Identification of signal substances in synapses made between primary afferents and their associated axon terminals in the rat trigeminal sensory nuclei

The relationships between primary afferent terminals (PATs) and their associated presynaptic terminals in the rat trigeminal sensory nuclear complex (TSNC) were examined with special reference to amino acid transmitters glutamate (Glu) and gamma-aminobutyric acid (GABA). Primary afferent terminals anterogradely labeled from the trigeminal ganglion with the B subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) were sectioned for electron microscopy. Serial sections from the principal nucleus (Vp), dorsomedial parts of the oral and interpolar nuclei (Vdm), and lamina III/IV of caudal nucleus (Vc) were immunostained for Glu and GABA by using a postembedding immunogold technique. The tracer, CTB-HRP to the trigeminal ganglion, preferentially labeled myelinated primary afferents. Sections immunostained with Glu antiserum showed that most labeled PATs were enriched with immunoreactivity (IR) for Glu. The Glu-IR PATs contained clear, round, synaptic vesicles and formed asymmetric synaptic contacts with somata or dendrites. They were frequently postsynaptic to, unlabeled axon terminals filled with a mixture of clear, round, oval, and flattened vesicles (p-endings), with symmetric synaptic junctions. The frequency of synapses onto somata or primary dendrites per Glu-IR PAT was higher in the Vdm than in either the Vp or Vc lamina III/IV. The frequency of contacts of the p-endings per Glu-IR PAT was higher in the Vp than in the Vdm and Vc lamina III/IV. Sections immunostained with GABA antiserum showed that most axon terminals presynaptic to PATs were enriched with GABA in the three nuclei. The GABA-IR axon terminals and their postsynaptic PATs had a similar ultrastructural character to p-endings and their postsynaptic Glu-IR PATs, respectively. The present study suggests that primary afferent neurons with large-caliber fibers use glutamate as a neurotransmitter and are subject to presynaptic modulation by GABAergic fibers.     

Quantitative analysis of tooth pulp afferent terminals in the rat brain stem

This study analyzed quantitatively the ultrastructural features of tooth pulp afferent terminals and their presynaptic axonal endings (p-endings) in the trigeminal principal (Vp), dorsomedial oral (Vdm), and caudal nuclei (Vc). Mitochondrial volume, active zone area, apposed surface area, and vesicle number were highly correlated with afferent bouton volume. The afferent bouton volume varied widely in Vp, compared to that in Vdm and Vc. The values of all parameters of p-endings were within a narrow range, and were smaller than those of afferent boutons. The afferent bouton volume correlated with the number of postsynaptic dendrites and p-endings. These results suggest that pulpal afferent information is regulated in a unique manner in the each trigeminal sensory nucleus.     

The synaptic microcircuitry associated with primary afferent terminals in the interpolaris and caudalis of trigeminal sensory nuclear complex

Previous ultrastructural studies indicating a higher number of axoaxonic contacts on individual low-threshold mechanoreceptive afferents in the principalis (Vp) than in the oralis (Vo) of cat trigeminal sensory nuclear complex (TSNC) suggest that the synaptic microcircuitry associated with primary afferents manifests unique differences across the sensory nuclei of TSNC. To address this issue, we analyzed synaptic microcircuits associated with fast adapting vibrissa afferent terminals in the interpolaris (Vi) and caudalis (Vc, laminae III/IV) by using intraaxonal injections of horseradish peroxidase (HRP) in cats. Forty-two and 65 HRP-labeled boutons were analyzed in the Vi and Vc, respectively. The labeled boutons contained clear, spherical vesicles. They most frequently formed asymmetric axodendritic synapses and were commonly postsynaptic to unlabeled axon terminals containing pleomorphic vesicles (p-endings) with symmetric junctions. The examination of synaptic contacts over the entire surface of individual boutons indicated that the afferent boutons made contacts with an average of two postsynaptic targets in the Vi and Vc. In contrast, axoaxonic contacts, and labeled boutons participating in synaptic triads, where p-endings contacted both the boutons and their postsynaptic targets, were, on average, higher in the Vi than in the Vc. These results suggest that the output of sensory information conveyed through low-threshold mechanoreceptive afferents is more strongly controlled at the level of the first synapse by presynaptic and postsynaptic mechanisms in the Vi responsible for sensory discriminative functions than in the Vc for sensorimotor reflexive functions.     

Quantitative ultrastructure of slowly adapting lingual afferent terminals in the principal and oral nuclei in the cat

Previous studies provide evidence that a structure/function correlation exists in the cytoarchitectonically different zones of the trigeminal sensory nuclei. To extend this relationship, we examined the ultrastructural features of trigeminal primary afferent neurons in the cat dorsal principal nucleus (Vpd) and the rostrodorsomedial oral nucleus (Vo.r) using intra-axonal labeling with horseradish peroxidase and morphometric analyses. All labeled boutons contained round synaptic vesicles. Eighty-two percent of the boutons in the Vo.r and 99% of the boutons in the Vpd were presynaptic to nonprimary dendrites. The remaining boutons in the Vo.r were presynaptic to somata (8%) or primary dendrites (10%). The average number of postsynaptic profiles per labeled bouton did not differ in the Vpd and Vo.r. Most labeled boutons in the two nuclei were postsynaptic to unlabeled axon terminals with pleomorphic vesicles (p-ending). The number of p-endings per labeled bouton was higher in the Vpd than Vo.r A morphometric analysis indicated that labeled bouton volume and apposed surface area were larger in the Vpd than Vo.r while active zone area and vesicle number did not differ. All these parameters were larger than those of p-endings in each nucleus. In both labeled boutons and p-endings, the parameters were positively correlated with bouton size. These results suggest that sensory information conveyed through trigeminal afferents is more strongly controlled at the level of the first synapse by presynaptic mechanisms in the Vpd than in the Vo.r, while the efficacy of transmission at primary afferent synapses does not differ.     

Ultrastructural analysis of the synaptic connectivity of TRPV1-expressing primary afferent terminals in the rat trigeminal caudal nucleus

Trigeminal primary afferents that express the transient receptor potential vanilloid 1 (TRPV1) are important for the transmission of orofacial nociception. However, little is known about how the TRPV1-mediated nociceptive information is processed at the first relay nucleus in the central nervous system (CNS). To address this issue, we studied the synaptic connectivity of TRPV1-positive (+) terminals in the rat trigeminal caudal nucleus (Vc) by using electron microscopic immunohistochemistry and analysis of serial thin sections. Whereas the large majority of TRPV1+ terminals made synaptic contacts of an asymmetric type with one or two postsynaptic dendrites, a considerable fraction also participated in complex glomerular synaptic arrangements. A few TRPV1+ terminals received axoaxonic contacts from synaptic endings that contained pleomorphic synaptic vesicles and were immunolabeled for glutamic acid decarboxylase, the synthesizing enzyme for the inhibitory neurotransmitter γ-aminobutyric acid (GABA). We classified the TRPV1+ terminals into an S-type, containing less than five dense-core vesicles (DCVs), and a DCV-type, containing five or more DCVs. The number of postsynaptic dendrites was similar between the two types of terminals; however, whereas axoaxonic contacts were frequent on the S-type, the DCV-type did not receive axoaxonic contacts. In the sensory root of the trigeminal ganglion, TRPV1+ axons were mostly unmyelinated, and a small fraction was small myelinated. These results suggest that the TRPV1-mediated nociceptive information from the orofacial region is processed in a specific manner by two distinct types of synaptic arrangements in the Vc, and that the central input of a few TRPV1+ afferents is presynaptically modulated via a GABA-mediated mechanism.     

Expression of vanilloid receptor TRPV1 in the rat trigeminal sensory nuclei

Little is known about the central projection patterns of trigeminal afferent neurons expressing the vanilloid receptor TRPV1 and their coexpression of neuromodulatory peptides. To address these issues, we examined the distribution of TRPV1-positive neurons in the trigeminal ganglion (TG) and trigeminal sensory nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc) in the rat via light and electron microscopy. In addition, we studied the colocalization of TRPV1-positive neurons with substance P (SP) and calcitonin gene-related peptide (CGRP) via confocal microscopy. In TG, only small and medium-sized neurons were immunopositive for TRPV1. The staining for TRPV1 was found in axon collaterals in the dorsal parts of Vp, Vo, and Vi and in terminals and fibers throughout lamina I and the outer zone of lamina II (IIo) of Vc. With electron microscopy, TRPV1-positive fibers in the ascending and descending trigeminal tracts were found to be unmyelinated. Almost all TRPV1-positive terminals in Vc contained numerous large dense-core vesicles and formed synaptic contacts with single small dendrites. Multiple immunofluorescence revealed a high degree of colocalization of TRPV1 with SP and CGRP in TG neurons as well as in fibers and terminals confined to laminae I and IIo of Vc. These results suggest that the central projections of unmyelinated (C) afferents sensitive to noxious heat and capsaicin are organized differently between Vc and the rostral trigeminal nuclei and that Vc may play a role in the development of hyperalgesia.     

Projection of jaw-muscle spindle afferents to the caudal brainstem in rats demonstrated using intracellular biotinamide

Intracellular staining with biotinamide was used to study the axonal projection and synaptic morphology of rat jaw-muscle spindle afferents. Intracellular recordings in the mesencephalic trigeminal nucleus (Vme) were identified as spindle afferent responses by their increased firing during stretching of the jaw-elevator muscles. Biotinamide-stained axon collaterals with boutons were found in the trigeminal motor nucleus (Vmo), Vme, the region dorsal to Vmo including the supratrigeminal region, the dorsomedial portion of the trigeminal principal sensory nucleus, and the dorsomedial part of the rostral spinal trigeminal subnucleus oralis. Additional, previously undescribed projections of jaw-muscle spindle afferents were found to the dorsomedial portiori of the caudal spinal trigeminal subnucleus oralis (Vodm), the dorsomedial part of the spinal trigeminal subnucleus interpolaris (Vidm), the caudal parvicellular reticular formation, laminae IV and V of the spinal trigeminal subnucleus caudalis (Vc), and the dorsal division of the medullary reticular field. Labeled spindle boutons in Vodm formed predominately axodendritic synapses. Some of these boutons received presynaptic inputs from unlabeled P-type boutons containing clear, spherical, or flattened vesicles. In Vidm, labeled collaterals and boutons were densely clustered into glomerular-like structures. Labeled boutons in Vidm made axodendritic, axosomatic, and axoaxonic synapses and received synaptic contacts from unlabeled boutons containng clear, spherical, or flat and pleomorphic vesicles. Unlabeled presynaptic boutons in Vidrn occasionally contained dense core vesicles. Labeled boutons in Vc mainly formed synaptic contacts with large diameter dendrites. This projection of jaw-muscle spindle afferents to caudal brainstem regions may play a significant role in masticatory-muscle stretch reflexes and in the integration of trigeminal proprioceptive information and its transmission to higher centers. © 1995 Wiley-Liss, Inc.     

Neuron numbers in the sensory trigeminal nuclei of the rat: A GABA- and glycine-immunocytochemical and stereological analysis

The volume, total neuron number, and number of GABA- and glycine-expressing neurons in the sensory trigeminal nuclei of the adult rat were estimated by stereological methods. The mean volume is 1.38+/-0.13 mm3 (mean+/-SD) for the principal nucleus (Vp), 1.59+/-0.06 for the n. oralis (Vo), 2.63+/-0.34 for the n. interpolaris (Vip), and 3.73+/-0.11 for the n. caudalis (Vc). The total neuron numbers are 31,900+/-2,200 (Vp), 21,100+/-3,300 (Vo), 61,600+/-8,300 (Vip), and 159,100+/-25,300 (Vc). Immunoreactive (-ir) neurons were classified as strongly stained or weakly stained, depending on qualitative criteria, cross-checked by a densitometric analysis. GABA-ir cells are most abundant in Vc, in an increasing rostrocaudal gradient within the nucleus. Lower densities are found in Vip and Vp. The mean total number of strongly labeled GABA-ir neurons ranges between 1,800 in Vp to 7,800 in Vip and 22,900 in Vc, and varies notably between subjects. Glycine-ir neurons are more numerous and display more homogeneous densities in all nuclei. Strongly labeled Gly-ir cells predominate in all nuclei, their total number ranging between 9,400 in Vp to 24,300 in Vip and 34,200 in Vc. A substantial fraction of immunolabeled neurons in all nuclei coexpress GABA and glycine. In general, all neurons strongly immunoreactive for GABA are small, while weakly GABA-ir cells which coexpress Gly are larger. In Vc, one-third of all neurons are immunoreactive: 16.6% of them are single-labeled for GABA and 31.6% are single-labeled for glycine. The remaining 51.8% express GABA and glycine in different combinations, with those showing strong double labeling accounting for 22.6%.     

Coexpression of VGLUT1 and VGLUT2 in trigeminothalamic projection neurons in the principal sensory trigeminal nucleus of the rat

VGLUT1 and VGLUT2 have been reported to show complementary distributions in most brain regions and have been assumed to define distinct functional elements. In the present study, we first investigated the expression of VGLUT1 and VGLUT2 in the trigeminal sensory nuclear complex of the rat by dual‐fluorescence in situ hybridization. Although VGLUT1 and/or VGLUT2 mRNA signals were detected in all the nuclei, colocalization was found only in the principal sensory trigeminal nucleus (Vp). About 64% of glutamatergic Vp neurons coexpressed VGLUT1 and VGLUT2, and the others expressed either VGLUT1 or VGLUT2, indicating that Vp neurons might be divided into three groups. We then injected retrograde tracer into the thalamic regions, including the posteromedial ventral nucleus (VPM) and posterior nuclei (Po), and observed that the majority of both VGLUT1‐ and VGLUT2‐expressing Vp neurons were retrogradely labeled with the tracer. We further performed anterograde labeling of Vp neurons and observed immunoreactivies for anterograde tracer, VGLUT1, and VGLUT2 in the VPM and Po. Most anterogradely labeled axon terminals showed immunoreactivities for both VGLUT1 and VGLUT2 in the VPM and made asymmetric synapses with dendritic profiles of VPM neurons. On the other hand, in the Po, only a few axon terminals were labeled with anterograde tracer, and they were positive only for VGLUT2. The results indicated that Vp neurons expressing VGLUT1 and VGLUT2 project to the VPM, but not to the Po, although the functional differences of three distinct populations of Vp neurons, VGLUT1‐, VGLUT2‐, and VGLUT1/VGLUT2‐expressing ones, remain unsettled. J. Comp. Neurol. 518:3149–3168, 2010. © 2010 Wiley‐Liss, Inc.     

Projections from subnucleus oralis of the spinal trigeminal nucleus to contralateral thalamus via the relay of juxtatrigeminal nucleus and dorsomedial part of the principal sensory trigeminal nucleus in the rat.

Following injection of HRP into contralateral thalamus, retrogradely labeled cells were observed in principal sensory trigeminal nucleus (Vp) and an area of juxtatrigeminal nucleus (JX) formerly described by John and Tracey (1987). When PHA-L was delivered to dorsomedial part of the subnucleus oralis (Vodm), PHA-L labeled terminals were seen in dorsomedial part of the Vp (Vpdm) and in the JX region. Comparing the distribution of PHA-L labeled terminal field with that of HRP labeled JX neurons showed that the labeled terminals and neurons were overlapped closely in the JX. The distribution patterns of the labeled terminals and JX neurons were also the same: viewed on the coronal planes caudal-rostrally, both of the labelings began to appear at the levels where the facial nerve root was just broken. Rostrally, at middle levels of the motor trigeminal nucleus (Vmo), the labelings showed their typical view covering dorsal and ventral JX (dJX, vJX). The labelings disappeared at rostral poles of the Vmo and Vp. When injections of PHA-L into the Vodm and HRP into the contralateral thalamus was made in one rat, the contacts between Vodm projecting terminals labeled with PHA-L and HRP labeled trigemino-thalamic neurons were seen in the JX and also in the Vpdm. Then, electron microscopic (EM) study was done, injections of kainic acid into the Vodm and HRP into the contralateral thalamus was performed simultaneously. After EM embedding, the JX and Vpdm regions were selected, ultrathin sections were cut and observed with EM. In both areas, axo-somatic and axo-dendritic synapses were seen between degenerated boutons and HRP labeled somata or dendrites. Namely, the Vodm projecting terminals synapsed on trigemino-thalamic neurons in the JX and Vpdm. Anyway, axo-dendritic synapses was the main type of observed synapses. Thus, the present work demonstrated 1. the JX containing a group of trigemno-thalamic neurons was a target of special projections froin the Vodm; 2. The Vodm neurons projected to the contralateral thalamus through the relay of JX and Vpdm neurons.     

Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat

Trigeminal primary afferents expressing P2X(3) receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X(3)-immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X(3) immunoreactivity was mainly in small and medium-sized somata, but also frequently in large somata. Although most P2X(3) (+) somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X(3) (+) fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X(3) immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X(3) (+) terminals contained numerous clear, round vesicles and sparse large, dense-core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X(3) (+) terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X(3) (+) afferents to all TBSN and that it may be processed differently in different TBSN.     

GABAergic neural elements in the rat basilar pons: electron microscopic immunochemistry

Previous light microscopic immunoperoxidase studies of glutamic acid decarboxylase (GAD)-immunoreactive neural elements in the rat basilar pontine nuclei revealed immunocytochemical reaction product in neuronal somata and axon terminals. In the present study, pre-embedding immunoperoxidase labeling of GAD or gamma-aminobutyric acid (GABA) and postembedding immunogold labeling of GABA allowed the ultrastructural visualization of these neural elements in the basilar pontine nuclei of colchicine-treated animals. At the electron microscopic level, immunolabeled neuronal somata exhibited smoothly contoured nuclei, whereas some dendrites also contained reaction product after immunocytochemical treatment and were postsynaptic to both immunoreactive and nonimmunoreactive axon terminals. Synaptic boutons immunoreactive for GAD or GABA exhibited cross-sectional areas that ranged from 0.1 to 3.8 microns 2 and generally appeared round or elongated in most sections. The majority (95%) of immunolabeled boutons contained pleomorphic synaptic vesicles and formed symmetric synapses at their postsynaptic loci; however, boutons exhibiting round vesicles and boutons forming asymmetric synapses (5%) were also immunopositive. Small (less than 1.5 microns 2) GAD- or GABA-labeled axon terminals formed synaptic contact mainly with small dendritic profiles, dendritic spines, and neuronal somata, whereas large labeled boutons (greater than 1.5 microns 2) formed synapses with all sizes of dendritic profiles. Occasionally, a single immunolabeled bouton formed synaptic contact with two separate postsynaptic dendrites. It is suggested that the immunolabeled neuronal somata and dendrites observed in the rat basilar pontine nuclei represent a population of pontine local circuit neurons; however, it is known that GABAergic cell groups extrinsic to the pontine gray provide afferent projections to the basilar pons, and therefore at least some immunoreactive axon terminals present in the pontine nuclei are derived from these extrinsic sources. The ultrastructural observation of GABAergic neural elements in the rat basilar pontine nuclei confirms previous light microscopic findings and provides an anatomical substrate through which GABAergic neurons, whether arising from an intrinsic or extrinsic source, might exert an inhibitory influence on target cells within the pontine nuclei.     

Ultrastructural observations of synaptic connections of vibrissa afferent terminals in cat principal sensory nucleus and morphometry of related synaptic elements

Previous work suggests that slowly adapting (SA) periodontal afferents have different synaptic arrangements in the principal (Vp) and oral trigeminal nuclei and that the synaptic structure associated with transmitter release may be related directly to bouton size. The present study examined the ultrastructures of SA and fast adapting (FA) vibrissa afferents and their associated unlabeled axonal endings in the cat Vp by using intra-axonal labeling with horseradish peroxidase and a morphometric analysis. All SA and FA afferent boutons contained clear, round, synaptic vesicles. All the FA and most SA boutons were presynaptic to dendrites, but a few SA boutons were axosomatic. Both types of bouton were frequently postsynaptic to unlabeled axonal ending(s) containing pleomorphic, synaptic vesicles (P-ending). The size of labeled boutons was larger in FA than SA afferents, but the size of dendrites postsynaptic to labeled boutons was larger for SA than FA afferents. Large-sized FA and SA boutons made synaptic contacts with small-diameter dendrites. The size of FA and SA boutons was larger than that of their associated P-endings. A morphometric analysis made on the pooled data of SA and FA boutons indicated that apposed surface area, active zone number, total active zone area, vesicle number, and mitochondrial volume were highly correlated in a positive linear manner with labeled bouton volume. These relationships were also applicable to unlabeled P-endings, but the range of each parameter was smaller than that of the labeled boutons. These observations provide evidence that the two functionally distinct types of vibrissa afferent manifest unique differences but share certain structural features in the synaptic organization and that the ultrastructural “size principle” proposed by Pierce and Mendell ([1993] J. Neurosci. 13:4748–4763) for Ia-motoneuron synapses is applicable to the somatosensory system. J. Comp. Neurol. 389:12–33, 1997. © 1997 Wiley-Liss, Inc.     

Ultrastructural features of synapse from dorsal parvocellular reticular formation neurons to hypoglossal motoneurons of the rat

The dorsal parvocellular reticular formation (PCRt) receives projection of the trigeminal mesencephalic nucleus neurons. It contains the dorsal group of interneurons that integrate and coordinate activity of the oral motor nuclei. Ultrastructural features of synaptic connection from the dorsal PCRt neurons to the motoneurons of the hypoglossal nucleus (XII) were examined at both the light and electron microscopic levels in rats. Biotinylated dextran amine (BDA) was initially iontophoresed into the dorsal part of PCRt unilaterally. Seven days later horseradish peroxidase (HRP) was injected into the body of the tongue. After histochemical reaction for visualization of HRP and BDA, the BDA-labeled fibers and terminals were seen distributing bilaterally in XII with ipsilateral predominance. BDA-labeled terminals were closely apposed upon HRP retrogradely labeled somata and dendrites of the XII motoneurons. A total of 1408 BDA-labeled boutons were examined ultrastructurally, which had mean size of 1.22+/-0.37 microm in diameter. Five hundred-ninety three of these boutons in both the ipsilateral (n=401) and contralateral (n=192) XII were seen to synapse on both the dendrites and somata of HRP-labeled motoneurons. The vast majorities of synapses were axodendritic (98%, 580/593), while 2% of them were axosomatic. Of the 1408 BDA-labeled boutons, 69.6% of them were S-type boutons containing small clear and spherical synaptic vesicles and 30.4% of them were PF-type boutons containing pleomorphic and flattened synaptic vesicles. Approximately 64% of synapses between BDA-labeled boutons and HRP-labeled motoneurons were asymmetric, and 33% of synapses were symmetric. No axoaxodendritic or axoaxosomatic synaptic triad was observed. The present study illustrated the anatomical pathway and synaptological characteristics of neuronal connection between the dorsal PCRt premotor neurons and the XII motoneurons. Its functional significance in coordinating activity of XII motoneurons during oral motor behaviors has been discussed.     

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.     

Ultrastructural analysis of glutamate‐, GABA‐, and glycine‐immunopositive boutons from supratrigeminal premotoneurons in the rat trigeminal motor nucleus

The supratrigeminal region (Vsup) is important for coordination of smooth jaw movement. However, little is known about the synaptic connections of the Vsup premotoneurons with the trigeminal motor neurons. In the present study, we examined axon terminals of Vsup premotoneurons in the contralateral trigeminal motor nucleus (Vmo) by a combination of anterograde tracing with cholera toxin B–horseradish peroxidase (CTB‐HRP), postembedding immunohistochemistry for the amino acid transmitters glutamate, GABA, and glycine, and electron microscopy. Tracer injections resulted in anterograde labeling of axon terminals of the Vsup premotoneurons in the motor trigeminal nucleus (Vmo). The labeled boutons in Vmo exhibited immunoreactivity for glutamate, GABA, or glycine: glutamate‐immunopositive boutons (69%) were more frequently observed than GABA‐ or glycine‐immunopositive boutons (19% and 12%, respectively). Although most labeled boutons (97%) made synaptic contacts with a single postsynaptic dendrite, a few glutamate‐immunopositive boutons (3%) showed synaptic contact with two dendrites. No labeled boutons participated in axoaxonic synaptic contacts. Most labeled boutons (78%) were presynaptic to dendritic shafts, and the remaining 22% were presynaptic to somata or primary dendrites. A large proportion of GABA‐ or glycine‐immunopositive boutons (40%) were presynaptic to somata or primary dendrites, whereas most glutamate‐immunopositive boutons (86%) were presynaptic to dendritic shafts. These results indicate that axon terminals of Vsup premotoneurons show simple synaptic connection with Vmo neurons. This may provide the anatomical basis for the neural information processing responsible for jaw movement control. © 2008 Wiley‐Liss, Inc.     

Central terminations of low-threshold mechanoreceptive afferents in the trigeminal nuclei interpolaris and caudalis of the cat

Previous studies indicate that vibrissa, nonvibrissa, guard hair, hairy skin, and periodontal ligament afferents give rise to morphologically distinct terminal arbors in the trigeminal nuclei principalis (Vp) and oralis (Vo) in the cat. The present study describes the extent to which morphological and functional relationships exist in the nuclei interpolaris (Vi) and caudalis (Vc). Twenty-two fibers were physiologically characterized and stained by intra-axonal HRP injection techniques. The fast adapting (FA) vibrissa (VF) afferents gave rise to compact and circumscribed arbors in Vj and Vc. These tended to be larger in Vc than in Vi. The slowly adapting (SA) vibrissa (VS) afferents in Vi and Vc had more widespread and larger arbors than those of the VF afferents. The VS arbors in Vc tended to be larger and less circular than those in Vi. Guard hair (GH) afferents gave rise to circumscribed arbors in both nuclei, but those in Vc tended to have larger and more circular arbors than those in Vi. Down hair (DH) afferents gave rise to small, circumscribed arbors or a few distinct patches of boutons within a small area in Vi; arbors in Vc were less extensive and “stringy.” Unlike other afferents, DH arbors were larger in Vi than in Vc, but smaller than those of GH afferents in either nuclei. The SA hairy skin (SS) afferents had arbors that were widespread with a few distinct patches of boutons; the arbors in Vc were larger than those in Vi. The arbors of SS afferents were smaller than those of VS and GH afferents in both nuclei. Like GH afferents, FA periodontal ligament (PF) afferents gave rise to large and circumscribed arbors in Vi, although the arbors in Vc were smaller and less dense. The present study demonstrated significant functional and morphological relationships for primary afferents in Vi and Vc, thus suggesting that sensory information from each of the distinct fiber or functional classes is processed in a characteristic manner in the individual nuclei. © Wiley-Liss, Inc.     

Parvalbumin immunoreactivity in the thalamus of guinea pig: Light and electron microscopic correlation with gamma-aminobutyric acid immunoreactivity

The relationship of the calcium binding protein parvalbumin (PV) with gamma-aminobutyric acidergic (GABAergic) neurons differs within different thalamic nuclei and animal species. In this study, the distribution of PV and GABA throughout the thalamus of the guinea pig was investigated at the light microscopic level by using immunoperoxidase methods. Intense PV labelling was found in all the GABAergic neurons of the reticular nucleus and in scattered GABAergic neurons in the anteroventral nucleus, whereas GABAergic interneurons in the ventrobasal and lateral geniculate nuclei were not PV labelled. At the electron microscopic level, preembedding immunuperoxidase for PV was combined with postembedding immunogold for GABA. In the ventrobasal nucleus, four types of profiles were recognized: 1) terminals with flattened vesicles and forming symmetric synapses, which were labelled with both PV and GABA and could therefore be identified as afferents from the reticular nucleus; 2) boutons morphologically similar to presynaptic dendrites of interneurons, labelled only with GABA; 3) large terminals with round vesicles and asymmetric synapses, labelled only with PV, which contacted GABAergic presynaptic dendrites in glomerular arrangements and resembled ascending excitatory afferents; and 4) terminals unlabelled by either antiserum. In the ventrobasal nucleus of the guinea pig a double immunocytochemical labelling permits therefore the differentiation of two populations of GABAergic vesicle-containing profiles, i. e., the terminals originating from reticular nucleus (that are double labelled) and the presynaptic dendrites originating from interneurons (that are GABA-labelled only). The possibility to differentiate GABAergic inputs from the reticular nucleus and from interneurons can shed light to the functional interpretation of synaptic circuits in thalamic sensory nuclei. © 1994 Wiley-Liss, Inc.     

Pallidal afferent territory of the Macaca mulatta thalamus: Neuronal and synaptic organization of the VAdc

Ventral anterior thalamic nucleus pars densicellularis (VAdc) as delineated earlier (Ilinsky and Kultas-Ilinsky [1987] J. Comp. Neurol. 262:331–364) was analyzed by using qualitative and quantitative neuroanatomical techniques. Projection neurons (PN), retrogradely labeled with wheat germ agglutinin conjugated horseradish peroxidase from the cortex, were small to medium in size (mean area, 312 μm²) with numerous primary dendrites displaying a tufted branching pattern. Local circuit neurons (LCN), immunoreactive for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase, were small (mean area, 110 μm²), and gave off few dendrites. Two subpopulations of GABA positive boutons (F1 type) were distinguished: large (mean area, 2.6 μm²) terminals with symmetric synapses containing few pleomorphic vesicles and numerous mitochondria densely covered proximal PN sites; smaller F1 boutons with a slightly different morphology contacted mostly distal PN dendrites. Two subpopulations of terminals containing round vesicles and forming asymmetric synapses were distinguished by bouton size (mean areas, 0.4 μm² and 1.6 μm², respectively). These targeted mainly distal PN dendrites, but some synapsed proximally next to large F1 boutons. On distal dendrites, representatives of both types were labeled from the cortex. The density of boutons with symmetric and asymmetric synapses (the number of boutons per 100 μm of PN membrane length) was 3.3:0.2 on primary, 2.5:1.2 on secondary, and 0.8:12 on distal dendrites. The numerical density of synapses formed by presynaptic LCN dendrites on all PN levels was 20 to 40 times less than that of axon terminals at the same sites. Afferent input to LCN from boutons of all types, including that from 50% of labeled cortical boutons, mainly targeted distal dendrites. Overall, the findings suggest that PN in VAdc receive massive inhibitory input proximally intermingled with some presumably excitatory input, and that LCN contribution to PN inhibition is modest. J. Comp. Neurol. 386:573–600, 1997. © 1997 Wiley-Liss, Inc.