Influences of entopeduncular nucleus stimulation upon electromyogram activity of masticatory muscles.

Influences of stimulation of the entopeduncular nucleus (Ep) upon electromyogram (EMG) activity of masticatory muscles were examined. In the rat lightly anesthetized with halothane, high frequency (HF) microstimulation (trains of 20, 333-Hz cathodal pulses at 30-60 microA) and GABA microinjection (0.2-0.6 microl of 10 mM GABA dissolved in physiological saline) were performed in the Ep by using a three-barreled microelectrode. EMG activity was recorded from the anterior digastrics and the anterior superficial masseter muscles by using two fine enamel-insulated copper wires. The EMG activity was also evoked by the GABA microinjection. The effect of the GABA microinjection was negated by the microinjection of bicuculline prior to the GABA microinjection. The EMG activity was classified into the tonic spike-type, burst-type, or mixed type on the basis of the waveform. In each rat, the location of the microelectrode tip was estimated by observing a series of serial frontal sections through the whole rostrocaudal extent of the Ep. The present data suggested that Ep neurons involved in elicitation of tonic spike-type activity in the jaw muscles might be located mainly in the rostral third of the Ep, and that Ep neurons implicated in provocation of burst-type activity in jaw muscles might be located in the caudal third of the Ep. Possible neuronal pathways from the Ep to motoneurons innervating the masticatory muscles were discussed. The present data shed new light on the control mechanisms of the basal ganglia upon jaw movements.

Control of oro-facio-lingual movements by the substantia nigra pars reticulata: high-frequency electrical microstimulation and GABA microinjection findings in rats.

To provide direct evidence for substantia nigra pars reticulata (SNr) control of oro-facio-lingual muscle activity, high-frequency electrical microstimulation (mainly trains of 20, 333-Hz cathodal pulses at 40-60 microA) and GABA microinjection (1-5 microl of 10 mM GABA in saline) were carried out using a three-barreled microelectrode at the same SNr site in lightly anesthetized, chronically decorticated rats (n=39). Decortication eliminated the possibility that SNr microstimulation might activate corticofugal fibers descending in the adjoining cerebral peduncle. When the most ventral layer of the SNr was approached, high-amplitude electromyographic (EMG) activity of up to 6 mV with a distinctive waveform appeared synchronously with electrical stimuli in the anterior digastric, masseter, genioglossus, and levator labii superioris muscles. This EMG activity was evoked bilaterally, with an ipsilateral predominance. Eye movements, mostly rotation of the eyeball vertically down in the orbit, were noted. Infrequent blinking was also noted. Histologic examination localized the effector site to the middle third of the mediolateral extent of the caudal SNr corresponding to between 5.8 mm (level of the oculomotor nerve) and 6.5mm (caudal end of the SNr) caudal to bregma; and to the ventralmost peripeduncular region of the SNr corresponding to 7.7 mm to 8.0 mm beneath the cortical surface. We referred to this site as the substantia nigra pars reticulata oro-facio-lingual (SNr-ofl) region. GABA injection produced tonic EMG discharge with consistent amplitude in all of the four muscles studied. The GABA effect was negated by a preceding microinjection of the GABA-A receptor antagonist bicuculline, whereas saline control injection had no effect. Changes in amplitude of evoked EMG activity according to location of the stimulating microelectrode reflected somatotopic organization of the SNr-ofl region. This extremely localized electrical and receptor microstimulation in the SNr produced synchronized powerful contraction of jaw, tongue, and facial muscles with different neural innervation. These findings advance our understanding of the mechanisms of the SNr concerning oro-facio-lingual movements.

Expression of c-Fos protein in the trigeminal nuclear complex resulting from quantified force application to the rat molar.

This study was conducted to investigate the expression and distribution of c-Fos-like immunoreactive neurones (Fos-neurones), in the rat trigeminal sensory nuclear complex, produced by mechanical forces with various magnitudes and durations applied to the left upper first molar. The magnitudes of forces applied to the tooth were 25, 50 and 100 g and the duration was 2 h. A quantified force of 100 g was also applied to the upper molar for varying durations [short-time (1-2 min)], 2, 4, 8 and 12 h. Fos-neurones distributed in the bilateral superficial laminae of the subnucleus caudalis, and the ipsilateral dorsomedial part of subnucleus oralis (Sp5Odm). The number of Fos-neurones increased in the subnucleus caudalis (Sp5C) according to the force magnitude. In the Sp5C, the number of Fos-neurones exhibited maximum level, 2 or 4 h after the application. In the Sp5Odm, however, the number of Fos-neurones reached the maximum level at 8 h. These data suggest that the change in the number of nociceptive neurones in Sp5C reflect changes in encoding the magnitude of force to tooth, and that the nature of pain response to orthodontic forces might have some relation to the delayed expression of c-Fos protein in the Sp5Odm.

Mesencephalic projections to the first cervical segment in the cat.

Mesencephalic neurons projecting to the upper cervical spinal cord were examined by mapping the distributions of labeled cells after injecting fluorescent tracers or wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the C1 segment. Injections into the central or deep regions of the ventral horn produced retrograde labeling in cells of several mesencephalic regions. The majority of cells were found contralaterally in the superior colliculus and red nucleus, and ipsilaterally in and around the interstitial nucleus of Cajal (INC), in the cuneiform region, and in the fields of Forel. Smaller numbers of cells were located in the periaqueductal gray matter, nucleus annularis, and magnocellular nucleus of the posterior commissure. Dorsomedial injections in the ventral horn near the ventral commissure labeled only a subset of these projections, including cells in the mesencephalic reticular formation adjacent to the INC and in the nucleus annularis. Dorsolateral injections labeled some cells in the superior colliculus and were particularly effective at labeling cells in the red nucleus. These results suggest that at least ten different cell groups project to the ventral horn of the first cervical segment. Most, but not all, groups originate from regions implicated previously in the control of eye or head movements.

Neostriatal stimulation activates tongue-protruder muscle, but not tongue-retractor or facial muscles: an electrical and chemical microstimulation study in rats.

Whether electrical microstimulation of a rat's striatal jaw region (SJR) in fact induced tongue or facial muscle activity in addition to jaw muscle activity was tested. Microstimulation of SJR-evoked EMG activity in a jaw-opener (anterior digastricus) and tongue-protruder (genioglossus). No activity was evoked in jaw-closers (temporalis or masseter), tongue-retractor (hyoglossus) or in facial muscles. In addition, the EMG effect could still be induced after extensive ablation of the neocortex; it was reproduced by microinjection of 50 mM kainic acid into SJR. The effective sites were histologically localized to a small central striatal region adjoining the anterior commissure. These findings may be of considerable value in understanding the striatal mechanism of orolingual dyskinesia involving involuntary jaw and tongue movements.

Maturation ameloblasts of the porcine tooth germ do not express amelogenin.

Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage.

Feline caudofemoralis muscle. Muscle fibre properties, architecture, and motor innervation.

Feline caudofemoralis (CF) is a promising preparation in which to study the properties of mammalian fast-twitch skeletal muscle, but little is known about its muscle fiber properties, architecture, and motor innervation. We used histochemical techniques to confirm that it contained predominantly type IIB fibers (95+/-2%, n=8, with six of eight muscles composed exclusively of type IIA and IIB fibers), but physiological experiments showed less fatiguability than for the type IIB component of medial gastrocnemius. This may be related to the surprisingly strong and regular recruitment of CF during repetitive tasks such as walking and trotting, which we demonstrated electromyographically. We measured muscle length over the anatomical range of motion for CF (approximately 0.6-1.2 L0) and estimated working length during walking and trotting (approximately 0.95-1.15 L0). The specific tension was similar to that of the exclusively slow-twitch soleus muscle (31.2+/-4.7 N/cm2 compared with 31.8+/-4.1 N/cm2; P>0.8). Single fiber dissections of CF revealed a series-fibered architecture with a mean of 2.3 fibers, each 2.5 cm long, required to span the fascicle length. We identified two neuromuscular compartments in CF by cutting one of the two nerve branches innervating CF and depleting the glycogen stores in the intact motor units. These compartments were in parallel and extended the length of the muscle; their electromyographic activity was similar during various natural behaviors. CF and gluteus maximus motoneurons were labeled concurrently with a combination of fluorescent, retrograde tracers including Fluororuby, Fluorogold and Fast Blue. The CF motor nucleus was located in L7-S1, overlapping and intermingling extensively with the nucleus of the adjacent gluteus maximus muscle. Distributions of CF motoneuron diameter revealed one large peak around 50-55 microm, with relatively few small-diameter (less than 35 microm) cells. Using estimates of the total number of fibers in three muscles and the estimated number of alpha-motoneurons for those same muscles, we calculated a mean innervation ratio of approximately 270, which is at the low end of the innervation ratios for type IIB motor units from other feline muscles and more similar to type IIA motor units. In general, CF appears to be a useful preparation in which to study the properties of fast-twitch muscle, but these properties may vary somewhat from type IIB fibers from different muscles.

Sheath proteins: synthesis, secretion, degradation and fate in forming enamel.

We investigated expression of ameloblastin and sheathlin, recently cloned enamel matrix proteins from the rat and pig, in forming enamel immunocytochemically and immunochemically, using region-specific antibodies. The results obtained from the rat and pig were essentially the same. Antibodies which recognize the N-terminal region stained the secretory machinery of the secretory ameloblast and the entire thickness of the enamel matrix, especially the peripheral region of the enamel rod. Immunostained protein bands were observed near 65 or 70 kDa and below 20 kDa. C-terminal-specific antibodies stained the secretory machinery of the ameloblast and the immature enamel adjacent to the secretion sites. Immunostained protein bands were found ranging from 25 to 70 kDa. Antibodies which recognize a region in the protein just prior to the C-terminal region stained the cis-side of the Golgi apparatus but not the enamel matrix. Immunostained protein bands were observed of about 55 kDa. These results suggest that post-translational and post-secretory modifications of ameloblastin and sheathlin are similar to each other, and further showed that their cleaved N-terminal polypeptides concentrate in the prism sheath. We propose that sheathlin and ameloblastin share the same role in amelogenesis and should be classified as sheath proteins.

Synthesis, secretion, degradation, and fate of ameloblastin during the matrix formation stage of the rat incisor as shown by immunocytochemistry and immunochemistry using region-specific antibodies.

Rat ameloblastin is a recently cloned tooth-specific enamel matrix protein containing 422 amino acid residues. We investigated the expression of this protein during the matrix formation stage of the rat incisor immunohistochemically and immunochemically, using anti-synthetic peptide antibodies that recognize residues 27-47 (Nt), 98-107 (M-1), 224-232 (M-2), 386-399 (M-3), and 406-419 (Ct) of ameloblastin. Immunohistochemical preparations using antibodies Nt and M-1 stained the Golgi apparatus and secretory granules of the secretory ameloblast and the entire thickness of the enamel matrix. Only M-1 intensely stained the peripheral region of the enamel rods. Immunostained protein bands were observed near 65, 55, and below 22 kD. Immunohistochemical preparations using antibodies M-2 and Ct stained the Golgi apparatus and secretory granules of the ameloblast and the immature enamel adjacent to the secretion sites, but not deeper enamel layers. Immunostaining using M-2 and Ct revealed protein bands near 65 and 40-56 kD, and 65, 55, 48, 36, and 25 kD, respectively. M-3 stained the cis side of the Golgi apparatus but not the enamel matrix. This antibody recognized a protein band near 55 kD, but none larger. After brefeldin A treatment, immunoreaction of the 55-kD protein band intensified, and dilated cisternae of rER of the secretory ameloblast contained immunoreactive material irrespective of the antibodies used. These data indicate that ameloblastin is synthesized as a 55-kD core protein and then is post-translationally modified with O-linked oligosaccharides to become the 65-kD secretory form. Initial cleavages of the 65-kD protein generate N-terminal polypeptides, some of which concentrate in the prism sheath, and C-terminal polypeptides, which are rapidly degraded and lost from the enamel matrix soon after secretion.

Immunochemical and immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine tooth germ.

Our previous report identified 27- and 29-kDa calcium-binding proteins in porcine immature dental enamel. In this study we revealed that the N-terminal amino acid sequences of the two proteins were identical: LLANPXGXIPNLARGPAGRSRGPPG. The sequence matches a portion of the amino acid sequence of the porcine sheath protein, sheathlin. Porcine tooth germs were investigated immunochemically and immunohistochemically using specific antibodies raised against synthetic peptide that included residues 13-25 of this sequence. The affinity-purified antibodies reacted with several proteins extracted from newly formed immature enamel in immunochemical analyses, especially protein bands migrating at 62, 35-45, 29, and 27 kDa in SDS-polyacrylamide gels. The largest protein detected was a weak band near 70 kDa. In immunochemical analyses of proteins extracted from the inner (old) immature enamel, the antibody reacted faintly with the 27- and 29-kDa proteins. In immunohistochemical preparations, the Golgi apparatus and secretory granules of the secretory ameloblast, and the surface layer of immature enamel showed immunoreactivity. The immunoreactivity of immature enamel just beneath the secretory face of the Tomes' process was intense. No immunoreactivity was found in the Golgi apparatus of the maturation ameloblast. These results suggest that the 70-kDa protein, whose degradation might be very fast, is the parent protein of the 27- and 29-kDa proteins.

The sites of origin and termination of afferent and efferent components in the lingual and pharyngeal branches of the glossopharyngeal nerve in the Japanese monkey (Macaca fuscata).

Afferent and efferent components in the lingual and pharyngeal branches of the glossopharyngeal nerve (Li and Ph) of the Japanese monkey (Macaca fuscata) were examined. After injecting wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) unilaterally into the central cut end of the Li and Ph, or into the stylopharyngeal muscle, labeled neuronal cell bodies and terminal labeling were observed in the medulla oblongata, peripheral ganglia of the glossopharyngeal and vagus nerves, and cervical ganglia of the sympathetic trunk. The following conclusions were deduced from the results. The Li contains efferent fibers from the inferior salivatory nucleus, and superior cervical ganglion. The afferent fibers in the Li are composed mainly of peripheral processes of ganglion neurons in the superior and petrous ganglia of the glossopharyngeal nerve, and additionally of those of ganglion neurons in the jugular ganglion of the vagus nerve. The afferent fibers in the Li terminate mainly in the lateral division of the nucleus of the solitary tract, and additionally in the dorsal aspect of the lateral marginal region of the interpolar spinal trigeminal nucleus. The Ph is mainly composed of efferent fibers from the ambiguous nucleus and superior cervical ganglion; only a small number of afferent fibers from the sensory ganglia of the glossopharyngeal and vagus nerves are contained in the Ph. Stylopharyngeal motoneurons are distributed in the retrofacial part of the ambiguous nucleus.

An anterograde-retrograde labeling study of the carotid sinus nerve of the Japanese monkey (Macaca fuscata).

The sites of origin and termination of efferent and afferent fibers in the carotid sinus nerve (CSN) were investigated in the Japanese monkey. After application of a mixture of horseradish peroxidase (HRP) and wheat germ aggulutinin-conjugated HRP to the central cut end of the CSN, sensory ganglion neurons were labeled in the jugular ganglion of the vagus nerve, as well as in the superior and petrosal ganglia of the glossopharyngeal nerve. Many sympathetic ganglion neurons were also labeled retrogradely in the superior cervical ganglion. In the brain, many labeled terminals were seen ipsilaterally in the lateral division of the nucleus of the solitary tract (NST). A few neuronal cell bodies were also labeled ipsilaterally in a reticular region dorsomedial to the caudal one-third of the facial nucleus. The results indicate that the CSN of the Japanese monkey is composed mainly of afferent fibers terminating in the NST, that the afferent fibers in the CSN originate not only from the superior and petrosal ganglia of the glossopharyngeal nerve but also from the jugular ganglion of the vagus nerve, and that efferent fibers contained in the CSN arise from the medullary reticular formation and superior cervical ganglion.

Distribution of GABA-immunoreactive premotor neurons projecting to the trigeminal motor nucleus in the rat.

The distribution of neuronal cell bodies with gamma-aminobutylic acid (GABA)-like immunoreactivity projecting to the trigeminal motor nucleus were examined in the rat brainstem by the retrograde transport of horseradish peroxidase (HRP) and immunofluorescence methods. After HRP injection into the unilateral trigeminal motor nucleus, retrogradely labeled neurons which showed GABA-like immunoreactivity were observed throughout the parvocellular reticular nucleus in the pons with ipsilateral predominance. A few neurons were encountered in the contralateral supratrigeminal nucleus, ipsilateral caudal pontine reticular nucleus. All labeled neuronal cell bodies were small or of medium size; no large cells were labeled. The results indicated that cell bodies of GABAergic premotor neurons projecting to the trigeminal motoneurons were mainly located in the bilateral parvocellular reticular nucleus of the pons.

Superior cervical ganglionic origin of sympathetic fibers in the facial nerve, and their preganglionic spinal inputs: a WGA-HRP study in Macaca fuscata.

The origin of sympathetic fibers in the facial nerve of Macaca fuscata was demonstrated using the HRP method. WGA-HRP injection into the facial nerve resulted in heavy labeling in the ipsilateral superior cervical ganglion. The majority of labeled neurons in the superior cervical ganglion had sizes ranging from 20 to 35 microns. In the present study it was of particular interest that labeled neurons are also found in the intermediolateral nucleus and lateral funiculus ipsilaterally at the level from the C8 to T4 spinal cord segments. Many labeled neurons in the lateral funiculus were located in close vicinity to the intermediolateral nucleus, and, characteristically, a few of these had a long dendrite extending to the lateral surface of the spinal cord. The labeling in the spinal cord appears to be caused by transneuronal transport of WGA-HRP.

Distribution of axons showing calcitonin gene-related peptide- and/or substance P-like immunoreactivity in the sensory trigeminal nuclei of the cat.

Distribution of axons with calcitonin gene-related peptide (CGRP)-like and/or substance P (SP)-like immunoreactivity (LI) within the sensory trigeminal nuclei was examined in the cat before and after trigeminal rhizotomy. Axons with CGRP-LI or SP-LI were seen throughout the principal sensory trigeminal nucleus (Vp) and spinal trigeminal nuclei, including the medullary dorsal horn (MDH). They were densely distributed particularly in the dorsolateral part of the dorsal subnucleus of the Vp, ventromedial marginal zone of the ventral subnucleus of the Vp, dorsomedial and ventromedial parts of the oral spinal trigeminal nucleus, ventromedial and lateral marginal zones of the interpolar spinal trigeminal nucleus, and lamina I, outer part of lamina II and lamina V of the MDH. Most of the CGRP-LI axons exhibited SP-LI, while many SP-LI axons did not show CGRP-LI. After trigeminal rhizotomy, almost all CGRP-LI axons disappeared from the ipsilateral sensory trigeminal nuclei, while a considerable number of SP-LI axons remained intact throughout the nuclei; these SP-LI axons did not show CGRP-LI. The results indicate that CGRP-LI axons within the sensory trigeminal nuclei exhibit SP-LI and are of peripheral origin, and that SP-LI axons without CGRP-LI are of central origin.

Retinofugal projections in the house musk shrew, Suncus murinus.

Retinofugal projections in the house musk shrew (Suncus murinus) were studied with the WGA-HRP method. After WGA-HRP injection into the vitreous cavity of one eye, terminal labeling was seen in the suprachiasmatic nucleus, dorsal and ventral lateral geniculate nuclei, pretectum and superficial layer of the superior colliculus. The terminal labeling in the suprachiasmatic nucleus was more marked on the side ipsilateral to the injection than on the contralateral side, whereas that in other regions was seen mainly on the contralateral side. A retino-intergeniculate leaflet projection was observed. No unequivocal terminal labeling was found in the lateroposterior thalamic nucleus.

Re-examination of the topographical distribution of motoneurons innervating the digastric muscle in the rabbit and guinea pig.

The topographical distribution of motoneurons innervating the digastric muscle in the rabbit and guinea pig was re-examined by the retrograde tracing method of HRP (horseradish peroxidase). Motoneurons innervating the anterior belly of the digastric muscle of the rabbit and guinea pig constituted a longitudinal cell column in the ventromedial part of the motor nucleus of the trigeminal nerve. Motoneurons innervating the posterior belly of the digastric muscle were localized in the accessory facial nucleus. No motoneurons supplying the digastric muscle were found within the main facial nucleus.

Representation of the tensor veli palatini muscle in the trigeminal motor nucleus of the Japanese monkey (Macaca fuscata).

Distribution of motoneurons supplying the tensor veli palatini (TVP) muscle was examined in the Japanese monkey (Macaca fuscata) by the retrograde horseradish peroxidase (HRP) method. Neurons labeled with HRP which was injected into the tensor veli palatini muscle were seen in the ventromedial aspects of the dorsolateral division of the trigeminal motor nucleus, at all rostrocaudal levels of the trigeminal motor nucleus. The vast majority of these TVP motoneurons were distributed around the margin, especially the dorsal margin, of the cluster of motoneurons which innervate the lateral pterygoid muscle.