the rVRG was located in the area ventral to the semicompact formation of the nucleus ambiguus (AmS). A cluster of the neurons moved dorsally and intermingled with the palatopharyngeal motor neurons at the caudal end of the AmS. The caudal half of the rVRG was located in the area including the loose formation of the nucleus ambiguus caudal to the AmS. We also labeled the rVRG neurons retrogradely with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to determine their ultrastructural characteristics. The neurons of the rVRG were medium to large (38.1 x 22.1 microm), oval or ellipsoid in shape, and had a dark cytoplasm containing numerous free ribosomes, rough endoplasmic reticulum (rER), mitochondria, Golgi apparatuses, lipofuscin granules and a round nucleus with an invaginated nuclear membrane. The average number of axosomatic terminals in a profile was 33.2. The number of axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Gray's type I) was almost equal to those containing pleomorphic vesicles and making symmetric synaptic contacts (Gray's type II). The axodendritic terminals were large (1.55 microm), and about 60% of them were Gray's type I. The rVRG neurons have ultrastructural characteristics, which are different from the palatopharyngeal motor neurons or the prorpiobulbar neurons.     

Nucleus reticularis thalami connections with the mediodorsal thalamic nucleus: A light and electron microscopic study in the monkey

Wheat germ agglutinin conjugated horseradish peroxidese (WGA-HRP) and biotinylated dextran amine (BDA) were used as tracers to study nucleus reticularis (NRT) connections with the mediodorsal nucleus (MD). Injections of WGA-HRP in the MO resulted in retrograde labeling of cells in the anteromedial segment of the NRT, the so-called rostral NRT pole. Injections of WGA-HRP and BDA in this NRT region resulted in dense anterograde labeling in the MD. Labeled NRT fibers gave off several collaterals to different MD regions ending with terminal plexuses of thin varicose fibers. In the neuropil, the varicosities were distributed at random, and no tendency to form pericellular baskets was noted. Postembedding immunocytochemistry for GABA was performed on the tissue containing anterograde WGA-HRP label for identification of NRT boutons under electron microscope. The double-labeled boutons were of small to medium size, contained a large number of pleomorphic vesicles, few mitochondria, and formed multiple symmetric synaptic contacts. The number of contacts established by one bouton ranged from 1 to 4 with an average of 1.8 per bouton. About 60% of these boutons made synapses on distal dendrites of GABAergic local circuit neurons; 33% of synaptic contacts were on distal dendrites of thalamocortical neurons, and the rest on their proximal dendrites and soma. NRT boutons were also found in serial synapses and triads. The results demonstrate that the NRT input to the MD is organized so that a single fiber innervates different MD regions and its terminals form numerous synaptic contacts mostly on the distal dendrites of a large number of local circuit neurons and projection neurons.     

Topographically organized projections from the nucleus subceruleus to the hypoglossal nucleus in the rat: a light and electron microscopic study with complementary axonal transport techniques.

Projections from the nucleus subceruleus (nSC) to the hypoglossal nucleus (XII) were investigated with complementary retrograde and anterograde axonal transport techniques at the light and electron microscopic level in the rat. Injections of WGA-HRP into XII resulted in labeling of neurons in and around the nSC. Labeled nSC neurons were few in number (less than 4 per 40-60 microns sections) and variable in size and shape. Most labeled nSC neurons were medium-sized (mean = 16.89 microns), fusiform, triangular, or oval, with 3-4 dendrites typically oriented dorsomedially and ventrolaterally. These neurons were found throughout the rostrocaudal extent of the nSC but were most numerous medial, dorsomedial, and ventromedial to the motor trigeminal nucleus. Others were observed rostral to the motor trigeminal nucleus and ventral to the parabrachial nuclear complex. Confirmation of retrograde results was obtained following injections of tritiated amino acids or WGA-HRP into the nSC. This resulted in labeling throughout the rostrocaudal extent of XII mainly ipsilaterally. Labeled fibers descended the brainstem in the dorsolateral and, to a lesser extent, in the ventromedial component of Probst's tract. Fibers entered XII mainly rostrally along the lateral border of the nucleus. All regions of XII were recipients of nSC afferents, but the caudoventromedial quadrant contained the greatest density of terminal labeling. Electron microscopic evaluation confirmed that nSC afferents synapsed on motoneurons in XII. Axon terminals containing WGA-HRP reaction product were found contacting dendrites and somata, but primarily the former (81.3% versus 10.6%). Axodendritic terminals synapsed mainly on medium-to-small sized dendrites (less than 3 microns in diameter). The majority of labeled axodendritic terminals (90.1%) contained small, round, and clear synaptic vesicles (S-type: 20-50 nm) and were associated with an asymmetric (60.6%), symmetric (11.4%), or no (18%) postsynaptic specialization. By contrast, most axosomatic terminals contained flattened vesicles (F-type) and formed a symmetric or no postsynaptic specialization (75%). Large dense core vesicles (55-90 nm) were observed within a small proportion of all labeled axon terminals (1.3%). The results from this study demonstrate that the nSC projects to XII, preferentially targets a specific subgrouping of protrusor motoneurons, and synapses on both somata and dendrites, although mainly on the latter. The implications of these data are discussed relative to tongue control.     

Distribution and ultrastructure of dopaminergic neurons in the dorsal motor nucleus of the vagus projecting to the stomach of the rat

Almost all parasympathetic preganglionic motor neurons contain acetylcholine, whereas quite a few motor neurons in the dorsal motor nucleus of the vagus (DMV) contain dopamine. We determined the distribution and ultrastructure of these dopaminergic neurons with double-labeling immunohistochemistry for tyrosine hydroxylase (TH) and the retrograde tracer cholera toxin subunit b (CTb) following its injection into the stomach. A few TH-immunoreactive (TH-ir) neurons were found in the rostral half of the DMV, while a moderate number of these neurons were found in the caudal half. Most of the TH-ir neurons (78.4%) were double-labeled for CTb in the half of the DMV caudal to the area postrema, but only a few TH-ir neurons (5.5%) were double-labeled in the rostral half. About 20% of gastric motor neurons showed TH-immunoreactivity in the caudal half of the DMV, but only 0.3% were TH-ir in the rostral half. In all gastric motor neurons, 8.1% were double-labeled for TH. The ultrastructure of the TH-ir neurons in the caudal DMV was determined with immuno-gold-silver labeling. The TH-ir neurons were small (20.4 x 12.4 microm), round or oval, and contained numerous mitochondria, many free ribosomes, several Golgi apparatuses, a round nucleus and a few Nissl bodies. The average number of axosomatic terminals per section was 4.0. More than half of them contained round synaptic vesicles and made asymmetric synaptic contacts (Gray's type I). Most of the axodendritic terminals contacting TH-ir dendrites were Gray's type I (90%), but a few contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II).     

Afferent synaptic contacts on glycine-immunoreactive neurons in the rat cuneate nucleus

This study was aimed to clarify whether the primary afferent terminals (PATs), GABAergic terminals, and glutamatergic terminals made direct synaptic contacts with glycine-IR neurons in the cuneate nucleus of rats. In this connection, injection of the anterograde tracer WGA-HRP into brachial plexus, antiglycine preembedding immunoperoxidase, and anti-GABA, along with antiglutamate postembedding immunogold labeling, were used to identify the PATs, glycine-IR neurons, GABA-IR terminals, and glutamate-IR terminals, respectively. The present results showed that HRP-labeled PATs, immunoperoxidase-labeled glycine-IR terminals, immunogold-labeled GABA-IR, and glutamate-IR terminals made axodendritic synaptic contacts with immunoperoxidase-labeled glycine-IR neurons. The latter three presynaptic elements also formed axosomatic synapses with glycine-IR neurons. Statistical analysis has shown that the minimum diameter of the glycine-IR dendrites postsynaptic to the above-mentioned four presynaptic elements did not differ significantly. In addition, the synaptic ratio of the glutamate-IR terminals on the glycine-IR dendrites was higher than that of GABA-IR terminals. The synaptic ratio of the GABA-IR terminals on glycine-IR dendrite was in turn higher than that of the PATs and glycine-IR terminals. It is suggested that the PATs and glutamate-IR terminals on the glycine-IR neurons may be involved in subsequent postsynaptic inhibition for spatial precision of lateral inhibition. On the other hand, the GABA-IR and glycine-IR terminals which make synaptic contacts with the dendrites of glycine-IR neurons may provide a putative means for disinhibition or facilitation to maintain the baseline neuronal activity in the rat cuneate nucleus. The results of quantitative analysis suggest that glutamate act as the primary excitatory neurotransmitter, while GABA, when compared with glycine, may serve as a more powerful inhibitory neurotransmitter on glycine-IR neurons in the rat cuneate nucleus.     

Fine structure, synaptology and immunocytochemistry of large neurons in the rat dorsal cochlear nucleus connected to the inferior colliculus

Neurons in the rat dorsal cochlear nucleus that project to the inferior colliculus (pyramidal and giant) were retrograde labelled with wheat germ agglutinin conjugated to horseradish peroxydase. Both cell types showed a similar ultrastructural feature, particularly the rough endoplasmic reticulum was well developed and sometimes surrounded the nucleus. The synaptological profile was similar in pyramidal and giant cells. Axo-somatic terminals covered 40-70% of the perimeter of pyramidal cells and 35-60% of the perimeter of giant neurons. Giant neurons featured bipolar or multipolar shape and different orientation but they possessed a similar synaptic profile. Most axo-somatic terminals contained flat and pleomorphic synaptic vesicles, some pleomorphic vesicles. Few terminals contained round vesicles. These cells were consistently immuno-negative for both glycine and GABA and variably positive for glutamate. The immunoelectron microcopic study of thin sections showed that glycine immunoreactivity was constantly present in terminals enriched with flat vesicles, which often did not show GABA immunoreactivity. Few anterograde labelled boutons containing flat vesicles were in contact with the proximal dendrites and the cell bodies of pyramidal and giant neurons. The origin of these terminals is discussed. No other cells of the dorsal cochlear nucleus, in particular cartwheel and tuberculo-ventral neurons, were in contact with labelled boutons. The present results suggest that descending inhibitory collicular projections are essentially directed to the large excitatory neurons of the dorsal cochlear nucleus.     

Afferent connections of Gudden's tegmental nuclei in the rabbit

The regions projecting to Gudden's tegmental nuclei were examined by retrograde transport of horseradish peroxidase or wheat-germ-agglutinin-conjugated horseradish peroxidase. Gudden's tegmental nuclei in the rabbit can be divided into a pars principalis of the ventral tegmental nucleus (TVP), a pars ventralis of the dorsal tegmental nucleus (TDV), and a pars dorsalis of the dorsal tegmental nucleus (TDD). The TVP receives many fibers from the medial division of the ipsilateral medial mammillary nucleus and bilaterally from the lateral habenular nucleus, and additionally some fibers from the posterior nucleus of the interpeduncular complex. The TDV receives many fibers from the ipsilateral lateral mammillary nucleus, from the ipsilateral prepositus hypoglossi nucleus, bilaterally from the lateral habenular nucleus, from the central and paramedian nuclei of the interpeduncular complex, from the bilateral gray matter along the floor of the fourth ventricle, and from the contralateral supragenual nucleus. The TDD receives a projection from the lateral habenular nucleus of both sides and from the central and paramedian nuclei of the interpeduncular complex, and a minor projection from the ipsilateral lateral mammillary nucleus, the posterior nucleus of the interpeduncular complex, the prepositus hypoglossi nucleus, and the contralateral supragenual nucleus.