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.     

Topographical and ultrastructural investigation of the habenulo-interpeduncular pathway in the rat: a wheat germ agglutinin-horseradish peroxidase anterograde study

The topographical and ultrastructural organization of the habenular projection to the interpeduncular nucleus (IPN) of the rat was examined employing the anterogradely transported tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and the chromogen tetramethylbenzidine (TMB). Unilateral placements of WGA-HRP in the habenular complex resulted in heavy terminal labelling in the rostral, central, and intermediate subnuclei bilaterally, and in the lateral subnuclei ipsilaterally. The apical subnucleus possessed only a sparse amount of label. Placements confined to the medial habenula (mH) produced similar results to those observed when the entire habenula was filled, suggesting that the afferent contribution made by the lateral habenula (lH) to the IPN is small. Unilateral placements of WGA-HRP in the dorsal portion of the mH resulted in heavy, predominantly ipsilateral labelling in the lateral subnucleus and the dorsal cap of the rostral subnucleus. In the lateral subnucleus labelled habenular terminals consistently contacted single dendritic processes shared by one or more other boutons, possibly of nonhabenular origin. Labelled habenular terminals in the rostral subnucleus normally contacted one or two dendrites. Labelled terminals in both subnuclei possessed clear, spherical vesicles and a variable number of dense-core vesicles. Unilateral placements of WGA-HRP in the ventral portion of the mH resulted in heavy labelling in the rostral half of the rostral subnucleus with a slight ipsilateral predominance, and in the central and intermediate subnuclei bilaterally. Terminal labelling was observed in crest and S synapses in the intermediate and central subnuclei respectively. Crest synapses, which consist of two parallel habenular terminals contacting an attenuated dendritic process, normally possessed label in only one of the two boutons. In the central subnucleus labelled horizontal axons formed several en passant S synapses with dendritic processes of small and medium diameter. These synaptic specializations of habenular axons contained numerous clear, spherical vesicles. This study demonstrates that a major topographically organized projection to the IPN originates from two distinct subpopulations of habenular neurons which comprise a dorsal sector and a ventral sector of the mH. Ultrastructural examination demonstrated that axons originating from neurons in the ventral and dorsal mH form characteristic contacts in the various IPN subnuclei.     

Immunocytochemical Visualization of the mG1uR1 a Metabotropic Glutamate Receptor at Synapses of Corticothalamic Terminals Originating from Area 17 of the Rat

Pre-embedding immunogold histochemistry was combined with Phaseolus vulgaris leucoagglutinin anterograde tract tracing in order to analyse the relationship between the subcellular localization of the mGluR1a metabotropic glutamate receptors and the distribution of corticothalamic synapses in the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior nucleus (LP) of the rat. The injection of the tracer into area 17 labelled two types of corticothalamic terminals: (i) the small boutons constituting the majority of the labelled fibres which form asymmetrical synapses both in the dLGN and LP; and (ii) the giant terminals typically participating in glomerulus-like synaptic arrangements and found exclusively in the lateral posterior nucleus. The small corticothalamic terminals often established synapses with mGluR1a-immunopositive dendrites, with immunometal particles concentrated at the periphery of their postsynaptic membranes. In contrast, the synapses formed by giant boutons in the lateral posterior nucleus were always mGluR1a-immunonegative. We conclude that the corticothalamic fibres forming the small synaptic terminals are the most likely candidates for the postulated mGluR-mediated modulation of visual information flow by corticothalamic feedback mechanisms.     

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.     

Ultrastructure of giant thalamic terminals in the auditory cortex

The auditory system comprises some very large axonal terminals like the endbulb and calyx of Held and “giant” corticothalamic synapses. Previously, we described a hitherto unknown population of giant thalamocortical boutons arising from the medial division of the medial geniculate body (MGm) in the Mongolian gerbil, which terminate over a wide cortical range but in a columnar manner particularly in the extragranular layers of the auditory cortex. As a first step towards an understanding of their potential functional role, we here describe their ultrastructure combining anterograde tract‐tracing with biocytin and electron microscopy. Quantitative ultrastructural analyses revealed that biocytin‐labelled MGm boutons reach much larger sizes than other, non‐labelled boutons. Also, mitochondria occupy more space within labelled boutons whereas synapses are of similar size. Labelled boutons are very heterogeneous in size but homogeneous with respect to their ultrastructural characteristics, with asymmetric synapses containing clear, round vesicles and targeting dendritic spines. Functionally, the ultrastructure of the MGm terminals indicates that they form excitatory contacts, which may transmit their information in a rapid, powerful and high‐fidelity manner onto strategically advantageous compartments of their cortical target cells.     

Fine structure of the ventral lateral nucleus (VL) of the Macaca mulatta thalamus: cell types and synaptology.

Ultrastructure of the major cerebellar territory of the monkey thalamus, or VL as delineated in sagittal maps by Ilinsky and Kultas-Ilinsky (J. Comp. Neurol. 262:331-364, '87), was analyzed by using neuroanatomical tracing, immunocytochemical, and quantitative morphometric techniques. The VL nucleus contains nerve cells of two types. Multipolar neurons (PN) retrogradely labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from the precentral gyrus display a tufted branching pattern of the proximal dendrites and have a range of soma areas from 200 to 1,000 microns2 (mean 535.2 microns2, SD = 159.5). Small glutamic acid decarboxylase (GAD) immunoreactive cells (LCN) exhibit sizes from 65 to 210 microns2 (mean 122.5 microns2, SD = 32.8) and remain unlabeled after cortical injections. The two cell types can be further distinguished by ultrastructural features. Unlike PN, LCN display little perikaryal cytoplasm, a small irregularly shaped nucleolus, and synaptic vesicles in proximal dendrites. The ratio of PN to LCN is 3:1. The LCN dendrites establish synaptic contacts on PN somata and all levels of dendritic arbor either singly or as a part of complex synaptic arrangements. They are also presynaptic to other LCN dendrites. Terminals known as LR type, i.e., large boutons containing round vesicles, are the most conspicuous in the neuropil. They form asymmetric contacts on somata and proximal dendrites of PN as well as on distal dendrites of LCN. The areas of these boutons range from 0.7 to 12 microns2 and the appositional length on PN dendrites ranges from 1.1 to 14 microns. All LR boutons except the largest ones become anterogradely labeled from large WGA-HRP injections in the deep cerebellar nuclei. These boutons are also encountered as part of triads and glomeruli, but very infrequently since the latter complex synaptic arrangements are rare. The most numerous axon terminals in the neuropil are the SR type, i.e., small terminals (mean area 0.42 micron2) containing round vesicles. The SR boutons become anterogradely labeled after WGA-HRP injections in the precentral gyrus. They form distinct asymmetric contacts predominantly on distal PN and LCN dendrites; however, their domain partially overlaps that of LR boutons at intermediate levels of PN dendrites. The SR boutons are components of serial synapses with LCN dendrites which, in turn, contact somata and all levels of dendritic arbors of PN. They also participate in complex arrangements that consist of sequences of LCN dendrites, serial synapses, and occasional boutons with symmetric contacts.(ABSTRACT TRUNCATED AT 400 WORDS)     

The striatum and the globus pallidus send convergent synaptic inputs onto single cells in the entopeduncular nucleus of the rat: a double anterograde labelling study combined with postembedding immunocytochemistry for GABA.

The entopeduncular nucleus is one of the major output stations of the basal ganglia. In order to better understand the role of this structure in information flow through the basal ganglia, experiments have been performed in the rat to examine the chemical nature, morphology, and synaptology of the projections from the globus pallidus and striatum to the entopeduncular nucleus. In order to examine the morphology and synaptology of pallidoentopeduncular terminals and striatoentopeduncular terminals, rats were subjected to a double anterograde labelling study. The globus pallidus was injected with Phaseolus vulgaris-leucoagglutinin (PHA-L), and on the same side of the brain, the striatum was injected with biocytin. The entopeduncular nuclei of these animals were then examined for anterogradely labelled pallidal and striatal terminals. Rich plexuses of PHA-L-labelled pallidal terminals and biocytin-labelled striatal terminals were identified throughout the entopeduncular nucleus. At the electron microscopic level, the pallidal boutons were classified as two types. The majority (Type 1), were large boutons that formed symmetrical synapses with the dendrites and perikarya of neurones in the entopeduncular nucleus. Type 2 PHA-L-labelled terminals were much rarer, slightly smaller, and formed asymmetrical synapses. It is suggested that the Type 2 boutons are not derived from the globus pallidus but from the subthalamic nucleus. The biocytin-labelled terminals from the striatum had the typical morphological features of striatal terminals and formed symmetrical synapses. The distribution of the postsynaptic targets of the pallidal terminals and the striatal terminals differed in that the pallidal terminals preferentially made synaptic contact with the more proximal regions of the neurones in the entopeduncular nucleus, whereas the striatal terminals were located more distally on the dendritic trees. Examination in the electron microscope of areas where there was an overlap of the two sets of anterogradely labelled boutons revealed that terminals from the globus pallidus and the striatum made convergent synaptic contact with the perikarya and dendrites of individual neurones in the entopeduncular nucleus. In order to examine the chemical nature of the input to the entopeduncular nucleus from the globus pallidus and the striatum, ultrathin sections were immunostained by the postembedding method to reveal endogenous GABA. Three classes of GABA-containing terminals were identified; two of them formed symmetrical synapses and one rare type formed asymmetrical synapses. The combination of the GABA immunocytochemistry and anterograde labelling revealed that both the striatal and pallidal afferents that make symmetrical synapses with neurones in the entopeduncular nucleus, including those involved in convergent inputs, are GABAergic.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic relationships between hair follicle afferents and neurones expressing GABA and glycine-like immunoreactivity in the spinal cord of the rat

gamma-Aminobutyric acid (GABA) and glycine have been implicated in the inhibition of sensory pathways in the dorsal horn of the spinal cord. The object of this study is to investigate the interactions between neurones immunoreactive for GABA and/or glycine and hair follicle afferent terminals labelled by intracellular injection with neurobiotin. GABA and glycine-like immunoreactivity in axons and dendrites in synaptic contact with the afferent terminals was demonstrated by using a postembedding immunogold method, and serial section reconstruction was used to show the distribution and nature of these interactions in lamina III of the dorsal horn. Most afferent boutons (94%) were postsynaptic at axo-axonic synapses: 67% of presynaptic boutons presynaptic to the afferent terminals were immunoreactive for GABA and glycine, 24% for GABA alone, and 7% for glycine alone. Only a small percentage of dendrites postsynaptic to afferent boutons appeared to belong to inhibitory interneurones: 3% were immunoreactive for GABA and glycine, 10% for glycine alone, but 87% were immunoreactive for neither antibody. Many afferent boutons were the central terminals of what appeared to be type IIb glomeruli and were involved triadic synaptic arrangements at which boutons presynaptic to an afferent terminal also made axodendritic contacts with dendrites postsynaptic to the afferent. Many of the presynaptic boutons involved in the triads were immunoreactive for GABA and glycine. Because afferent terminals do not themselves express glycine receptors (Mitchell et al. [1993] J. Neurosci. 13:2371-2381), glycine may therefore act on dendrites postsynaptic to hair follicle afferent terminals at these triads.     

Fine structure of the magnocellular subdivision of the ventral anterior thalamic nucleus (VAmc) of Macaca mulatta: I. Cell types and synaptology

The nucleus ventralis anterior pars magnocellularis (VAmc) is recognized only in primates and is the major recipient of the nigrothalamic projections. The neuronal and synaptic composition of this nucleus in the rhesus monkey was studied with the use of a variety of neuroanatomical techniques that included quantitative morphometry, anterograde and retrograde labeling with WGA-HRP from the prefrontal cortex, and immunocytochemistry for glutamic acid decarboxylase (GAD). Two major cell types were identified in the nucleus: thalamocortical projection neurons (PN) that were multipolar cells of various sizes, and small GAD-immunoreactive cells, apparently local circuit neurons (LCN). The approximate ratio of the two types of cells was 10:1. The major type of bouton encountered in the neuropil was of medium to large-sized (areas from 1.5 to 12 microns 2) and mostly of en passant type. These terminals formed symmetric contacts, contained moderate amounts of pleomorphic or mostly flat synaptic vesicles and large numbers of mitochondria, and displayed numerous puncta adhaerentia. All of these boutons exhibited positive GAD immunoreactivity. These boutons constituted the only synaptic population on somata and primary dendrites of PN and formed an overwhelming majority on the secondary PN dendrites. There were fewer of these axon terminals on tertiary PN and LCN dendrites. Additionally, boutons with similar features formed synapses on axon hillocks or initial axonal segments of PN, and somata or very proximal parts of primary dendrites of LCN. With the exception of the boutons in the last two locations, all of the other boutons in this group were shown to be terminals of the nigrothalamic afferents in the parallel EM autoradiographic study (Kultas-Ilinsky and Ilinsky: J. Comp Neurol. 294:479-489, '90). The second major bouton population in the VAmc was represented by small to medium-sized terminals (areas range from 0.2 to 2.0 microns 2) that formed distinct asymmetric contacts and contained large numbers of round vesicles and few or no mitochondria. These boutons were labeled anterogradely from the cortex and dominated on distal PN and LCN dendrites. Some of them were found on secondary PN dendrites where they formed synapses either directly or indirectly via LCN dendrites and dendro-dendritic contacts. The latter arrangements, i.e., serial synapses, were also formed between the cortical boutons and PN somata or tertiary dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complete Axon Arborization of a Single CA3 Pyramidal Cell in the Rat Hippocampus, and its Relationship With Postsynaptic Parvalbumin-containing Interneurons

The complete axon arborization of a single CA3 pyramidal cell has been reconstructed from 32 (60 /μm thick) sections from the rat hippocampus following in vivo intracellular injection of neurobiotin. The same sections were double-immunostained for parvalbumin-a calcium-binding protein selectively present in two types of GABAergic interneurons, the basket and chandelier cells-in order to map boutons of the pyramidal cell in contact with dendrites and somata of these specific subsets of interneurons visualized in a Golgi-like manner. The axon of the pyramidal cell formed 15 295 boutons, 63.8% of which were in stratum oriens, 15.4% in stratum pyramidale and 20.8% in stratum radiatum. Only 2.1% of the axon terminals contacted parvalbumin-positive neurons. Most of these were single contacts (84.7%), but double or triple contacts (15.3%) were also found. The majority of the boutons terminated on dendrites (84.1%) of parvalbumin-positive cells, less frequently on cell bodies (15.9%). In order to estimate the proportion of contacts representing synapses, 16 light microscopically identified contacts between boutons of the filled pyramidal cell axon and the parvalbumin-positive targets were examined by correlated electron microscopy. Thirteen of them were found to be asymmetrical synapses, and in the remaining three cases synapses between the labelled profiles could not be confirmed. We conclude that the physiologically effective excitatory connections between single pyramidal cells and postsynaptic inhibitory neurons are mediated by a small number of contacts, mostly by a single synapse. This results in a high degree of convergence and divergence in hippocampal networks.     

Synaptic organization of tooth pulp afferent terminals in the rat trigeminal sensory nuclei

Previous studies provide evidence that a structure/function correlation exists in the distinct zones of the trigeminal sensory nuclei. To evaluate this relationship, we examined the ultrastructure of afferent terminals from the tooth pulp in the rat trigeminal sensory nuclei: the principalis (Vp), the dorsomedial part of oral nucleus (Vdm), and the superficial layers of caudalis (Vc), by using transganglionic transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). A total of 93 labeled boutons were serially sectioned, in which some sections were incubated with gamma-aminobutyric acid (GABA) antiserum. Almost all labeled boutons formed asymmetric contact with nonprimary dendrites, in which more than half of labeled boutons in the Vc made synapses with their spines. The labeled boutons could be divided into two types on the basis of numbers of dense-cored vesicles (DCVs) in a boutons: S-type and DCV-type. Almost all labeled boutons in the Vp and Vdm were S-type, whereas two types were distributed evenly in the Vc. In contrast to DCV-type boutons, the S-type was frequently postsynaptic to unlabeled axon terminals containing a mixture of round, oval, and flattened vesicles (p-endings) and forming symmetrical synapses. Most p-endings examined were immunoreactive to GABA. The frequency of axoaxonic contacts was higher for labeled boutons in the Vp than in the Vdm and Vc. These results suggest that the three structures of trigeminal sensory nuclei serve distinct functions in nociceptive processing.     

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 innervation of neurons in the internal pallidal segment by the subthalamic nucleus and the external pallidum in monkeys

In order to better understand the way by which the subthalamic nucleus interacts with the globus pallidus to control the output of the basal ganglia, we carried out a series of experiments to investigate the pattern of synaptic innervation of the pallidal neurons by the subthalamic terminals in the squirrel monkey. To address this problem we used the anterograde transport of biocytin. Following injections of biocytin in the subthalamic nucleus, rich plexuses of labelled fibres and varicosities formed bands that lay along the medullary lamina in both segments of the ipsilateral pallidum. At the electron microscopic level, two populations of bioctyin-containing terminals were identified in the internal pallidum (GPi). A first group of small to medium-sized terminals (type 1) mean cross-sectional area ±S. D. = 0.41 ± 0.04 μm² contained round vesicles and formed asymmetric synapses with dendritic shafts (95%) of mixed sizes (maximum diameter ranging from 0.3 to 4.0 μm) and spine-like structures (5%). This second group of terminals (type 2) contained pleiomorphic vesicles, had a larger cross-sectional area (mean ± S. D. = 0.9 ± 0.4 μm²) and formed symmetric synapses predominantly with perikarya (41%) and large dendrites (57%). In some cases, the two types of terminals converged at the level of single GPi neurons. Postembedding immunogold method revealed that the type 2 terminals displayed gamma-aminobutyric acid (GABA) immunoreactivity, whereas the type 1 terminals did not. In the external pallidum (GPe), injections in the subthalamic nucleus labelled both type 1 or type 2 terminals. However, the labelled type 2 boutons were much less abundant in GPe than in GPi. The presence of biocytin-labelled perikarya in GPe and the fact that the type 2 terminals displayed GABA immunoreactivity led us to suspect that these terminals were derived from axons of GPe neurons. In agreement with this hypothesis, injctions of Phaseolus vulgaris-leucoagglutinin (PHA-L) in GPe labelled terminals in GPi that displayed the morphological features and a pattern of synaptic organization similar to the type 2 terminals. In conclusion, the results of our study demonstrate that the subthalamopallidal terminals form asymmetric synapses that are distributed along the dendritic tree of GPe and Gpi neurons. In contrast, the GPe projection to GPi give rise to large GABA-containing terminals that form symmetric synapses predominantly with the proximal region of pallidal neurons. Because the GABAergic axon terminals from GPe form synapses onto the perikarya and proximal dendrites of GPi neurons, the Gpe input is in a strategic position to reduce the excitatory influence generated more distally on the dendritic tree by the subthalamic nucleus. © 1994 Wiley-Liss, Inc.     

Neuropeptide Y-immunoreactive terminals form axo-axonic synaptic arrangements in the substantia gelatinosa (lamina II) of the cat spinal dorsal horn

The ultrastructural organization of nerve terminals containing neuropeptide Y-immunoreactivity was studied in the substantia gelatinosa of the cat spinal dorsal horn. Seventy immunoreactive boutons were examined through serial sections and 67 of them were found to form between one and five synaptic junctions with dendrites (59.5% of synapses), somata (3% of synapses) and other axon terminals (37.5% of synapses). The postsynaptic axon terminals were often the central boutons of glomeruli. These findings suggest that neuropeptide Y regulates spinal sensory transmission through both a postsynaptic action upon dorsal horn neurons and a presynaptic action upon primary afferent terminals.     

Synaptic innervation of midbrain dopaminergic neurons by glutamate-enriched terminals in the squirrel monkey

The excitatory amino acid, glutamate, has long been thought to be a transmitter that plays a major role in the control of the firing pattern of midbrain dopaminergic neurons. The present study was aimed at elucidating the anatomical substrate that underlies the functional interaction between glutamatergic afferents and midbrain dopaminergic neurons in the squirrel monkey. To do this, we combined preembedding immunocytochemistry for tyrosine hydroxylase and calbindin D-28k with postembedding immunostaining for glutamate. On the basis of their ultrastructural features, three types (so-called types I, II, and III) of glutamate-enriched terminals were found to form asymmetric synapses with dendrites and perikarya of midbrain dopaminergic neurons. The type I terminals accounted for more than 70% of the total population of glutamate-enriched boutons in contact with dopaminergic cells in the dorsal and ventral tiers of the substantia nigra pars compacta as well as in the ventral tegmental area, whereas 5–20% of the glutamatergic synapses with dopaminergic neurons involved the two other types of terminals. The major finding of our study is that the glutamate-enriched boutons were involved in 70% of the axodendritic synapses in the ventral tegmental area. In contrast, less than 40% of the boutons in contact with dopaminergic dendrites were immunoreactive for glutamate in the dorsal and ventral tiers of the substantia nigra pars compacta. Approximately 50% of the terminals in contact with the perikarya of the different populations of midbrain dopaminergic neurons displayed glutamate immunoreactivity. In conclusion, our findings provide the first evidence that glutamate-enriched terminals form synapses with midbrain dopaminergic neurons in primates. The fact that the proportion of glutamatergic boutons in contact with dopaminergic cells is higher in the ventral tegmental area than in the substantia nigra pars compacta suggests that the different groups of midbrain dopaminergic neurons are modulated differently by extrinsic glutamatergic afferents in primates. © 1996 Wiley-Liss, Inc.     

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.     

Neuronal circuitry and synaptic organization of trigeminal proprioceptive afferents mediating tongue movement and jaw-tongue coordination via hypoglossal premotor neurons

The neural framework and synaptic organization of trigeminal proprioceptive afferent-mediated jaw-tongue coordination were studied in rats using multiple electrophysiological and neuroanatomical approaches. Electrostimulation of the masseter nerve evoked short-latency responses (5.86 +/- 2.59 ms) in hypoglossal premotor pools including the parvocellular (PCRt) and intermediate (IRt) reticular nuclei and the dorsomedial part of the spinal trigeminal nucleus oralis (Vodm) and interpolaris (Vidm). Biocytin-labelled axon terminals from these areas traveled into the hypoglossal nucleus (XII) and contacted motoneurons. Double labelling of biotinylated dextran amine (BDA) tracing and cholera toxin B (CTB) transport demonstrated that labelled axons and terminals from the mesencephalic trigeminal nucleus (Vme) overlapped with XII premotor neurons in the alpha division and in PCRt, IRt, Vodm and Vidm. Confocal microscopic observations revealed that Vme terminals closely contacted XII premotor neurons. Dual labelling of intracellular neurobiotin staining of jaw-muscle spindle afferents (JMSAs) combined with horseradish peroxidase (HRP) retrograde transport revealed that 498 JMSA boutons apposed to 146 HRP-labelled premotor neurons. Electron microscopic observations demonstrated that 127 JMSA boutons made both axodendritic (68%) and axosomatic (32%) synapses with XII premotor neurons. Eighty-three per cent of synapses were asymmetric and the rest (17%) were symmetric. Thirty-nine per cent of JMSA boutons received presynaptic contacts from P-type terminals. Varieties of synaptic organizations were found. These results provide evidence that trigeminal proprioceptive afferents mediate jaw-tongue coordination through XII premotor neurons. Ultrastructural findings demonstrated that synapses between JMSA boutons and XII premotor neurons are predominantly excitatory, and synaptic transmission to XII motoneurons is modified on XII premotor neurons by presynaptic mechanisms. These frameworks and synaptic organizations are most probably the neural substrate for trigeminal proprioceptive afferent-mediated jaw-tongue coordination.     

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and γ-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%) of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.     

Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. II. Synaptic organization on reinnervated Purkinje cells

A salient feature of the cerebellar Purkinje cells is the highly ordered distribution of their excitatory afferents on the dendritic tree. Climbing fibres synapse exclusively on the proximal dendrites, whereas parallel fibres articulate with the distal branches, the so-called spiny branchlets. This input organization is lost following the removal of climbing fibres. Such denervation results in the formation of a large number of new spines on the proximal dendrites, and these become contacted by sprouting parallel fibres, which thereby extend their domain of innervation. We have previously shown that the climbing fibres surviving a subtotal lesion of the inferior olive sprout and reinnervate neighbouring Purkinje cells. In the present ultrastructural study, we have investigated the features of Purkinje cells reinnervated by sprouting climbing fibres. The objectives were to examine the fine morphology of the newly formed synapses and to determine whether the modifications of Purkinje cell morphology and afferent organization are reversed by this reinnervation. Surviving climbing fibres were labelled by the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and immunohistochemically visualized by means of the gold-substituted silver peroxidase technique, 2 and 6 months after 3-acetylpyridine lesions of the inferior olive in adult rats. Sprouting climbing fibres and newly formed arborizations were identified in the light microscope, isolated, and cut in serial ultrathin sections for electron microscopic analysis. The labelled boutons belonging to newly formed terminal plexuses exhibited the typical morphological features of climbing fibre terminals, i.e., a high number of round synaptic vesicles and a few small mitochondria. Most frequently they formed asymmetric synapses on stubby thorns protruding from the proximal Purkinje cell dendrites. In some instances, however, the postsynaptic element consisted of long slender spines or spines showing an atypical morphology. A number of labelled boutons was also in contact with the perikarya of reinnervated Purkinje cells, either articulating with spines or synapsing directly on the smooth somatic surface. The proximal dendrites of denervated Purkinje cells were characterized by large numbers of spines, which were frequently postsynaptic to parallel fibres. By contrast, Purkinje cells reinnervated by the sprouting climbing fibres generally showed a lower number of spines on their proximal dendrites, indicating a reversal of this morphological change. The aberrant parallel fibre input was also decreased on reinnervated dendrites or had completely disappeared. Nevertheless, some reinnervated Purkinje cells showed the persistence of some parallel fibre synapses on their proximal dendrites. On occasion, climbing fibre and parallel fibre boutons synapsed on the same spine.     

The ultrastructural characteristics of the pyramidal callosal neurons of layer III in the primary auditory area (A1) of the cat cortex]

An electron microscope study of retrogradely labelled pyramidal neurons in layer III of the primary auditory cortex (AI) after HRP injections into the contralateral AI has been carried out in cats. From 4 to 10 synapses were usually revealed on somatic profiles of these callosal neurons. Synapses occupied 20.0% of the somatic surface of these neurons. All of the revealed synapses on the somata of callosal neurons had symmetric contacts and were formed by axon terminals with small elongated synaptic vesicles. Average length of these synaptic contacts was 1.6 microns. In layer III anterogradely labelled terminals of callosal fibres were also revealed. The majority of them contained large round synaptic vesicles and formed asymmetric contacts on spines. Three labelled axon terminals with small elongated vesicles were found to form symmetric axo-somatic synapses on callosal neurons of layer III.