Corticofugal projections to trigeminal motoneurons innervating antagonistic jaw muscles in rats as demonstrated by anterograde and retrograde tract tracing.

Little is known about the organization of corticofugal projections controlling antagonistic jaw muscles. To address this issue, we employed retrograde (Fluorogold; FG) and anterograde (biotinylated dextran amine; BDA) tracing techniques in rats. Three groups of premotoneurons were identified by injecting FG into the jaw-closing (JC) and -opening (JO) subdivisions of the trigeminal motor nucleus (Vmo). These were 1) the intertrigeminal region (Vint) and principal trigeminal sensory nucleus for JC nucleus; 2) the reticular region medial to JO nucleus (RmJO) for JO nucleus; and 3) the parabrachial (Pb) and supratrigeminal (Vsup) nuclei, reticular regions medial and ventral to JC nucleus, rostrodorsomedial oralis (Vor), and juxtatrigeminal region (Vjuxt) containing a mixture of premotoneurons to both the nuclei. Subsequently, FG was injected into the representative premotoneuron structures. The JC and JO premotoneurons received main afferents from the lateral and medial agranular fields of motor cortex (Agl and Agm), respectively, whereas afferents to the nuclei with both JC and JO premotoneurons arose from Agl also and from primary somatosensory cortex (S1). Finally, BDA was injected into each of the three cortical areas representing the premotoneuron structures to complement the FG data. The Agl and Agm projected to reticular regions around the Vmo, whereas the Pb, Vsup, Vor, and Vjuxt received input from Agl. The S1 projected to the trigeminal sensory nuclei as well as to the Pb, Vsup, and Vjuxt. These results suggest that corticofugal projections to Vmo via premotoneuron structures consist of multiple pathways, which influence distinct patterns of jaw movements.

Developmental changes in distribution of gamma-aminobutyric acid- and glycine-immunoreactive boutons on rat trigeminal motoneurons. I. Jaw-closing motoneurons.

We have previously described the distribution pattern of inhibitory synapses on rat jaw-closing (JC) alpha- and gamma-motoneurons. In the present study, we investigated developmental changes in inhibitory synapses on JC motoneurons. We performed a quantitative ultrastructural analysis of putative inhibitory synaptic boutons on JC motoneuron somata by using postembedding immunogold labeling for GABA and glycine. In total, 206, 350, and 497 boutons contacting JC motoneuron somata were analyzed at postnatal days 2 (P2), 11 (P11) and 31 (P31), respectively. The size of the somata increased significantly during postnatal development. The size distribution was bimodal at P31. Mean length of the boutons and percentage of synaptic covering also increased during postnatal development, whereas bouton density did not differ significantly among the three age groups. Synaptic boutons on the somata of JC alpha-motoneurons could be classified into four types: boutons immunoreactive for 1) GABA only, 2) glycine only, 3) both GABA and glycine, and 4) neither GABA nor glycine. There was no developmental change in the proportion of putative inhibitory boutons to the total number of studied boutons. However, the glycine-only boutons increased significantly (15.1% to 27.3%), and the GABA-only boutons decreased significantly (17.7% to 2.6%) during the period from P11 to P31. Our ultrastructural data indicate that the inhibitory synaptic input to JC motoneurons is developmentally regulated and that there is a postnatal switch from GABA to glycine. The postnatal changes revealed in the present study could play an important role in the maturation of the oral motor system.

The somatotopic organization of trigeminal premotoneurons in the cat brainstem.

This study was performed to complement the results of prior intracellular recording and labeling studies by investigating the general distribution pattern of trigeminal premotoneurons in the cat brainstem using the retrograde tracing methods. The results of the present study reconfirmed the presence of premotoneurons in the trigeminal principal and oral nuclei following horseradish peroxidase injections into the jaw-opening (JO) or jaw-closing (JC) nucleus. Furthermore, we found that labeled cells from the JO nucleus and JC nucleus located in the reticular regions surrounding the trigeminal motor nucleus (Vmo; Vmo shell region) were arranged in a topographic fashion, while those in the parabrachial nucleus, supratrigeminal nucleus, lateral reticular formation caudal to the shell region and raphe nuclei were intermingled with each other. The labeling in the individual nuclei was bilateral with an ipsilateral predominance to each injection site, with the exception of the mesencephalic trigeminal nucleus, where the labeling was ipsilateral to the injection site in the JC nucleus. These results, combined with the data of the previous intracellular tracing studies, indicate that based on the presence of somatotopic organization, premotoneurons can be largely divided into two groups; those projecting to either the JO or the JC nucleus and those projecting to the two nuclei, and we offer the suggestion that roles of premotoneurons for jaw movements differ among the individual nuclei.

Spatial distribution patterns of excitatory and inhibitory synapses in the dendritic tree differ between jaw-closing and -opening motoneurons.

This paper reviews recent data on the spatial distribution of inhibitory and excitatory synapses on the dendritic tree of jaw-closing (JC) and -opening (JO) motoneurons in the cat, in which a combination of techniques employing intracellular injections of horseradish peroxidase and postembedding immunogold labelling was used. The dendritic tree is divided into three segments: primary and distal dendrites and intermediate dendrites between the two segments. The proportion of inhibitory boutons (immunoreactive for GABA and/or glycine) is slightly higher than proportion of excitatory boutons (immunoreactive for glutamate) in JC motoneurons, but this trend is reversed in JO motoneurons. In the two kinds of motoneuron, boutons immunoreactive to glycine alone are more numerous than boutons double-labelled to GABA and glycine, which, in turn, occur more frequently than boutons immunoreactive to GABA alone. In JC motoneurons, the packing density (number of boutons per 100 microm(2)) of the inhibitory boutons decreases somatofugally, but this trend is not applicable to the excitatory boutons. In contrast, the packing density of the inhibitory and excitatory boutons in JO motoneurons does not significantly differ among the three dendritic compartments, though it is slightly higher for the excitatory than the inhibitory ones on each dendritic segment. These differences have important implications for synaptic integration in JC and JO motoneurons.

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.

Bilateral projection of functionally characterized trigeminal oralis neurons to trigeminal motoneurons in cats.

Intracellular Neurobiotin-injections were used to label functionally identified neurons in the rostro-dorsomedial part of the trigeminal oral nucleus (Vo.r) in the cat. The labeled Vo.r neurons with the mechanoreceptive field in oral tissues innervated bilaterally either jaw-opening motoneurons or jaw-closing motoneurons. This result suggests that Vo.r neurons play an important role in sensory-motor reflexes responsible for coordination of bilaterally symmetrical jaw movements.

GABA and glycine in synaptic microcircuits associated with physiologically characterized primary afferents of cat trigeminal principal nucleus.

Previous studies suggest that sensory information conveyed through trigeminal afferents is more strongly controlled at the level of the first synapse by GABA-mediated presynaptic mechanisms in the trigeminal principal sensory nucleus (Vp) than other sensory nuclei. However, it is unknown if such a mechanism is common to functionally different classes of primary afferent in the same nucleus or across the nuclei. To address these issues, the present study focused on synaptic microcircuits associated with slowly adapting (SA) mechanosensory afferents innervating the periodontal ligaments in the cat Vp and attempted to examine GABA, glycine, and glutamate immunoreactivity in axon terminals involved in the circuits. Afferents were physiologically characterized before injection of horseradish peroxidase (HRP) and preparation for electron microscopy. HRP-labeled afferent boutons were serially sectioned and immunostained with antibodies against GABA, glycine, and glutamate using a postembedding immunogold method. All the afferent boutons examined contacted non-primary dendrites and they were frequently postsynaptic to unlabeled axons (p-endings). Axodendritic and axoaxonic contacts per afferent bouton were 1.3 (46/35) and 2.0 (70/35), respectively. Most p-endings were immunoreactive for GABA (63/70) and also glycine was co-stained in the majority of the p-endings (49/63). Thirty percent of p-endings with the colocalization of GABA and glycine participated in synaptic triads where a p-ending formed a synapse with the same dendrite as the afferent bouton. None of the p-endings was immunoreactive for glutamate. Most afferent boutons were enriched with glutamate but were immunonegative for GABA and glycine. This study provides evidence suggesting that transmission from SA afferents is strongly controlled presynaptically by GABAergic interneurons with colocalized glycine, and that a proportion of these interneurons, involved in synaptic triads, may also have postsynaptic inhibitory actions on target neurons of the SA afferents.

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.

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

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

Quantitative analysis of the dendritic architectures of single jaw-closing and jaw-opening motoneurons in cats.

Little is known about the dendritic architectures of trigeminal motoneurons innervating antagonistic muscles. Thus, the aim of the present study was to provide a quantitative description of jaw-closing (JC) and jaw-opening (JO) alpha motoneurons and to determine geometrical similarities and differences of the dendritic tree between the two. Seven JC alpha motoneurons and four JO alpha motoneurons were intracellularly labeled with horseradish peroxidase (HRP) in the cat and quantitatively analyzed with a computer-assisted three-dimensional system. The dendritic tree of JC alpha motoneurons was confined within the JC motor nucleus, despite locations of the cell body. In contrast, JO alpha motoneurons generated extensive extranuclear dendrites in the reticular formation. The branching pattern of proximal dendritic segments was simpler in the JC than in the JO alpha motoneurons. Despite these differences, the mean values of dendritic parameters examined per neuron were not different between the two kinds of alpha motoneurons, and the stem dendrite diameter was positively correlated with several dendritic parameters in a linear manner. The present study provides new evidence that underlying design principles of the geometry of the dendritic tree are not concerned with the differences in configuration and branching pattern of the dendritic tree of trigeminal alpha motoneurons innervating antagonistic muscles. In addition, we estimated the number of excitatory and inhibitory synapses covering dendrites of single JC alpha motoneurons.

NADPH-diaphorase and calcium binding proteins in the trigeminal nucleus oralis of rats.

We have examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the calcium binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV), in the trigeminal nucleus oralis (Sp5O). NADPH-d was detected by histochemistry while CBP was detected by immunohistochemistry. NADPH-d-positive neurons were distributed in the medial rostro-dorsomedial part (RDMsp5O) and dorsomedial part (DMsp5O) of Sp5O, and the rostrolateral part of the nucleus of the solitary tract (NTS). CB- and CR-positive neurons were mainly distributed in the dorsal part of Sp5O. In contrast, PV-positive neurons were mainly distributed in the ventral part of Sp5O. NADPH-d colocalized with CB (40%) and CR (20%) but not with PV in neurons of DMsp5O/ NTS. The mean cell sizes of neurons in RDMsp5O were larger than those in DMsp5O/NTS. PV-positive neurons were larger than NADPH-d-positive neurons. NADPH-d-, CB- and CR-positive neurons were generally small in RDMsp5O and DMsp5O/NTS. Few neurons were retrogradely labeled in RDMsp5O and DMsp5O from the thalamus, when numerous labeled neurons were in the principal and interpolar nuclei. These data indicate that NADPH-d histochemistry and CB, CR and PV immunohistochemistry identify a discrete cell population in Sp5O. Those labeled neurons in RDMsp5O and DMsp5O/NTS were considered to be involved in sensorimotor reflexive function of the intra-oral structures.

Effects of somatosensory cortical stimulation on expression of c-Fos in rat medullary dorsal horn in response to formalin-induced noxious stimulation.

We examined the effects of epidural electrical stimulation of primary (SI) and secondary (SII) somatosensory cortex on expression of c-Fos protein in rat medullary dorsal horn neurons (Vc; trigeminal nucleus caudalis) in response to formalin-induced noxious stimulation. Epidural electrical stimulation (single pulse, 0.2 msec duration at 10 Hz) was applied to the left facial region SI or SII at three different stimulus intensities, 0.1, 0.5, and 1.0 mA for 60 min 0 or 2 hr after bilateral injection of formalin into the lower lip. SII stimulation at 1.0 mA immediately after injection of formalin, significantly decreased the number of Fos-positive cells in the right VcI/II by 32.4%. There was no significant change in the number of Fos-positive cells in the VcIII/IV. SII stimulation at 0.5 and 1.0 mA 2 hr after injection of formalin, significantly decreased the number of Fos-positive cells in the right VcI/II by 47.9% and 40.8%, but significantly increased the number of Fos-positive cells in the right VcIII/IV by 178.8% and 324.3%, respectively. In contrast, SI stimulation had no effect on expression of c-Fos in Vc. Possible direct corticotrigeminal projections were labeled anterogradely by injection of WGA-HRP into the SI and SII. In the Vc, labeled terminals were distributed mostly in the contralateral medial half of VcIII/IV and medullary reticular nucleus dorsalis but rarely in VcI/II. These results suggest that activation of SII-medullary fibers suppress nociceptive information from the oro-facial regions.

Quantitative ultrastructural analysis of glycine- and gamma-aminobutyric acid-immunoreactive terminals on trigeminal alpha- and gamma-motoneuron somata in the rat.

Detailed knowledge of the inhibitory input to trigeminal motoneurons is needed to understand better the central mechanisms of jaw movements. Here a quantitative analysis of terminals contacting somata of jaw-closing (JC) and jaw-opening (JO) alpha-motoneurons, and of JC gamma-motoneurons, was performed by use of serial sectioning and postembedding immunogold cytochemistry. For each type of motoneuron, the synaptic boutons were classified into four groups, i.e., immunonegative boutons or boutons immunoreactive to glycine only, to gamma-aminobutyric acid (GABA) only, or to both glycine and GABA. The density of immunolabeled boutons was much higher for the alpha- than for the gamma-motoneurons. In the alpha-motoneuron populations, the immunolabeled boutons were subdivided into one large group of boutons containing glycine-like immunoreactivity only, one group of intermediate size harboring both glycine- and GABA-like immunoreactivity, and a small group of boutons containing GABA-like immunoreactivity only. The percentage of immunolabeled boutons was higher for JC than JO alpha-motoneurons, the most pronounced difference being observed for glycine-like immunoreactivity. In contrast, on the somatic membrane of gamma-motoneurons, the three types of immunoreactive bouton occurred at similar frequencies. These results indicate that trigeminal motoneurons are strongly and differentially controlled by premotoneurons containing glycine and/or GABA and suggest that these neurons play an important role for the generation of masticatory patterns.