Quantitative analysis of synaptic contacts made between functionally identified oralis neurons and trigeminal motoneurons in cats.

A previous study revealed that rostrodorsomedial oralis (Vo.r) neurons synapsing on trigeminal motoneurons use GABA and/or glycine as neurotransmitters. To determine the number and spatial distribution of contacts, injections of biotinamide and horseradish peroxidase were made into a Vo.r neuron and an alpha-motoneuron in the jaw-closing (JC) and jaw-opening (JO) motor nucleus, respectively, in 39 cats. All Vo.r neurons responded to low-threshold mechanical stimulation of the oral tissues. Single Vo.r neurons terminating in the JC nucleus (Vo.r-dl neurons; n = 5) issued, on average, 10 times more boutons than Vo.r neurons terminating in the JO nucleus (Vo.r-vm neurons; n = 5; 4437 vs 445). The Vo.r-dl neuron-JC alpha-motoneuron pairs (n = 4) made contacts on either the soma-dendritic compartment or dendrites, and the Vo.r-vm neuron-JO motoneuron pairs (n = 2) made contacts on dendrites, with a range of two to seven contacts. In five of the six pairs, individual or groups of two to three terminals contacted different dendritic branches of a postsynaptic cell. The Vo.r-dl neurons innervated a greater number of counter-stained motoneuronal somata than did the Vo.r-vm neurons (216 vs 26). Total number of contacts per Vo.r neuron was higher for the Vo.r-dl than Vo.r-vm neurons (786 vs 72). The present study demonstrates that axonal branches of Vo.r neurons are divided into two types with different innervation domains on the postsynaptic neuron and that they are highly divergent. The overall effect exerted by these neurons is predicted to be much greater within the JC than JO motoneuron pool.

The distribution of inhibitory and excitatory synapses on single, reconstructed jaw-opening motoneurons in the cat.

In a previous study, we reported that the distribution of inhibitory input, in contrast to excitatory input, decreased somatofugally along dendrites of cat jaw-closing alpha-motoneurons [J Comp Neurol 414 (1999) 454]. The present study examined the distribution of GABA, glycine, and glutamate immunopositive boutons covering horseradish peroxidase-labeled cat jaw-opening motoneurons. The motoneurons were divided into four compartments: the soma, and primary, intermediate, and distal dendrites. Ninety-seven percent of the total number of studied boutons had immunoreactivity for at least one of the three amino acids. The proportion of boutons immunoreactive for GABA and/or glycine was lower than the proportion of boutons immunoreactive for glutamate. Boutons immunoreactive to glycine alone were more numerous than boutons double-labeled for GABA and glycine, which, in turn, occurred more frequently than boutons immunoreactive to GABA alone. The percentage synaptic covering (proportion of membrane covered by synaptic boutons) of the putatively excitatory (glutamate containing) and putatively inhibitory (GABA and/or glycine containing) boutons decreased somatofugally along the dendrites. Such systematic variations were not seen in the packing density (number of boutons per 100 microm(2)); the packing density showed a distinct drop between the soma and primary dendrites but did not differ significantly among the three dendritic compartments. Overall, the packing density was slightly higher for the putatively excitatory boutons than for the inhibitory ones. When taken together with previous analyses of jaw-closing alpha-motoneurons the present data on jaw-opening alpha-motoneurons indicate that the two types of neuron differ in regard to the nature of synaptic integration in the dendritic tree.

The central projection of masticatory afferent fibers to the trigeminal sensory nuclear complex and upper cervical spinal cord.

Retrograde and anterograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) conjugate was used to study the organization of primary afferent neurons innervating the masticatory muscles. HRP applied to the nerves of jaw-closing muscles--the deep temporal (DT), masseter (Ma), and medial pterygoid (MP)--labeled cells in the trigeminal ganglion and the mesencephalic trigeminal nucleus (Vmes), whereas HRP applied to nerves of the jaw-opening muscles--anterior digastric (AD) and mylohyoid (My)--labeled cells only in the trigeminal ganglion. Cell bodies innervating the jaw-closing muscles were found with greater frequency in the intermediate region of the mandibular subdivision, while somata supplying the jaw-opening muscles were predominant posterolaterally. The distribution of their somatic sizes was unimodal and limited to a subpopulation of smaller cells. Projections of the muscle afferents of ganglionic origin to the trigeminal sensory nuclear complex (TSNC) were confined primarily to the caudal half of pars interpolaris (Vi), and the medullary and upper cervical dorsal horns. In the Vi, Ma, MP, AD, and My nerves terminated in the lateral-most part of the nucleus with an extensive overlap in projections, save for the DT nerve, which projected to the interstitial nucleus or paratrigeminal nucleus. In the medullary and upper cervical dorsal horns, the main terminal fields of individual branches were confined to laminae I/V, but the density of the terminals in lamina V was very sparse. The rostrocaudal extent of the terminal field in lamina I differed among the muscle afferents of origin, whereas in the mediolateral or dorsoventral axis, a remarkable overlap in projections was noted between or among muscle afferents. The terminals of DT afferents were most broadly extended from the rostral level of the pars caudalis to the C3 segment, whereas the MP nerve showed limited projection to the middle one-third of the pars caudalis. Terminal fields of the Ma, AD, and My nerves appeared in the caudal two-thirds of the pars caudalis including the first two cervical segments, the caudal half of the pars caudalis and the C1 segment, and in the caudal part of the pars caudalis including the rostral C1 segment, respectively. This rostrocaudal arrangement in the projections of muscle nerves, which corresponds to the anteroposterior length of the muscles and their positions, indicates that representation of the masticatory muscles in lamina I reflects an onion-skin organization. These results suggest that primary muscle afferent neurons of ganglionic origin primarily mediate muscle pain.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphology and synaptic connections of slowly adapting periodontal afferent terminals in the trigeminal subnuclei principalis and oralis of the cat.

Previous studies suggest that sensory information from primary afferent fibers is processed in a distinct manner in the individual subnuclei of trigeminal sensory nuclear complex. The present study has addressed this issue by using intra-axonal labeling with horseradish peroxidase to examine the ultrastructure and synaptic organization of axon terminals from slowly adapting (SA) periodontal afferents in the ventral subdivision (Vpv) of principalis and the rostro-dorsomedial part (Vo.r) of oralis. Our observations are based on complete or near-complete reconstructions of 139 synaptic boutons in Vpv and 105 in Vo.r. All the labeled boutons contained clear, spherical, synaptic vesicles and were presynaptic to unlabeled dendrites, and they were frequently postsynaptic to unlabeled axon terminals containing pleomorphic synaptic vesicles (P-endings). The P-endings frequently formed axodendritic synapses on dendrites which received axodendritic synapses from labeled boutons (synaptic triads). On the basis of the number of contacts, synaptic arrangements associated with the labeled boutons could be subgrouped into simple (one or two contacts), intermediate (three or four contacts), and complex (five or more contacts) types. The labeled boutons varied from round to elongated forms with smooth to more irregular or scalloped contours. The boutons with scalloped contour were much more frequent in the complex type. The boutons of the intermediate type were significantly smaller than the complex type and larger than the simple type. The SA periodontal afferent terminals participated in each type of synaptic arrangements in Vpv, but were mostly of the simple type in Vo.r. The size of labeled boutons was significantly larger in Vpv than in Vo.r. The total number of axodendritic and axoaxonic contacts per labeled bouton was significantly higher in Vpv than in Vo.r. Another difference was the more frequent occurrence of synaptic triads in Vpv than in Vo.r. These observations provide evidence that sensory information from primary afferent fibers is processed in a different manner in the two subnuclei.

Organization of the descending projections from the parabrachial nucleus to the trigeminal sensory nuclear complex and spinal dorsal horn in the rat.

To clarify direct descending projections from the parabrachial nucleus (PB) to the trigeminal sensory nuclear complex (TSNC) and spinal dorsal horn (SpDH), the origin and termination of descending tract cells were examined by the anterograde and retrograde transport methods. Phaseolus vulgaris leucoagglutinin (PHA-L) and Fluorogold (FG) or dextran-tetramethylrhodamine (Rho) were used as neuronal tracers for the anterograde and retrograde transport, respectively. The ventrolateral PB, including Kölliker-Fuse nucleus (KF), sent axons terminating mainly in the ventrolateral parts of rostral trigeminal nuclei of the principalis (Vp), oralis (Vo), and interpolaris (Vi) as well as in the inner lamina II of the medullary (nucleus caudalis, Vc) and SpDH. Although the descending projections were bilateral with an ipsilateral dominance, TSNC received a more dominant ipsilateral projection than SpDH. The cells of origin of the descending tracts were located mainly in KF, but TSNC received fewer projections from the KF than SpDH. Namely, TSNC received a considerable projection from the medial subnucleus of PB and the ventral parts of lateral subnuclei of PB, such as the central lateral subnucleus and lateral crescent area. The other difference noted between TSNC and SpDH was that the former received projections mainly from the caudal two thirds of KF and the latter from the rostral two thirds of KF. These results demonstrate the existence of direct parabrachial projections to TSNC and SpDH that are organized in a distinct manner and suggest that both pathways are involved in the control of nociception.

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 Vi 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.

Quantitative ultrastructure of physiologically identified premotoneuron terminals in the trigeminal motor nucleus in the cat.

Little is known about the ultrastructure of synaptic boutons contacting trigeminal motoneurons. To address this issue, physiologically identified premotor neurons (n = 5) in the rostrodorsomedial part of the oral nucleus (Vo.r) were labeled by intracellular injections of horseradish peroxidase (HRP) in cats. The ultrastructure of 182 serially sectioned axon terminals from the five neurons was both qualitatively and quantitatively analyzed. In addition, the effects of the glycine antagonist strychnine, GABA(A) antagonist bicuculline, NMDA antagonist 2-amino-5-phosphonovalerate (APV), and non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on Vo.r-induced postsynaptic potentials in trigeminal motoneurons (n = 11) were examined to evaluate potential signaling substances of the premotor neurons. Labeled boutons made synaptic contacts with either jaw-closing or -opening motoneurons. All the boutons contained pleomorphic vesicles, and most formed a single symmetric synapse either on the somata or on primary dendrites. Morphometric analyses indicated that bouton volume, bouton surface area, apposed surface area, total active zone area, and mitochondrial volume were not different between boutons on jaw-closing and -opening motoneurons. Vesicle number and density, however, were higher for boutons on jaw-closing motoneurons. The five morphological parameters were positively correlated with bouton volume. Vesicle density was the exception, which tending to be negatively correlated. Intravenous infusion of strychnine or bicuculline suppressed Vo.r-induced inhibitory postsynaptic potentials (IPSPs) in jaw-closing motoneurons. Abolition of Vo. r-induced excitatory postsynaptic potentials in jaw-opening motoneurons with APV and CNQX unmasked IPSPs. The present results suggest that premotor neurons in the Vo.r are inhibitory and that positive correlations between the ultrastructural parameters associated with synaptic release and bouton size are applicable to the interneurons, as they are in primary afferents.

Light microscopic observations of the contacts made between two spindle afferent types and alpha-motoneurons in the cat trigeminal motor nucleus.

Previous studies indicate that cat jaw-muscle spindle afferents can be divided into two types (type I and II) on the basis of their axonal trajectories. The present study examined the relationship between spindle afferent fibers and their target masseter alpha-motoneurons in the cat by using the intracellular horseradish peroxidase (HRP) injection technique, and provided several new findings on the synaptic organization generated between the two. Five type I afferent fiber-motoneuron pairs and nine type II afferent-motoneuron pairs were well stained with HRP. The following conclusions were drawn: 1) A motoneuron received contacts from only one collateral of any given spindle afferent. 2) The number of contacts made between an afferent and a motoneuron ranged from one to three. 3) The contacts made by a spindle afferent were on the same dendrite or dendrites branching from the same primary dendrite. 4) The vast majority of the contacts made by an afferent on a motoneuron were distributed in the dendritic tree within 600 microns from the soma, i.e., in the proximal three fourths of the dendritic tree. The differences observed between the two afferent types were as follows. First, type II afferent terminals made contacts on more distal dendrites of the motoneurons than did type I afferent terminals. Second, the contacts made between a type I afferent and a motoneuron were clustered together, but those made between a type II afferent and a motoneuron were widely dispersed. The present results provided the general rules of synaptic contacts between the spindle afferents and masseter alpha-motoneurons, and demonstrated that the spatial distribution of synaptic contacts on the dendritic tree was different between type I and type II afferents.

Quantitative analysis of the dendritic architectures of cat hypoglossal motoneurons stained intracellularly with horseradish peroxidase.

Little is known about the dendritic architecture of cat hypoglossal motoneurons. Thus, the present study was done to provide quantitative descriptions of hypoglossal motoneurons and to determine correlations between dendritic size parameters by using the intracellular horseradish peroxidase (HRP) injection technique in the cat. Twelve hypoglossal motoneurons stained with HRP were antidromically activated by stimulation applied to the medial branch of hypoglossal nerve. Eight (type I) and four (type II) of the 12 motoneurons were located in the ventral and dorsal parts of the ventromedial subnucleus of hypoglossal nucleus, respectively. The somatodendritic morphology of the two types of neurons was remarkably different, especially in the dendritic arborization pattern. The type I neurons established an egg-shaped dendritic tree that was distributed through the nucleus to the reticular formation; the type II dendritic tree was confined within the nucleus and presented a rostrocaudally oriented, mirror-image, fan-shape appearance. The total dendritic area and length and the number of terminations and branch points were significantly larger for type I than for type II neurons. For the two types of neuron, there was a positive correlation between stem dendritic diameter and several dendritic size parameters. Although the slopes of the regression lines were slightly different between the two, these were not statistically significant. The present study provides evidence that hypoglossal motoneurons located in the ventromedial subnucleus could be divided into two types according to the dendritic arborization pattern and quantitative analysis of the dendritic tree and according to neuronal location and suggests that the two types of hypoglossal motoneurons can be viewed as intrinsically distinct cell types: type I and type II, which innervate extrinsic and intrinsic muscles, respectively. In addition, the morphometric analysis made it possible to estimate the size of the dendritic tree by measuring the stem dendritic diameter.

Serotoninergic axonal contacts on identified cat spinal dorsal horn neurons and their correlation with nucleus raphe magnus stimulation.

This study examined the distribution of serotoninergic (5-HT) immunoreactive axonal contacts on spinal laminae I and II neurons by combining the intracellular horseradish peroxidase (HRP) method with immunocytochemistry. In addition, the 5-HT distribution was correlated with effects produced by electrical stimulation within the nucleus raphe magnus (NRM). Responses of lamina I neurons and lamina II stalked cells to noxious stimulation were markedly suppressed during NRM stimulation. In contrast, responses of nociceptive lamina IIa islet or non-nociceptive lamina IIb islet cells remained unchanged during nucleus raphe magnus stimulation. These inhibitory influences were positively correlated with the distribution of 5-HT immunoreactive contacts on these neurons. Nociceptive lamina I neurons and lamina II stalked cells received a significantly greater number of contacts (average of 74 and 63, respectively) than either nociceptive lamina IIa islet or non-nociceptive lamina IIb islet cells (average of 25 and eight contacts, respectively). Irrespective of cell type, most 5-HT contacts occurred on dendritic shafts rather than spines. These data reveal a differential distribution of 5-HT contacts on neurons in spinal laminae I and II, and indicate that at least a portion of the NRM modulation of dorsal horn neuronal activity is serotoninergic and concentrated on the dendritic shafts of nociceptive lamina I neurons and lamina II stalked cells.

Morphological differences between fast and slowly adapting lingual afferent terminations in the principal and oral nuclei in the cat.

Previous studies indicated that fast-adapting (FA) and slowly adapting (SA) mechanoreceptive afferents innervating the facial or intraoral structures give rise to morphologically distinct terminal arbors in the individual subdivisions of the trigeminal sensory nuclear complex. The present study examined the collateral morphologies of lingual afferents in the nuclei principalis (Vp) and oralis (Vo) of the cat. Seven FA and six SA lingual afferents were physiologically characterized and stained by the intra-axonal horseradish peroxidase (HRP) injection technique. The two types of afferents established terminal arbors in the dorsomedial subdivision (Vpd) of the Vp, and the rostrodorsomedial (Vo.r) and dorsomedial subdivisions (Vo.dm) of the Vo, but the collateral morphologies are different between the two types. The FA afferents gave rise to mediolaterally extended oblong arbors in each subdivision, but the arbors were better developed in the Vo.r than in the Vpd and Vo.dm. The number of collaterals, intercollateral distance, number of boutons per collateral, and bouton size were also different among the subdivisions. The SA afferents were divided into two subtypes; one had a preferential projection into the Vpd or the Vo.r and Vo.dm, and others lacked a selected projection. Although the shape of their arbors varied from a stringy form to a roundish form, the general profile was denser, better developed, and rounder than that of FA afferents in each subdivision. The intercollateral distance and bouton size were different among the subdivisions. The number of boutons per collateral, bouton density, and bouton size were larger in SA than FA afferents in each subdivision. The present study demonstrated that two functionally distinct lingual afferents manifest unique morphological differences in the Vpd and Vo.

Two major types of premotoneurons in the feline trigeminal nucleus oralis as demonstrated by intracellular staining with horseradish peroxidase.

Previous studies suggest that neurons in the dorsomedial subdivisions of trigeminal nucleus oralis (Vo) may contribute to reflex control of jaw movements and to modulation of sensory information. The present study has addressed this possibility by the use of intracellular staining with horseradish peroxidase of physiologically identified neurons in Vo to examine functional and morphological properties of these neurons. Of 14 labeled neurons, eight had axon collaterals terminating exclusively in the dorsolateral subdivision of the trigeminal motor nucleus (DL neurons) and four in its ventromedial subdivision (VM neurons); axon collaterals of two neurons were not traced. Both groups of neurons sent terminal arbors into other nuclei of the lower brainstem. The DL neurons were distinguishable from the VM neurons in their receptive field (RF) location, neuronal position, somadendritic architecture, and projections to other brainstem nuclei. All neurons, except for two that were exclusively activated by noxious stimuli applied to the tongue, were responsive to light mechanical stimulation of peri- and intraoral structures. The RFs of the DL neurons were located in more posterior oral structures than those of the VM neurons. The RF of nearly all low-threshold DL neurons was located in the maxillary region, and that of the VM neurons, in contrast, involved the mandibular region. The VM neurons were located medial or ventral to the DL neurons. The soma size of the VM neurons was significantly larger than that of the DL neurons. Dendritic arbors of both groups could be separated into medial and lateral components. The ratio of the dendritic transverse areas in the medial vs. lateral component was significantly higher in the VM neurons than in the DL neurons. The DL neurons also issued collaterals that terminated in larger brainstem areas than those of the VM neurons. These observations provide new evidence on the morphological and functional properties of Vo neurons that contribute to reflex control of jaw and facial movements and modulation of sensory information.

Oral and facial representation within the medullary and upper cervical dorsal horns in the cat.

Transganglionic transport of HRP was used to study the patterns of termination of somatic afferent fibers innervating oral and facial structures within the trigeminal nucleus caudalis and upper cervical dorsal horn of the cat. In separate animals, the superior alveolar, pterygopalatine, buccal, inferior alveolar, lingual, frontal, corneal, zygomatic, infraorbital, mental, mylohyoid, and auriculotemporal branches of the trigeminal nerve were traced in this experiment. The organization of the primary afferents innervating the oral structures is not uniform across laminae and at different rostrocaudal levels of the nucleus caudalis. The superior alveolar and pterygopalatine nerves mainly terminate in laminae I, II, and V at the level of the rostral one-third of the caudalis. By contrast, the lingual, inferior alveolar, and buccal nerve terminate in laminae I-V of, respectively, the rostral third, the entire length, and caudal two-thirds of the caudalis. In addition, the lingual, buccal, and pterygopalatine nerves terminate in the dorsal and middle parts of the interstitial islands or pockets of lamina I neuropil extending to the rostral levels parallel to the nucleus interpolaris. Mediolaterally, in laminae I, II, and V of the rostral third an extensive overlap of projections was found between the branches from each trigeminal division, and some overlap was observed between projections from the mandibular and maxillary divisions. On the other hand, the projections of primary afferents innervating the facial structures are arranged in a somatotopic fashion in rostrocaudal and mediolateral axes over the laminae (I-IV) through the nucleus caudalis and upper cervical dorsal horn. Fibers from the perioral and perinasal regions terminate most rostrally in caudalis, and fibers from progressively more posterior facial regions terminate at successively lower levels. A mediolateral somatotopic arrangement was observed, with fibers from the ventral parts of face ending in the medial regions and fibers from the progressively more dorsal parts of the face ending in successively more lateral regions of the medullary and upper cervical dorsal horns. Corneal afferent terminals are concentrated in the outer parts of lamina II at the levels of the rostral parts of the caudal two-thirds of the caudalis and the interstitial islands of lamina I. The maxillary division terminates first at the most caudal level of the caudalis, followed by the ophthalmic division descending as far as the C2 segment and the mandibular division reaching the most caudal level of the C2 segment.(ABSTRACT TRUNCATED AT 400 WORDS)

Oral and facial representation in the trigeminal principal and rostral spinal nuclei of the cat.

Transganglionic transport of horseradish peroxidase (HRP) was used to study the patterns of termination of somatic afferent fibers innervating oral and facial structures within the principal nucleus (Vp), nucleus oralis (Vo), and nucleus interpolaris (Vi). The primary trigeminal afferent fibers that innervate the oral cavity supplied by the pterygopalatine, superior alveolar, lingual, buccal, and inferior alveolar branches, as well as the facial skin supplied by the frontal, corneal, zygomatic, infraorbital, auriculotemporal, mylohyoid, and mental branches, were traced in this experiment. The results show that trigeminal afferent nerves that innervate the oral cavity project mainly to the principal nucleus, the rostrodorsomedial part (Vo.r) and dorsomedial division (Vo.dm) of pars oralis, and the dorsomedial region of pars interpolaris, while an extensive overlap of projections is found in the Vo.r, Vo.dm, and rostral Vi. The central processes of fibers innervating the anterior face (i.e., mental, infraorbital, and frontal nerves) terminate in the ventral division of principalis (Vpv), caudal region pars oralis (Vo.c), and ventrolateral Vi, with the largest numbers of terminals being found in the Vpv and Vi. In contrast, the central projection patterns of the corneal, zygomatic, mylohyoid, and auriculotemporal afferents are different from those of other afferent nerves examined, and present a discrete projection to the trigeminal sensory nuclear complex (TSNC). The corneal, mylohyoid, and auriculotemporal afferents mainly project to the restricted regions of principalis and caudal Vi, while zygomatic afferent nerve fibers project to the caudal third of pars interpolaris. The typical somatotopic organization with the face of the mouth open inverted is represented in the rostrocaudal midlevels of the Vpv and caudal pars interpolaris. The Vpd receives topographical projection from primary afferent nerves that innervate the oral structure only, while this projection was organized in a complicated manner. The relationship between the functional segregation and the cytoarchitectonic differentiation of the TSNC is discussed, particularly with respect to this somatotopic organization, combined with the characteristics of projecting cells in the TSNC.

Topographic representation of lower and upper teeth within the trigeminal sensory nuclei of adult cat as demonstrated by the transganglionic transport of horseradish peroxidase.

Transganglionic transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) entrapped in hypoallergenic polyacrylamide gel was used to study the patterns of termination of primary afferents that innervate the lower and upper tooth pulps within the trigeminal sensory nuclear complex (TSNC). HRP injections were made into the inferior and superior alveolar nerves in order to compare the central projections of the whole nerve with those from tooth pulps. In addition, the relationship between the distribution of the trigeminothalamic tract cells and the projection sites of the tooth pulp afferents was investigated by injecting HRP into the posterior ventral thalamus. HRP-labeled tooth pulp afferent fibers innervating the lower and upper teeth projected to the subnucleus dorsalis (Vpd) of pars principalis, the rostrodorsomedial part (Vo.r) and nucleus dorsomedialis (Vo.dm) of pars oralis, the medial regions of pars interpolaris, and laminae I, II, and V of pars caudalis. Terminal fields of the lower tooth pulp afferents formed a rostrocaudally running, uninterrupted column from the midlevel of Vpd to the caudal tip of caudalis. In contrast, the column of termination of upper tooth pulp afferents was discontinuous at the Vpd/Vo.r transition, and ended at the more rostral level of the caudalis than that of the lower tooth pulp afferents. The representation of the lower and upper teeth in the TSNC was organized in a somatotopic fashion which varied from one subdivision to the next, although terminal zones of the inferior and superior alveolar nerves overlapped within the Vo.r, Vo.dm, and dorsomedial part of rostral pars interpolaris. The lower and upper teeth were represented in the Vpd, Vo.r, Vo.dm, medial region of pars interpolaris, and laminae I, II, and V, in a ventrodorsal or caudorostral, dorsoventral, lateromedial, dorsoventral, and mediolateral or dorsomedial-ventrolateral sequence, respectively. The smaller, more focal terminal areas of the teeth contrasted sharply with more extensive terminal fields of the alveolar nerves. The HRP injections within the thalamus indicated that neurons in Vpd, the caudal pars interpolaris, and laminae I/V of caudalis, which are subdivisions of TSNC that receive pulpal projections, sent their axons to the ipsilateral and contralateral posterior ventral thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonergic axonal contacts on identified cat trigeminal motoneurons and their correlation with medullary raphe nucleus stimulation.

The innervation of the trigeminal motor nucleus by serotonergic fibers with cell bodies in the raphe nuclei pallidus and obscurus suggests that activation of this pathway may alter the excitability of trigeminal motoneurons. Thus, we recorded intracellular responses from cat jaw-closing (JC) andjaw-opening (JO) alpha-motoneurons evoked by raphe stimulation and used a combination of intracellular staining of horseradish peroxidase (HRP) and immunohistochemistry at the light and electron microscopic levels to examine the distribution of contacts made by serotonin (5-HT)-immunoreactive boutons on the two motoneurons types. Electrical stimulation applied to the nucleus raphe pallidus-obscurus complex induced a monosynaptic excitatory postsynaptic potential (EPSP) in JC (masseter) alpha-motoneurons and an EPSP with an action potential in JO (mylohyoid) alpha-motoneurons. The EPSP rise-times (time to peak) and half widths were significantly longer in the JC than in the JO motoneurons. The EPSPs were suppressed by systemic administration of methysergide (2 mg/kg). Six JC and seven JO alpha-motoneurons were well stained with HRP. Contacts were seen between 5-HT-immunoreactive boutons and the motoneurons. The JC motoneurons received a significantly larger number of the contacts than did the JO motoneurons. The contacts were distributed widely in the proximal three-fourths of the dendritic tree of JC motoneurons but were distributed on more proximal dendrites in the JO motoneurons. At the electron microscopic level, synaptic contacts made by 5-HT-immunoreactive boutons on motoneurons were identified. The present study demonstrated that JC motoneurons receive stronger 5-HT innervation, and this correlates with the fact that raphe stimulation caused larger EPSPs among these neurons than among JO motoneurons.

Central distribution of synaptic contacts of primary and secondary jaw muscle spindle afferents in the trigeminal motor nucleus of the cat.

Little is known about the differences of the terminations of group Ia and group II afferents within the brainstem or spinal cord. The present study was performed to classify cat jaw muscle spindle afferents by the use of succinylcholine (SCh) and to examine the morphological characteristics of the physiologically classified afferents at the light and electron microscopic levels through the use of the intra-axonal horseradish peroxidase (HRP) injection technique. The effects of SCh on stretch responses of 119 jaw muscle spindle afferents from the masseter were examined. The SCh converted the single skew distribution of the values for dynamic index (DI) into a bimodal one. Fifty-eight and 61 afferents were classified as group Ia and group II afferents, respectively. The central projections of 17 intra-axonally stained afferents (10 group Ia and 7 group II afferents) were examined. The spindle afferents terminated mainly in the supratrigeminal nucleus (Vsup), region h, and the dorsolateral subdivision of trigeminal motor nucleus (Vmo.dl) but differed in the pattern of projections of group Ia and group II afferents. The proportion of group Ia afferent terminals was higher in Vmo.dl but lower in Vsup than that of group II afferents. In Vmo.dl, the proportion of group Ia afferent terminals was higher in the central region but lower in the more outer regions than that of group II afferents. The ultrastructure of serially sectioned afferent boutons (63 group Ia and 72 group II boutons) also was examined. The boutons from the two groups were distributed widely from the soma to small-diameter dendrites, but the frequency of synaptic contacts on proximal dendrites was higher in group Ia than group II afferents. The present study provides evidence that the two groups of jaw muscle spindle afferents differ in their central projection and the spatial distribution of their synaptic contacts on Vmo.dl neurons.

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.

Electron microscopic observation of synaptic connections of jaw-muscle spindle and periodontal afferent terminals in the trigeminal motor and supratrigeminal nuclei in the cat.

Previous studies indicate that the trigeminal motor nucleus (Vmo) and supratrigeminal nucleus (Vsup) receive direct projections from muscle spindle (MS) and periodontal ligament (PL) afferents. The aim of the present study is to examine the ultrastructural characteristics of the two kinds of afferent in both nuclei using the intracellular horseradish peroxidase (HRP) injection technique in the cat. Our observations are based on complete or near-complete reconstructions of 288 MS (six fibers) and 69 PL (eight fibers) afferent boutons in Vmo, and of 93 MS (four fibers) and 188 PL (four fibers) afferent boutons in Vsup. All the labeled boutons contained spherical synaptic vesicles and were presynaptic to neuronal elements, and some were postsynaptic to axon terminals containing pleomorphic, synaptic vesicles (P-endings). In Vmo neuropil, MS afferent boutons were distributed widely from soma to distal dendrites, but PL afferent boutons predominated on distal dendrites. Most MS afferent boutons (87%) formed synaptic specialization(s) with one postsynaptic target while some (13%) contacting two or three dendritic profiles; PL afferents had a higher number of boutons (43%) contacting two or more dendritic profiles. A small but significant number of MS afferent boutons (12%) received contacts from P-endings, but PL afferent boutons (36%) received three times as many contacts from P-endings as MS afferents. In Vsup neuropil, most MS (72%) and PL (87%) afferent boutons formed two contacts presynaptic to one dendrite and postsynaptic to one P-ending, and their participation in synaptic triads was much more frequent than in Vmo neuropil. The present study indicates that MS and PL afferent terminals have a distinct characteristic in synaptic arrangements in Vmo and Vsup and provides evidence that the synaptic organization of primary afferents differs between the neuropils containing motoneurons and their interneurons.

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.