GABA- and glycine-like immunoreactivity in axonal endings presynaptic to the vibrissa afferents in the cat trigeminal interpolar nucleus.

The goal of this study was to analyze the synaptic interaction of primary afferents with GABA- and/or glycine-immunopositive presynaptic endings in the cat trigeminal interpolar nucleus (Vi). Fast adapting vibrissa afferents were labeled by intra-axonal injections of horseradish peroxidase. Postembedding immunogold labeling on serially cut ultrathin sections and quantitative ultrastructural analysis of the labeled boutons and their presynaptic endings (p-endings) in the Vi were performed. The majority of p-endings presynaptic to labeled boutons (83%) were immunopositive for both GABA and glycine and 8% were immunopositive for glycine alone. A small fraction of p-endings were immunopositive for GABA alone (4%) or immunonegative for both GABA and glycine (4%). Ultrastructural parameters related to synaptic release, i.e. bouton volume, mitochondrial volume, and active zone area, were significantly larger in the labeled boutons of primary afferents than in the p-endings. The volume of labeled boutons was positively correlated with the number of the postsynaptic dendrites and p-endings. In addition, fairly large-sized labeled boutons and p-endings were frequently observed in the Vi. These results reveal that large majority of vibrissa afferents in the Vi are presynaptically modulated by interneurons immunopositive for both GABA and glycine, and suggest that the Vi plays a distinct role in the processing of orofacial sensory information, different from that of other trigeminal sensory nuclei.

The association of genetic variants in Krüppel-like factor 11 and Type 2 diabetes in the Japanese population.

AIMS: Krüppel-like factor 11 (KLF11) is a transcriptional factor of the zinc finger domain family that regulates the expression of insulin. In North European populations, its common functional variant Q62R (rs35927125) is a strong genetic factor for Type 2 diabetes (P = 0.00033, odds ratio for G allele = 1.29, 95% CI 1.12-1.49). We examined the contribution of KLF11 variants to the susceptibility to Type 2 diabetes in a Japanese population. METHODS: By re-sequencing Japanese individuals (n = 24, partly 96), we screened all four exons, exon/intron boundaries and flanking regions of KLF11. Verified single nucleotide polymorphisms (SNPs) were genotyped in 731 initial samples (369 control and 362 case subjects). Subsequently, we tested for association in 1087 samples (524 control and 563 case subjects), which were collected in different districts of Japan from the initial samples. RESULTS: We identified eight variants, including a novel A/C variant on intron 3, but no mis-sense mutations. In an association study, we failed to find any significant result of SNPs (minor allele frequency 8.2-46.2%) after correcting for multiple testing. Similarly, no haplotypes were associated with Type 2 diabetes. It is notable that the G allele in rs35927125 was completely absent in 1818 Japanese individuals. CONCLUSIONS: Genetic variants in KLF11 are unlikely to have a major effect of Type 2 diabetes in the Japanese population, although they were significantly associated in North European populations. These observations might help to determine the role of KLF11 variants in Type 2 diabetes in different populations.

Prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes. A gene therapy model for autoimmune diabetes.

Four pancreatic islet-specific CD4+ helper T (Th) 1 (Th1) clones and two Th1 clones transduced with an SRalpha promoter-linked murine IL-10 (mIL-10) cDNA of 2.0-6.0 x 10(6) cells were adoptively transferred to nonobese diabetic (NOD) mice at age 8 d. Cyclophosphamide (CY) was administered at age 37 d (plus CY), and the incidence of diabetes and the histological grade of insulitis were examined at age 47 d. After the adoptive transfer of IL-10-transduced Th1 cells, polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR detected the neo gene and the retrovirus vector-mediated IL-10 mRNA in situ in recipient islets, respectively. RT-PCR detected the decrease of IFN-gamma mRNA relative to IL-10 mRNA in IL-10-transduced Th1 clones in vitro and also in recipient islets. All four wild type Th1 clones plus CY induced the insulitis grade of 2.75 and diabetes in 66% of recipient NOD mice. IL-10-transduced two Th1 clones plus CY induced periinsulitis with the grade of 1.43 and diabetes in 8.0%. The 1:1 mixture of wild type Th1 cells and IL-10-transduced Th1 cells plus CY induced periinsulitis with the grade of 1.85 and diabetes in 20%. The suppression of diabetes through decreasing IFN-gamma mRNA by the tissue-specific delivery of IL-10 to pancreatic islets with IL-10-transduced Th1 cells affords us the starting basis to develop the gene therapy for autoimmune diabetes.

Abrogation of autoimmune diabetes in nonobese diabetic mice and protection against effector lymphocytes by transgenic paracrine TGF-beta1.

Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.

Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.

Molecular cloning and tissue-specific expression of a new member of the regenerating protein family, islet neogenesis-associated protein-related protein.

Islet neogenesis-associated protein (INGAP) is a protein expressed during islet neogenesis. We have cloned a novel cDNA having a similar sequence to INGAP cDNA. The cDNA encodes 175 amino acids designated INGAP-related protein (INGAPrP). INGAP is expressed in cellophane-wrapped pancreas, but not in normal pancreas, whereas INGAPrP was abundantly expressed in normal pancreas.

Rat genomic structure of amidophosphoribosyltransferase, cDNA sequence of aminoimidazole ribonucleotide carboxylase, and cell cycle-dependent expression of these two physically linked genes.

Genomic structure of rat amidophosphoribosyltransferase (ATase; EC 2.4.2.14), which catalyzes the first committed step in de novo purine nucleotide synthesis, was determined by polymerase chain reaction (PCR)-based methods. There are 11 exons and all exon-intron boundaries were conserved among rat, human, and chicken ATase genes. A rat aminoimidazole ribonucleotide carboxylase (AIRC) cDNA encoding a bifunctional enzyme of AIRC (EC 4.1.1.21) at step 6 and SAICAR synthetase (EC 6.3.2.6) at step 7 in de novo purine nucleotide synthesis was cloned and sequenced. The size of the cloned rat AIRC cDNA was 1329 bp, and amino acid identity with human and chicken AIRC was 96 and 85%, respectively. The intergenic sequence using a phage clone and the PCR product disclosed that ATase and AIRC genes are physically linked with the 736 bp sequence between the translation start sites, and the determination of the transcriptional start sites by the primer extension assay for these genes disclosed that distance between the two major transcriptional start sites is 585 bp. The amount of mRNAs of both genes showed approx. 5-6-fold increase in G1/S phase of the cell cycle over those in G0 phase in synchronized rat 3Y1 fibroblasts.

Elimination of neurokinin-1 receptor neurons in caudal nucleus reverses the effects of systemic bicuculline on c-Fos expression in rat trigeminal sensory nucleus: I. High intensity electrical stimulation of the trigeminal ganglion.

Although neurokinin-1 receptor (NK-1)-bearing neurons are distributed in lamina I of the trigeminal caudal nucleus (Vc) and constitute major projection neurons, little is known about their fundamental role(s) in nociceptive processing. This study examines the effect of intra cisterna magna injection of substance P (SP) conjugated to saporin (SP-Sap; 5 microM, 5 microl) [with/without systemic administration of bicuculline] on c-Fos expression in the trigeminal sensory nucleus (TSN) induced 2 h after 10 min repetitive electrical stimulation of the trigeminal ganglion (TG) at high intensity (1.0 mA, 5 Hz, 5 ms) in the urethane-anesthetized rat. In the SP-Sap-treated rats, the numbers of NK-1-immunopositive neurons in laminae I and III of the Vc decreased compared with rats similarly pretreated with saline (Sal; 5 microl) or blank-saporin (Bl-Sap; 5 microM, 5 microl). In Sal- or Bl-Sap-treated controls, high intensity stimulation induced c-Fos expression in neurons throughout the full extent of ipsilateral superficial layers of the Vc (VcI/II), magnocellular zone of the Vc (VcIII/IV) and the dorsal or dorsomedial subdivisions of the rostral TSN above the obex (trigeminal principal, oral (Vo) and interpolar nuclei). Preadministration of bicuculline (2 mg/kg, i.p.) decreased the numbers of c-Fos-immunopositive neurons in the VcI/II, VcIII/IV and Vo in Sal- or Bl-Sap-treated controls. In contrast, high intensity stimulation induced less c-Fos-immunopositive neurons in the VcI/II and Vo of rats treated with SP-Sap compared with those in Sal- or Bl-Sap-treated controls. In SP-Sap-treated rats preadministered with bicuculline, the numbers of c-Fos-immunopositive neurons in the VcI/II and Vo were increased compared with the SP-Sap-treated rats preadministered with Sal. These results suggest that NK-1-immunopositive neurons in laminae I and III of Vc play a pivotal role in the nociceptive specific processing in the TSN through GABA(A) receptors.

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.

Jaw movement-related primary somatosensory cortical area in the rat.

It has anatomically been revealed that the rostral part of the rat primary somatosensory cortex (S1) directly projects to the dorsal part of the trigeminal oral subnucleus (dorVo) and the dorsal part of juxtatrigeminal region (dorVjuxt), and that the dorVo and dorVjuxt contain premotoneurons projecting directly to the jaw-opening or jaw-closing motoneurons in the trigeminal motor nucleus (Vmo). However, little is known about how the rostral S1 regulates jaw movements in relation to its corticofugal projections. To address this issue, we performed intracortical microstimulation of the rat rostral S1 by monitoring jaw movements and electromyographic (EMG) activities. We for the first time found that low-frequency long-train stimulation of the rostral S1 induced single sustained opening of the jaw with elevated EMG activities of the anterior digastric muscles (jaw-opener). The effective sites for the low-frequency long-train stimulation overlapped the S1 sites where traditional high-frequency short-train stimulation was effective to induce single twitch-like jaw movement. We also found that the effective sites for the two kinds of train stimuli were included in the rostral S1 area, which has previously been identified to send direct projections to the dorVo or the dorVjuxt. Specifically, the most effective stimulation sites for the two kinds of train stimuli were located in the rostralmost part of S1 which has been reported to emanate strong direct projections to the dorVjuxt but less to the dorVo. Therefore, the present study suggests that the rat rostral S1, especially its rostralmost part, plays an important role in controlling jaw movements by activation of direct descending projections from the rostral S1 to the trigeminal premotoneuron pools, especially to the dorVjuxt.

Infrequent mutations of p27Kip1 gene and trisomy 12 in a subset of human pituitary adenomas.

To study the etiologic roles of genes on chromosome 12 for the pituitary tumorigenesis of adenomas, mutations of the p27Kip1 gene and allelic ratios of 18 microsatellite markers on the entire chromosome 12 were studied in 33 pituitary adenomas. The p27Kip1 gene on chromosome 12p12-p13 encoding an inhibitor of complexes between cyclins and cyclin-dependent kinases is supposed to function as the tumor suppressor gene. Among 31 sporadic and 2 familial pituitary adenomas, PCR-single strand conformation polymorphism analysis detected three polymorphic changes but no tumor-specific mutations of the p27Kip1 gene. Genotyping of 18 microsatellite markers on the entire chromosome 12 detected the uniformly decreased allelic ratios ranging from 54-66% in 8 of 33 pituitary adenomas (24%), although no loss of heterozygosity was detected. Fluorescence in situ hybridization confirmed trisomy 12 in all 5 available samples out of these 8 samples. Based on these, we conclude that not mutations of the p27Kip1 gene, but trisomy 12 may be etiologically important in a subgroup of pituitary adenomas.

Absence of germ-line mutations of the multiple endocrine neoplasia type 1 (MEN1) gene in familial pituitary adenoma in contrast to MEN1 in Japanese.

Germ-line mutations of the MEN1 gene were analyzed in five cases of familial and four cases of sporadic multiple endocrine neoplasia type 1 (MEN-1), six cases in three independent pedigrees of familial pituitary adenoma without MEN-1, and three cases of familial isolated primary hyperparathyroidism (FIHP) in Japanese. Eight different types of germ-line mutations in all nine cases of MEN-1 were distributed in exons 2, 3, 7, and 10 and intron 7 of the MEN1 gene. Loss of heterozygosity (LOH) on 11q13 was detected in all nine tumors of these cases with microsatellite analysis. No germ-line mutation of the MEN1 gene was detected in three pedigrees of familial pituitary adenoma and three cases of FIHP. LOH on 11q13 was detected in two cases in one pedigree of familial pituitary adenoma, and one of them showed a heterozygous somatic mutation of the MEN1 gene. No LOH on 11q13 was detected in three cases of FIHP. Based on these, we conclude that the loss of function of menin is etiological for familial or sporadic MEN-1, but not for FIHP or most familial pituitary adenoma without MEN-1.

Analysis of loss of heterozygosity on chromosome 11 and infrequent inactivation of the MEN1 gene in sporadic pituitary adenomas.

To investigate the role of tumor suppressor genes in sporadic pituitary adenomas, we first analyzed loss of heterozygosity on 11q13 with microsatellite analysis in 31 tumors. Loss of heterozygosity on 11q13 was detected in 1 mixed GH/PRL adenoma, and the somatic 22-bp deletion of the multiple endocrine neoplasia type 1 (MEN1) gene encoding menin was detected in this tumor. Trisomy 11 suggested by the decreased mean allelic ratios of 66% or 65% for 16 or 13 microsatellite markers, respectively, in 2 of 31 pituitary adenomas was confirmed by interphase fluorescence in situ hybridization. Screening for mutations of the MEN1 gene did not find mutations with PCR-single strand conformation polymorphism analysis in other pituitary adenomas retaining heterozygosity on 11q13. Based on these, it is concluded that inactivation of the MEN1 gene comprises a rare etiology for tumorigenesis of the pituitary gland, and that trisomy 11 or another gene(s) may contribute to the pathogenesis of sporadic pituitary adenomas.

Jaw-muscle spindle afferent pathways to the trigeminal motor nucleus in the rat.

Neural pathways conveying proprioceptive feedback from the jaw muscles were studied in rats by combining retrograde and intracellular neuronal labeling. Initially, horseradish peroxidase was iontophoresed unilaterally into the trigeminal motor nucleus (Vmo). Two days later, 1-5 jaw-muscle spindle afferent axons located in the mesencephalic trigeminal nucleus were physiologically identified and intracellularly stained with biotinamide. Stained mesencephalic trigeminal jaw-muscle spindle afferent axon collaterals and boutons were predominantly distributed in the supratrigeminal region (Vsup), Vmo, dorsomedial trigeminal principal sensory nucleus (Vpdm), parvicellular reticular formation (PCRt), alpha division of the parvicellular reticular formation (PCRtA), and dorsomedial portions of the spinal trigeminal subnuclei oralis (Vodm), and interpolaris (Vidm). Numerous neurons retrogradely labeled with horseradish peroxidase from the trigeminal motor nucleus were found bilaterally in the PCRt, PCRtA, Vodm, and Vidm. Retrogradely labeled neurons were also present contralaterally in the Vsup, Vpdm, Vmo, peritrigeminal zone, and bilaterally in the dorsal medullary reticular field. Putative contacts between intracellularly stained mesencephalic trigeminal jaw-muscle spindle afferent boutons and trigeminal premotor neurons retrogradely labeled with horseradish peroxidase were found in the ipsilateral Vodm, PCRtA, and PCRt, as well as the contralateral Vsup, Vmo, Vodm, PCRt, and PCRtA. Thus, multiple disynaptic jaw-muscle spindle afferent-motoneuron circuits exist. These pathways are likely to convey long-latency jaw-muscle stretch reflexes and may contribute to stiffness regulation of the masticatory muscles.

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.

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.

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.

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.

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.

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.