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.     

Morphology and synaptic connections of myelinated primary axons in the ventrolateral region of rat trigeminal nucleus oralis

Neurons in the ventrolateral (VL) subdivision of rat trigeminal nucleus oralis (Vo) have most of their dendritic arbors confined within this region. This study examines the morphology and synaptic connections of a population of myelinated primary trigeminal axons that arborize within VL and are in a position to provide input directly to VL neurons. Primary axons were visualized for light and electron microscopic analysis by injecting 30% horseradish peroxidase (HRP) in 2% dimethylsulfoxide (DMSO) into the sensory root of the trigeminal nerve and allowing 24-36 hours for the anterograde transport of HRP into the terminal axonal arbors. This population is characterized by its cone-shaped terminal arbors, which generate many axonal endings (2-8 micron in diameter) along unmyelinated terminal strands. These arbors arise from collaterals emanating from thinly myelinated (2-5 micron in diameter) parent branches descending in the spinal V tract, which, on the basis of their size, are considered to be small myelinated (A sigma) primary trigeminal axons. HRP-labeled P endings belonging to this population of primary axons are scalloped, filled with spherical to ovoid (40-70 nm in diameter) synaptic vesicles, and lie centrally in glomeruli where they make asymmetrical axodendritic synapses on dendritic shafts and spine heads. It is at these synapses that this population of primary trigeminal axons is probably transferring its input directly to the dendritic arbors of VL neurons. The dendritic shafts and spine heads also receive symmetrical to intermediate axodendritic synapses from endings containing flattened (70 X 29 nm) synaptic vesicles. These terminals also establish axo-axonic synapses on the P ending. Other synaptic components found less often in the glomeruli include small terminals containing oval (14-23 nm) synaptic vesicles that establish symmetrical to intermediate synapses on the P ending, boutons containing pleomorphic (35-80 nm) synaptic vesicles that form symmetrical to intermediate synapses on the P ending as well as on dendritic shafts, and small peripheral endings containing round (20-40 nm) synaptic vesicles that establish asymmetrical synapses on dendritic shafts.     

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.     

The fine structure of the lateral superior olivary nucleus of the cat

The synaptic organization of the lateral superior olivary nucleus of the cat was analyzed under the electron microscope. The predominant cell type, the fusiform cell, has dendrites that extend from opposite poles of the cell body toward the margins of the nucleus, where they terminate in spinous branches. The fusiform cells are contacted by three types of synaptic terminals that can be distinguished by the size and shape of their synaptic vesicles. The somatic and proximal dendritic surfaces are apposed by synaptic terminals containing small, flat synaptic vesicles. Further from the cell body, the dendrites form numerous synaptic contacts with terminals containing large round vesicles as well as with the terminals containing small, flat vesicles. The most distal dendritic branches and their spiny appendages appear to form synapses almost exclusively with the terminals with large, round vesicles. A relatively rare type of terminal that contains small, round vesicles may form synapses with either the somatic or dendritic surfaces. A few small cells are interspersed among the fusiform cells, but they are more commonly located around the margins of the nucleus. The small cells form few axosomatic contacts. The simplest interpretation of the findings is that the terminals with small, flat vesicles arise in the medial nucleus of the trapezoid body and are inhibitory in function, whereas the terminals with large, round vesicles arise in the anteroventral cochlear nucleus and are excitatory; however, this remains to be demonstrated experimentally. In any case, the differential distribution of these two types of inputs on the somatic and dendritic surfaces must be an important determinant of the physiological response properties of the fusiform cells to binaural acoustic stimuli.     

Dopamine-immunoreactivity in the rat mesencephalic trigeminal nucleus: an ultrastructural analysis

The ultrastructure and distribution of dopaminergic boutons within the rat mesencephalic trigeminal (Me5) nucleus was examined with the use of electronmicroscopic immunocytochemistry. A total of 5102 boutons, comprising axosomatic and axodendritic synaptic terminals as well as non-synaptic boutons (or varicosities), located in the ventrocaudal portion of Me5 was analysed. Approximately 20% of these boutons were dopamine-immunoreactive. Morphological analysis showed that the dopaminergic synaptic terminals, axodendritic as well as axosomatic, were exclusively of the S- and G-bouton type; they contained, respectively, small spherical vesicles or small pleomorphic vesicles in combination with large granular dense-cored vesicles. All dopaminergic varicosities in the Me5 were of the G-bouton type. Quantitative analysis revealed that most of the dopaminergic synaptic terminals in the Me5 nucleus contacted dendrites, while only a minority (12%) contacted Me5 somata. This dopaminergic somatic input comprised about half (52%) of the total axosomatic input on Me5 neurons. The present results and previous findings with respect to the prominent serotonergic component of the axosomatic input to Me5 neurons indicate that dopamine and serotonin account for most of the axosomatic input in the ventrocaudal part of the Me5 nucleus. In fact, the present results seem to support previous observations regarding the existence of a population of afferent neurons in which dopamine and serotonin are colocalized.     

Electron microscopic analysis of the mesencephalic ventromedial tegmentum in the cat

We have studied the normal ultrastructure of the ventral mesencephalic tegmentum (VMT) in the cat, particularly the morphology and distribution of presynaptic terminals and the types of synaptic junctions. The following subnuclei of the region were examined: n. linearis rostralis (LR), n. paranigralis (PN), and n. interfascicularis (IF). The qualitative and quantitative data revealed significant ultrastructural differences between these subnuclei. Each subnucleus had a characteristic dendritic structure. In LR the dendrites were nonspinous and cylindrical and had presynaptic terminals randomly distributed over their surface. In PN we observed varicose dendrites with spines; the presynaptic terminals formed clusters on the narrow segments of the dendrites and around the spines. Dendrodendritic synapses were also observed in this nucleus. In IF, there was an internal division regarding dendritic structure: in the rostral part of the nucleus there were cylindrical dendrites while in the caudal part irregularly shaped dendrites bearing long spines were found. In IF and LR some of the cylindrical dendrites were seen to be in direct contact with the basal lamina of blood vessels. Four types of presynaptic terminals were distinguished by the morphology of their vesicles, and the proportion of each type in the total terminal population was determined. On this basis the compositions of the presynaptic terminal population in the three subnuclei were found to be very similar. Most terminals contained clear, round vesicles (62.6%), or both clear and dense-cored vesicles (35.1%). Few terminals were seen with dense-cored vesicles only (1.4%) or with pleomorphic vesicles (0.9%). The majority of synapses in the VMT were found to have symmetrical densities. LR had twice as many asymmetrical synapses as the other two subnuclei. Eighty percent of the terminals formed synapses with dendrites, although axosomatic and axoaxonic synapses were also seen. The density of the terminals was significantly different for each subnucleus: 191/1,000 micrometers 2 in IF, 120/1,000 micrometers 2 in PN, and 81/1,000 micrometers 2 in LR. These data indicate that while the subnuclei of the VMT receive morphologically similar afferents, each has a unique way of processing the information provided by them, through a different internal circuitry.     

Ultrastructural and neurochemical analysis of synaptic input to trigemino-thalamic projection neurones in lamina I of the rat: a combined immunocytochemical and retrograde labelling study

The synaptology of lamina I thalamic projection neurones in the spinal trigeminal nucleus of the rat was investigated by combining electron microscopic immunocytochemistry with the retrograde transport of horseradish peroxidase. Fifteen retrogradely labelled neurones were serially sectioned and their dendrites were traced for up to 160 microns in order to characterise the synaptic input to their cell bodies and proximal dendrites. Projection neurones receive synapses from dome-shaped substance P and enkephalin immunoreactive terminals, which make simple axosomatic or axodendritic synapses. In addition, the cells receive synapses from numerous nonimmunoreactive terminals including a wide range of different dome-shaped terminals and various scalloped or glomerular terminals. Dome-shaped terminals synapse with small stubby spines in addition to cell bodies or dendritic shafts and they are probably derived from lamina II interneurones and from descending bulbospinal pathways. Glomerular terminals occur in two main classes: small type A terminals with dark axoplasm and larger type B terminals. Type B terminals participate in synaptic triads in which a peripheral terminal synapses both axoaxonically with the glomerular terminal and axodendritically with the projection neurone. Type A and type B terminals closely resemble the central terminals of spinal cord lamina II glomeruli and are probably derived from C and A delta I degrees afferent fibers. The results indicate that lamina I projection neurones are under pre- and postsynaptic control from diverse sources. Their complex synaptic organisation highlights the key role that such cells play in the rostrad transmission of somatosensory information.     

Termination in trigeminal nucleus oralis of ascending intratrigeminal axons originating from neurons in the medullary dorsal horn: an HRP study in the rat employing light and electron microscopy

The anterograde horseradish peroxidase (HRP) technique was used to identify ascending intratrigeminal axons originating from neurons in the medullary dorsal horn (MDH) which terminate in trigeminal nucleus oralis (Vo). HRP injections into the MDH labeled two populations of axons ascending ipsilaterally within the spinal trigeminal nucleus. The first population was composed of parent branches which each gave off a single branching collateral strand to Vo as they ascended. These collaterals were characterized by boutons filled with small, round synaptic vesicles and forming asymmetrical synaptic contacts with large diameter dendritic shafts. The second axonal population was made up of parent branches which terminated directly in Vo. Their short terminal strands were distinguished by axonal endings containing pleomorphic synaptic vesicles and forming symmetrical synaptic junctions with small diameter dendritic shafts and spines.     

Immunocytoehemical localization of substance P in the spinal trigeminal nucleus of the rat: A light and electron microscopic study

The neuropeptide substance P is a transmitter candidate for certain primary afferent fibers which terminate in the substantia gelatinosa. In this study the light and electron microscopic localization of substance P in the substantia gelatinosa of the spinal trigeminal nucleus of the rat has been studied using immunocytochemical procedures. Substance P immunoreactive fibers were observed mainly in lamina I and outer lamina II. Ultrastructural analysis revealed immunoreactivity in unmyelinated fibers and in axon terminals which contained agranular spherical vesicles and large dense-cored vesicles and which made predominantly simple asymmetric axodendritic synaptic contacts. Immunoreactive terminals only rarely formed the central terminal of synaptic glomeruli and in only one example was a stained terminal possibly postsynaptic to an unstained terminal. The majority of synapses were onto small dendrites in outer lamina II and in some cases these dendrites were themselves presynaptic to other dendrites. Immunoreactive terminals also synapsed with the soma and proximal dendrites of large neurons on the border of laminae I and II. The results show that there are at least two distinct targets for substance P immunoreactive terminals in the substantia gelatinosa, namely the large lamina I neurons and lamina II probable intemeurons. Some of the former may be projection neurons while some of the latter may correspond to the inhibitory islet cells described by Gobel and colleagues in the cat. In addition the results indicate that few substance P immunoreactive terminals receive axoaxonic synapses and emphasize instead the role of postsynaptic interactions. In particular the results suggest several sites at which substance P might interact postsynaptically with the neuropeptide enkephalin.     

Axonal endings terminating on dendrites of identified large trigeminospinal projection neurons in rat trigeminal nucleus oralis

The retrograde horseradish peroxidase technique was used to: (1) identify and assess the overall morphology of large neurons in the ventrolateral portion (VL) of rat trigeminal nucleus oralis projecting to cervical, thoracic and lumbosacral levels of the spinal cord; and (2) characterize the synaptic endings terminating on their dendrites. The morphology of large VL neurons projecting to all spinal levels is similar. They have 25–50 μm pyramidal-shaped somata which emit 3–6 primary dendrites. These primary dendrites give rise to spherical to elliptical-shaped dendritic arbors measuring up to 700 μm in diameter. Labeled axons enter either a deep axon bundle or the medial portion of the spinal V tract. Dendrites of labeled neurons are contacted by axonal endings of 3 types. The most numerous endings are filled with clear, spherical synaptic vesicles and usually form a single asymmetrical contacts along the entire length of dendritic shafts. Synapsing less frequently on dendritic shafts are endings containing pleomorphic synaptic vesicles and forming single symmetrical synaptic contacts. The least frequently encountered synaptic terminal contains flattened synaptic vesicles and makes a single symmetrical synaptic contact with a dendritic shaft.     

Trigeminal projections to hypoglossal and facial motor nuclei in the rat

This study was undertaken to identify the trigeminal nuclear regions connected to the hypoglossal (XII) and facial (VII) motor nuclei in rats. Anterogradely transported tracers (biotinylated dextran amine, biocytin) were injected into the various subdivisions of the sensory trigeminal complex, and labeled fibers and terminals were searched for in the XII and VII. In a second series of experiments, injections of retrogradely transported tracers (biotinylated dextran amine, gold-horseradish peroxidase complex, fluoro-red, fluoro-green) were made into the XII and the VII, and labeled cells were searched for in the principal sensory trigeminal nucleus, and in the pars oralis, interpolaris, and caudalis of the spinal trigeminal nucleus. Trigeminohypoglossal projections were distributed throughout the ventral and dorsal region of the XII. Neurons projecting to the XII were found in all subdivisions of the sensory trigeminal complex with the greatest concentration in the dorsal part of each spinal subnucleus and exclusively in the dorsal part of the principal nucleus. Trigeminofacial projections reached all subdivisions of the VII, with a gradual decreasing density from lateral to medial cell groups. They mainly originated from the ventral part of the principal nucleus. In the spinal nucleus, most of the neurons projecting to the VII were in the dorsal part of the nucleus, but some were also found in its central and ventral parts. By using retrograde double labeling after injections of different tracers in the XII and VII on the same side, we examined whether neurons in the trigeminal complex project to both motor nuclei. These experiments demonstrate that in the spinal trigeminal nucleus, neurons located in the pars caudalis and pars interpolaris project by axon collaterals to XII and VII. J. Comp. Neurol. 415:91–104, 1999. © 1999 Wiley-Liss, Inc.     

Distribution of gastrin‐releasing peptide in the rat trigeminal and spinal somatosensory systems

Gastrin‐releasing peptide (GRP) has recently been identified as an itch‐specific neuropeptide in the spinal sensory system in mice, but there are no reports of the expression and distribution of GRP in the trigeminal sensory system in mammals. We characterized and compared GRP‐immunoreactive (ir) neurons in the trigeminal ganglion (TG) with those in the rat spinal dorsal root ganglion (DRG). GRP immunoreactivity was expressed in 12% of TG and 6% of DRG neurons and was restricted to the small‐ and medium‐sized type cells. In both the TG and DRG, many GRP‐ir neurons also expressed substance P and calcitonin gene‐related peptide, but not isolectin B₄. The different proportions of GRP and transient receptor potential vanilloid 1 double‐positive neurons in the TG and DRG imply that itch sensations via the TG and DRG pathways are transmitted through distinct mechanisms. The distribution of the axon terminals of GRP‐ir primary afferents and their synaptic connectivity with the rat trigeminal sensory nuclei and spinal dorsal horn were investigated by using light and electron microscopic histochemistry. Although GRP‐ir fibers were rarely observed in the trigeminal sensory nucleus principalis, oralis, and interpolaris, they were predominant in the superficial layers of the trigeminal sensory nucleus caudalis (Vc), similar to the spinal dorsal horn. Ultrastructural analysis revealed that GRP‐ir terminals contained clear microvesicles and large dense‐cored vesicles, and formed asymmetric synaptic contacts with a few dendrites in the Vc and spinal dorsal horn. These results suggest that GRP‐dependent orofacial and spinal pruriceptive inputs are processed mainly in the superficial laminae of the Vc and spinal dorsal horn. J. Comp. Neurol. 522:1858–1873, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

Reorganization of corticorubral synapses following cross-innervation of flexor and extensor nerves of adult cat: a quantitative electron microscopic study

A quantitative electron microscopic study of corticorubral synapses was performed in the red nucleus (RN) of adult cats to determine the morphological correlates for the changes in time course of corticorubral excitatory post-synaptic potentials, which occur following cross-innervation of forelimb extensor and flexor nerves. Corticorubral synaptic endings were identified by anterograde degeneration after lesions of the ipsilateral sensorimotor cortex. Rubrospinal neurons innervating upper spinal segments were electrophysiologically identified and filled with horseradish peroxidase (HRP). These cells were mainly situated in the dorsomedial part of RN. Electron micrographs of the degenerating corticorubral synaptic endings were taken in the region surrounding HRP-filled neurons and the diameter of the dendrites contacted by such terminals was measured.In the cross-innervated animals many degenerating terminals were found to synapse on dendrites with large diameter and the somata of neurons in RN. This is in contrast to the previous observations in normal cats, in which very few corticorubral synapses were found to synapse on proximal dendrites and somata of RN neurons. The diameter of HRP-filled neurons in cats which were cross-innervated was slightly smaller than those observed in normal animals. These results indicate that new corticorubral synapses were formed on proximal dendrites and somata of RN neurons as a consequence of cross-innervation.     

The central projections of tooth pulp afferent neurons in the rat as determined by the transganglionic transport of horseradish peroxidase

Transganglionic transport of horseradish peroxidase (HRP) or horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) was used to map in detail the central projections of trigeminal primary afferent neurons that innervate the dental pulp organ of the rat. In each of ten animals, 0.5-2.0 microliters of enzyme solution was injected into the pulp chamber of the first maxillary molar tooth. Postmortem examination of the decalcified teeth in all cases showed that the HRP/HRP-WGA remained confined to the pulp chamber and pulp roots, with no spread of enzyme into periapical tissues. HRP-labeled tooth pulp afferent fibers projected to all four rostrocaudal subdivisions of the ipsilateral trigeminal brainstem nuclear complex (TBNC) and to the upper cervical spinal cord. The labeled terminal fields formed a column that stretched relatively uninterrupted from just caudal to the rostromedial tip of the trigeminal principal sensory nucleus to at least the C2 segment of the spinal cord. The density of the afferent projection varied markedly from one rostrocaudal level of the TBNC to the next but was heaviest in an area encompassing the caudal one-half of the principal sensory nucleus and the rostral two-thirds of pars oralis. Fibers projected only lightly to pars caudalis, where they terminated preferentially in laminae I, IIa, and the junctional zone between laminae IV and V. HRP-labeled terminals in C1 and C2 were located almost exclusively in laminae I. In the dorsoventral axis, the terminal fields in the TBNC were located in a surprisingly dorsal part of the complex, well within what has been shown by others to be largely an area of termination for mandibular division fibers. Most fibers ended in medial parts of the TBNC, with the exception of two modestly labeled terminal fields located in the lateral aspects of rostral pars oralis and rostral pars caudalis. No labeled fibers terminated in the contralateral TBNC or contralateral cervical spinal cord.     

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.     

Ultrastructural study of cholinergic and noncholinergic neurons in the pars compacta of the rat pedunculopontine tegmental nucleus

A group of medium-to-large cholinergic neurons situated in the dorsolateral mesopontine tegmentum comprises the pedunculopontine tegmental nucleus (PPT). The PPT pars compacta (PPT-pc), which occupies the lateral part of the caudal two-thirds of the nucleus, contains a dense aggregation of cholinergic neurons. In the present study, we have employed immunohistochemistry for choline acetyltransferase (ChAT) and electron microscopy to investigate the ultrastructure and synaptic organization of neuronal elements in the PPT-pc. Our results demonstrate that: (1) ChAT-immunoreactive (i.e., cholinergic) PPT-pc neurons are characterized by abundant cytoplasm and organelles, and have few axosomatic synapses (both asymmetric and symmetric); (2) ChAT-immunoreactive dendrites comprise 6-15% of total dendritic elements in the neuropil; the mean percentage of dendritic membrane covered by synaptic terminals is approximately 15%, and nearly all synapses with ChAT-immunoreactive dendrites are asymmetric; (3) within the boundaries described by cholinergic PPT-pc, there are noncholinergic neurons which, in contrast, exhibit a lucent cytoplasm and a higher frequency of axosomatic synapses (10.5% versus 3.7% for cholinergic neurons); and (4) noncholinergic neurons are morphologically heterogeneous with one subpopulation exhibiting a mean diameter that approximates that of cholinergic cells (i.e., >15 μm and <20 μm) and a very high frequency of axosomatic synapses (>20%). Only 0.2-0.7% of terminal elements in the neuropil were ChAT-immunoreactive and these were not observed to synapse with cholinergic dendrites or somata. This relative paucity of terminal labeling and lack of cholinergic-cholinergic interactions seems inconsistent with the recognized and prominent physiological actions of acetylcholine on cholinergic PPT-pc neurons, and suggests a methodological limitation and/or a potential paracrine-like action of nonsynaptically released acetylcholine in the PPT region. J. Comp. Neurol. 382:285-301, 1997. © 1997 Wiley-Liss, Inc.     

Expression of transient receptor potential ankyrin 1 (TRPA1) in the rat trigeminal sensory afferents and spinal dorsal horn

Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold and pungent compounds, is implicated in the mediation of nociception, but little is known about the processing of the TRPA1‐mediated nociceptive information within the trigeminal sensory nuclei (TSN) and the spinal dorsal horn (DH). To address this issue, we characterized the TRPA1‐positive (+) neurons in the trigeminal ganglion (TG) and investigated the distribution of TRPA1⁺ afferent fibers and their synaptic connectivity within the rat TSN and DH by using light and electron microscopic immunohistochemistry. In the TG, TRPA1 was expressed in unmyelinated and small myelinated axons and also occasionally in large myelinated axons. Many TRPA1⁺ neurons costained for the marker for peptidergic neurons substance P (26.8%) or the marker for nonpeptidergic neurons IB4 (44.5%). In the CNS, small numbers of axons and terminals were immunopositive for TRPA1 throughout the rostral TSN, in contrast to the dense network of positive fibers and terminals in the superficial laminae of the trigeminal caudal nucleus (Vc) and DH. The TRPA1⁺ terminals contained clear round vesicles, were presynaptic to one or two dendrites, and rarely participated in axoaxonic contacts, suggesting involvement in relatively simple synaptic circuitry with a small degree of synaptic divergence and little presynaptic modulation. Immunoreactivity for TRPA1 was also occasionally observed in postsynaptic dendrites. These results suggest that TRPA1‐dependent orofacial and spinal nociceptive input is processed mainly in the superficial laminae of the Vc and DH in a specific manner and may be processed differently between the rostral TSN and Vc. J. Comp. Neurol. 518:687–698, 2010. © 2009 Wiley‐Liss, Inc.     

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

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

An electron microscopic description of glutamate-like immunoreactive axon terminals in the rat principal sensory and spinal trigeminal nuclei.

The spinal and principal sensory trigeminal nuclei relay noxious and nonnoxious stimuli from the orofacial region to the thalamus. Physiological studies have implicated glutamate as an important neurotransmitter in this region. Despite its importance as a potential transmitter, few studies have examined the anatomical distribution of glutamate within these nuclei. We therefore chose to use a monoclonal antibody raised against glutamate conjugated to a carrier protein to identify and describe glutamate-like immunoreactive processes at the electron microscopic level. Glutamate-like immunoreactive axon terminals were identified throughout the spinal trigeminal and principal sensory trigeminal nucleus. In subnucleus caudalis glutamate-like immunoreactive terminals occurred frequently in all laminae and were morphologically heterogeneous. In lamina I, glutamate-like immunoreactive terminals were primarily ovoid, contained spherical synaptic vesicles, and participated in synaptic complexes with both dendritic and axonal profiles. In laminae II and III many glutamate-like immunoreactive axon terminals were identified as the central element in synaptic glomeruli. Within discrete patches of lamina II, large numbers of glutamate-like immunoreactive terminals contained dense core vesicles. The majority of glutamate-like immunoreactive terminals in subnucleus interpolaris, subnucleus oralis, and principal sensory trigeminal nucleus were similar in morphology and synaptic interaction to the glutamate-like immunoreactive terminals found in subnucleus caudalis. Glutamate-like immunoreactive terminals that were the central presynaptic element in glomerular complexes were seen in all subnuclei. In sections from subnucleus interpolaris and subnucleus oralis central glutamate-like immunoreactive terminations within glomerular complexes had much smoother profiles, and in subnucleus interpolaris participated primarily in axodendritic synaptic junctions. In the principal sensory trigeminal nucleus central glutamate-like immunoreactive terminations were highly scalloped and participated in numerous axoaxonic synaptic junctions. The above observations are consistent with the hypothesis that glutamate-like immunoreactivity is present in some primary afferent terminations and functions as an important excitatory transmitter involved in the relay of sensory information to the spinal trigeminal and principal sensory trigeminal nucleus.