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Detailed information about the development of excitatory and inhibitory synapses on the genioglossal (GG) motoneuron may help to understand the mechanism of fine control of GG motoneuron firing and the coordinated tongue movement during postnatal development. For this, we investigated the development of γ-aminobutyric acid (GABA)-immunopositive (GABA +), glycine + (Gly +), and glutamate + (Glut +) axon terminals (boutons) on the somata of rat GG motoneurons at a postnatal day 2 (P2), P6 and P18 by retrograde labeling of GG motoneurons with horseradish peroxidase, electron microscopic postembedding immunogold staining with GABA, Gly, and Glut antisera, and quantitative analysis. The number of boutons per GG motoneuron somata and the mean length of bouton apposition, measures of bouton size and synaptic covering percentage, were significantly increased from P2/P6 to P18. The number and fraction of GABA + only boutons of all boutons decreased significantly, whereas those of Gly + only boutons increased significantly from P2/P6 to P18, suggesting developmental switch from GABAergic to glycinergic synaptic transmission. The fraction of mixed GABA +/Gly + boutons of all boutons was the highest among inhibitory bouton types throughout the postnatal development. The fractions of excitatory and inhibitory boutons of all boutons remained unchanged during postnatal development. These findings reveal a distinct developmental pattern of inhibitory synapses on the GG motoneurons different from that on spinal or trigeminal motoneurons, which may have an important role in the regulation of the precise and coordinated movements of the tongue during the maturation of the oral motor system.

Detailed information about the excitatory and inhibitory synapses on the hypoglossal motoneurons may help understand the neural mechanism for control of the hypoglossal motoneuron excitability and hence the precise and coordinated movements of the tongue during chewing, swallowing and licking. For this, we investigated the distribution of GABA-, glycine (Gly)- and glutamate (Glut)-immunopositive (+) axon terminals on the genioglossal (GG) motoneurons by retrograde tracing, electron microscopic immunohistochemistry, and quantitative analysis. Small GG motoneurons ( 400 μm2). The fraction of inhibitory boutons (GABA + only, Gly + only, and mixed GABA +/Gly + boutons) of all boutons was significantly higher for small GG motoneurons than for large ones, whereas the fraction of Glut + boutons was significantly higher for large GG motoneurons than for small ones. Almost all boutons (> 95%) on both small and large GG motoneurons were GABA + , Gly + or Glut + . The frequency of mixed GABA +/Gly + boutons was the highest among inhibitory boutons types for both small and large GG motoneurons. These findings may elucidate the anatomical substrate for precise regulation of the motoneuron firing required for the fine movements of the tongue, and also suggest that the excitability of small and large GG motoneurons may be regulated differently.

To provide information on the glutamatergic synapses on the trigeminal motoneurons, which may be important for understanding the mechanism of control of jaw movements, we investigated the distribution of vesicular glutamate transporter (VGLUT)1-immunopositive (+) and VGLUT2 + axon terminals (boutons) on the rat jaw-closing (JC) and jaw-opening (JO) motoneurons, and their morphological determinants of synaptic strength by retrograde tracing, electron microscopic immunohistochemistry, and quantitative ultrastructural analysis. We found that (1) the large majority of VGLUT + boutons on JC and JO motoneurons were VGLUT2+, (2) the density of VGLUT1 + boutons terminating on JC motoneurons was significantly higher than that on JO motoneurons, (3) the density of VGLUT1 + boutons terminating on non-primary dendrites of JC motoneurons was significantly higher than that on somata or primary dendrites, whereas the density of VGLUT2 + boutons was not significantly different between JC and JO motoneurons and among various compartments of the postsynaptic neurons, and (4) the bouton volume, mitochondrial volume, and active zone area of the VGLUT1 + boutons forming synapses on JC motoneurons were significantly bigger than those of VGLUT2 + boutons. These findings suggest that JC and JO motoneurons receive glutamatergic input primarily from VGLUT2-expressing intrinsic neurons (premotoneurons), and may be controlled differently by neurons in the trigeminal mesencephalic nucleus and by glutamatergic premotoneurons.

Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold (‹17� C) and pungent compounds, is implicated in nociception, but little is known about the coexpression of TRPA1 and other channels or receptors involved in the nociception in craniofacial regions. To address this issue, we characterized the TRPA1-immunopositive (+) neurons in the rat trigeminal ganglion (TG) and investigated their colocalization with other proteins known to be expressed in nociceptive neurons, such as transient receptor potential vanilloid (TRPV1) and P2X3 receptor, using light microscopic immunofluorescence labeling method with TRPA1 and TRPV1 or P2X3 antisera. The majority of TRPA1+ neurons costained for TRPV1 (TRPV1+/TRPA1+; 58.8%, 328/558) and 41.2% only expressed TRPA1 but not TRPV1. The TRPV1+/TRPA1+ neurons were small and medium sized. In addition, we investigated the colocalization of TRPA1 with P2X3, a nonselective cation channel activated by ATP that may be released in the extracellular space as a result of tissue damage and inflammation. Among all TRPA1+ TG neurons, 26.1% (310/1186) costained for P2X3, whereas 73.9% (876/1186) of TRPA1+ neurons did not coexpress P2X3. P2X3 +/TRPA1+ neurons were predominantly small and medium sized. These results suggest that TRPA1+ neurons coexpressing TRPV1 or P2X3 are involved in specific roles in the transmission and processing of orofacial nociceptive information by noxious cold, heat, and inflammation.

The goal of this study was to identify neurotransmitters in endings (p-endings) presynaptic to low-threshold mechanoreceptive vibrissa afferents in the laminae III/IV of cat trigeminal caudal nucleus (Vc). Rapidly-adapting vibrissa afferents were intra-axonally labeled after electrophysiological identification, and postembedding immunogold staining with antisera against -aminobutyric acid (GABA) and glycine was performed, followed by quantitative ultrastructural analysis of p-endings presynaptic to the labeled vibrissa afferent terminals. Sixteen p-endings, which are presynaptic to the HRP-labeled vibrissa afferent terminals, were analyzed in this study: Eight p-endings (50%, 8/16) were immunopositive to GABA but immunonegative to glycine (GABA+ p-ending), and remaining 8 p-endings (50%, 8/16) exhibited immunoreactivity to both GABA and glycine. Bouton volume of the p-endings was not significantly different between the two groups. However, the p-endings differed from each other in relative content of GABA and glycine. These findings suggest that low-threshold mechanoreceptive information conveyed through vibrissa afferent at Vc is presynaptically modulated by GABA and/or glycine, and that degree of presynaptic modulation may differ among each vibrissa afferent terminal.

Inhibitory and excitatory synaptic inputs onto trigeminal motoneurons play an important role in coordinating jaw movements. Previously, we reported that the phenotype of the inhibitory boutons apposing the somata of jaw-closing (JC) motoneurons changes from γ-aminobutyric acid (GABA)-positive (GABA+) to predominantly glycine-positive (Gly+) during development. In the present study, we investigated the development of inhibitory and excitatory boutons apposing antagonistic jaw-opening (JO) motoneurons (anterior digastric motoneurons) at postnatal day 2 (P2), P11, and P31 in the rat. JO motoneurons were retrogradely labeled with horseradish peroxidase. Postembedding immunogold staining with antisera against GABA, Gly, and glutamate (Glut) was performed and followed by quantitative ultrastructural analysis. The size of both small and large JO motoneurons increased during development. The number of excitatory (Glut+) and inhibitory (GABA+, Gly+, and GABA+/Gly+) boutons per JO motoneuron increased significantly from P2 to P11 and then remained unchanged until P31. The time course of inhibitory synapse formation differed between JO and JC motoneurons, whereas that of excitatory synapse formation was similar between the two neuronal populations. The fraction of GABA+ boutons decreased by 86% and the fraction of GABA+/Gly+ boutons increased by 200% from P11 to P31, suggesting a switch from GABA+ to GABA+/Gly+ phenotype. The fraction of Gly+ boutons remained unchanged. These results indicate that inhibitory synapses onto somata of JO motoneurons exhibit a developmental pattern distinct from that of synapses onto JC motoneurons, which may reflect distinctive maturation of oral motor system. J. Comp. Neurol. 520:1212–1226, 2012. © 2011 Wiley Periodicals, Inc.

Ultrastructural parameters related to synaptic release and their correlation with synaptic connectivity were analyzed in the low-threshold mechanoreceptive vibrissa afferent boutons in laminae III and IV of the trigeminal caudal nucleus (Vc). Rapidly adapting vibrissa afferents were intra-axonally labeled, and quantitative ultrastructural analyses with serial sections were performed on the labeled boutons and their presynaptic endings (p-endings). The volume of the labeled boutons was widely distributed from small to large ones (0.8~12.3 µm(3)), whereas the p-endings were small and uniform in size. The volume of the labeled boutons was positively correlated with the ultrastructural parameters such as mitochondrial volume (correlation coefficient, r=0.96), active zone area (r=0.82) and apposed surface area (r=0.79). Vesicle density (r=-0.18) showed little correlation to the volume of labeled boutons, suggesting that the total vesicle number of a bouton is proportional to its volume. In addition, the bouton volume was positively correlated with the number of p-endings (r=0.52) and with the number of dendrites postsynaptic to the labeled bouton (r=0.83). These findings suggest that low-threshold mechanoreception conveyed through vibrissa afferents is processed in a bouton size-dependent manner in the Vc, which may contribute to the sensory-motor function of laminae III/IV in Vc.

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι−/− synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι−/− synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

Introduction Previous studies have suggested that myelinated axons lose their myelin and become thinner in their peripheral course to the target organ. In this study, we investigated the morphologic changes of pulpal myelinated axons between their root portion (radicular pulp) and their terminal area (peripheral pulp). Methods Sections of pulp of the rat upper molar teeth were immunostained for the marker of myelinated axons neurofilament (NF) 200. The proportion of NF200+ myelinated and unmyelinated fibers and their sizes were analyzed by using quantitative electron microscopy. Results The axon area, myelin thickness, and fraction of NF200+ myelinated axons of all NF200+ axons were significantly lower in peripheral than in radicular pulp. In addition, large unmyelinated axons were frequently observed in peripheral pulp. Conclusions These results suggest that pulpal innervation originates predominantly from myelinated axons, and the myelinated axons undergo extensive morphologic changes during their course from the radicular to the peripheral pulp.

Inhibition of Cdc42 by dRich induces postsynaptic release of the BMP ligand Glass bottom boat., Retrograde bone morphogenetic protein signaling mediated by the Glass bottom boat (Gbb) ligand modulates structural and functional synaptogenesis at the Drosophila melanogaster neuromuscular junction. However, the molecular mechanisms regulating postsynaptic Gbb release are poorly understood. In this study, we show that Drosophila Rich (dRich), a conserved Cdc42-selective guanosine triphosphatase–activating protein (GAP), inhibits the Cdc42–Wsp pathway to stimulate postsynaptic Gbb release. Loss of dRich causes synaptic undergrowth and strongly impairs neurotransmitter release. These presynaptic defects are rescued by targeted postsynaptic expression of wild-type dRich but not a GAP-deficient mutant. dRich inhibits the postsynaptic localization of the Cdc42 effector Wsp (Drosophila orthologue of mammalian Wiskott-Aldrich syndrome protein, WASp), and manifestation of synaptogenesis defects in drich mutants requires Wsp signaling. In addition, dRich regulates postsynaptic organization independently of Cdc42. Importantly, dRich increases Gbb release and elevates presynaptic phosphorylated Mad levels. We propose that dRich coordinates the Gbb-dependent modulation of synaptic growth and function with postsynaptic development.

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

The morphology of intradental nerve fibers of permanent teeth and of continuously growing rodent incisors has been studied in detail but little information is available on the parent axons that give rise to these fibers. Here we examined the axons and somata of trigeminal neurons that innervate the rat upper molar and lower incisor pulp using tracing with horseradish peroxidase and light and electron microscopic analysis. The majority (approximately 80%) of the parent axons in the proximal root of the trigeminal ganglion that innervated either molar or incisor pulp were small myelinated fibers (20 microm(2) cross-sectional area). The remaining approximately 20% of the fibers were almost exclusively large myelinated for the molar pulp and unmyelinated for the incisor pulp. The majority of neuronal somata in the trigeminal ganglion that innervated either molar (48%) or incisor pulp (62%) were medium in size (300-600 microm(2) cross-sectional area). Large somata (600 microm(2)) constituted 34% and 20% of the trigeminal neurons innervating molar and incisor pulp, respectively, while small somata (300 microm(2)) constituted 17% of the molar and 18% of the incisor neurons. The present study revealed that the morphology of parent axons of dental primary sensory neurons may differ from that of their intradental branches, and also suggests that the nerve fiber function may be carried out differently in the molar and incisor pulp in the rat.

The supratrigeminal region (Vsup) is important for coordination of smooth jaw movement. However, little is known about the synaptic connections of the Vsup premotoneurons with the trigeminal motor neurons. In the present study, we examined axon terminals of Vsup premotoneurons in the contralateral trigeminal motor nucleus (Vmo) by a combination of anterograde tracing with cholera toxin B-horseradish peroxidase (CTB-HRP), postembedding immunohistochemistry for the amino acid transmitters glutamate, GABA, and glycine, and electron microscopy. Tracer injections resulted in anterograde labeling of axon terminals of the Vsup premotoneurons in the motor trigeminal nucleus (Vmo). The labeled boutons in Vmo exhibited immunoreactivity for glutamate, GABA, or glycine: glutamate-immunopositive boutons (69%) were more frequently observed than GABA- or glycine-immunopositive boutons (19% and 12%, respectively). Although most labeled boutons (97%) made synaptic contacts with a single postsynaptic dendrite, a few glutamate-immunopositive boutons (3%) showed synaptic contact with two dendrites. No labeled boutons participated in axoaxonic synaptic contacts. Most labeled boutons (78%) were presynaptic to dendritic shafts, and the remaining 22% were presynaptic to somata or primary dendrites. A large proportion of GABA- or glycine-immunopositive boutons (40%) were presynaptic to somata or primary dendrites, whereas most glutamate-immunopositive boutons (86%) were presynaptic to dendritic shafts. These results indicate that axon terminals of Vsup premotoneurons show simple synaptic connection with Vmo neurons. This may provide the anatomical basis for the neural information processing responsible for jaw movement control.

Trigeminal primary afferents expressing P2X3 receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X3-immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X3 immunoreactivity was mainly in small and medium-sized somata, but also frequently in large somata. Although most P2X3+ somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X3+ fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X3 immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X3+ terminals contained numerous clear, round vesicles and sparse large, dense-core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X3+ terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X3+ afferents to all TBSN and that it may be processed differently in different TBSN. J. Comp. Neurol. 506:627–639, 2008. © 2007 Wiley-Liss, Inc.

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.

This study examined the ultrastructures of neuronal elements within trigeminal mesencephalic nucleus by labeling masseteric mesencephalic neurons and masseter motoneurons with injection of horseradish peroxidase into masseteric muscle. Of eight horseradish peroxidase-labeled muscle spindle afferents examined, four terminals showed synaptic contact with labeled dendrites of masseteric motoneurons, two with labeled somata, and the remaining two with unlabeled dendrites. A few of the labeled dendrites showed intimate contact with the somata of the trigeminal mesencephalic nucleus neurons. These results provide morphological evidence of synaptic contact of recurring masseteric muscle spindle afferents with the trigeminal mesencephalic nucleus somata and also suggest the presence of electrical synapses between the somata of the trigeminal mesencephalic nucleus neurons and dendrites of jaw-closing motoneurons.

Introduction Vesicular glutamate transporters (VGLUTs) are involved in the transport of transmitter glutamate into synaptic vesicles and are used as markers for glutamatergic neurons. Methods To assess which types of VGLUTs are involved in the glutamate signaling in pulpal axons and to investigate their distribution, we performed light microscopic immunohistochemistry by using antibodies against VGLUT1, VGLUT2, calcitonin gene-related peptide, and Western blot analysis in human dental pulp. Results VGLUT1 was expressed in a large number of pulpal axons, especially in the peripheral pulp where the axons branch extensively. The VGLUT1 immunopositive axons showed bead-like appearance, and the majority of these also expressed calcitonin gene-related peptide. VGLUT2 was expressed in few axons throughout the pulp. Conclusions Our findings suggest that VGLUT1 is involved mainly in the glutamate-mediated signaling of pain, primarily at the level of the peripheral pulp.

The excitatory synapses on the jaw-closing (JC) motoneurons mediate the neuronal input that ensures smooth and rhythmic movements of the jaw. Recently, we have shown that the neurotransmitter phenotype of the inhibitory boutons onto JC motoneurons shifts from GABA to glycine, and new inhibitory synapses onto JC motoneurons are continuously formed during postnatal development (Paik et al. [2007] J. Comp. Neurol. 503:779–789). To test whether the developmental pattern of the excitatory synapses onto JC motoneurons differs from that of the inhibitory synapses, we studied the distribution of glutamate-immunopositive boutons onto the rat JC motoneurons during postnatal development by using a combination of retrograde labeling with horseradish peroxidase (HRP), postembedding immunogold staining, and quantitative ultrastructural analysis. The analysis of 175, 281, and 465 boutons contacting somata of JC motoneurons at postnatal days P2, P11, and P31, respectively, revealed that the number of glutamate-immunopositive (Glut+) boutons increased by 2.6 times from P2 to P11 and showed no significant change after that, whereas the length of apposition of these boutons increased continuously from P2 to P31, suggesting that the time course for the development of Glut+ boutons differed from that for Glut− boutons, most of which were immunopositive for GABA and/or glycine. Our findings indicate that excitatory and inhibitory synapses onto JC motoneurons exhibit distinctly different developmental patterns that may be closely related to the maturation of the masticatory system. © 2010 Wiley-Liss, Inc.

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

Accumulating evidence indicates that the metabotropic glutamate receptor mGluR5 is involved in the peripheral mechanisms of inflammatory nociception. To investigate whether mGluR5 may mediate the inflammatory pain and thermal hyperalgesia in the dental pulp, we examined the expression of mGluR5 and transient receptor potential vanilloid 1 (TRPV1) in human dental pulp by immunohistochemistry and electron microscopy; mGluR5-immunopositive (+) axons were observed in nerve bundles and branched extensively within the peripheral coronal pulp. Most of the mGluR5+ axons were unmyelinated. A large fraction of these axons (36.5%) were immunostained for TRPV1. Immunoreactivity for mGluR5 and TRPV1 was also observed in odontoblasts. These results support the possibility that the nerve fibers in the dental pulp mediate inflammatory pain and thermal hyperalgesia through coactivation of mGluR5 and TRPV1 and also suggest a possible role for odontoblasts in the transduction of nociceptive signals via mGluR5-mediated mechanism.

Hereditary spastic paraplegia (HSP) is an inherited neurological disorder characterized by progressive spasticity and weakness of the lower extremities. The most common early-onset form of HSP is caused by mutations in the human gene that encodes the dynamin-family GTPase Atlastin-1 (Atl-1). Recently, loss of the Drosophila ortholog of Atl-1 (Atl) has been found to induce locomotor impairments from the earliest adult stages, suggesting the developmental role of atlastin-subfamily GTPases. Here, we provide evidence that Atl is required for normal growth of muscles and synapses at the neuromuscular junction (NMJ). Atl protein is highly expressed in larval body-wall muscles. Loss-of-function mutations in the atl gene reduce the size of muscles and increase the number of synaptic boutons. Rescue of these defects is accomplished by muscular, but not neuronal expression of Atl. Loss of Atl also disrupts ER and Golgi morphogenesis in muscles and reduces the synaptic levels of the scaffold proteins Dlg and α-spectrin. We also provide evidence that Atl functions with the microtubule-severing protein Spastin to disassemble microtubules in muscles. Finally, we demonstrate that the microtubule-destabilizing drug vinblastine alleviates synapse and muscle defects in atl mutants. Together, our results suggest that Atl controls synapse development and ER and Golgi morphogenesis by regulating microtubule stability.

The microarchitecture of collagen fibrils in the articular disc of the temporomandibular joint (TMJ) plays an important role in dissipating the mechanical load during jaw movement. However, little information is available on its adaptations to the biomechanical environment during development. To address this issue, we analyzed the diameter of collagen fibrils of the articular disc of the rat TMJ with quantitative ultrastructural analysis during postnatal development. The mean diameter of the collagen fibrils significantly increased and the arrangement of the collagen fiber networks became compact during development. Articular discs of suckling rat pups were composed of thin, uniformly sized collagen fibrils (range: 30-60 nm, peak: 40-50 nm). At the age of 4 weeks, thicker collagen fibrils began to appear in articular discs, shortly after weaning (range: 20-70 nm, peak: 40-50 nm). In articular discs of adult rats, collagen fibrils varied widely in diameter, with thick fibrils predominating (range: 10-120 nm, peak: 40-70 nm). These age-related changes in the microarchitecture of collagen fibrils in articular discs may reflect changes in their biomechanical environment during development.

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.

Signal substances of axon terminals presynaptic to jaw spindle Ia afferents and their ultrastructural features were examined using a combination of intra-axonal horseradish peroxidase injection and postembedding immunogold-labeling techniques in cats. A total of 35 axon terminals presynaptic to 22 horseradish peroxidase-labeled Ia boutons were examined. Of the 35 presynaptic axon terminals, 14 (40%) were immunoreactive for both gamma-aminobutyric acid and glycine, 9 (26%) for gamma-aminobutyric acid alone and 9 (26%) for glycine alone. The bouton volume, mitochondrial volume, active zone area, and apposed surface area were larger for Ia boutons than for presynaptic axon terminals, while each of the values is similar among the three types of presynaptic axon terminals. These results suggest that gamma-aminobutyric acid and glycine play an important role for modulating the jaw-jerk reflex presynaptically and that the smaller size of presynaptic axon terminals is important to prevent action potential generation from Ia afferents.

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.

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.

This study analyzed quantitatively the ultrastructural features of tooth pulp afferent terminals and their presynaptic axonal endings (p-endings) in the trigeminal principal (Vp), dorsomedial oral (Vdm), and caudal nuclei (Vc). Mitochondrial volume, active zone area, apposed surface area, and vesicle number were highly correlated with afferent bouton volume. The afferent bouton volume varied widely in Vp, compared to that in Vdm and Vc. The values of all parameters of p-endings were within a narrow range, and were smaller than those of afferent boutons. The afferent bouton volume correlated with the number of postsynaptic dendrites and p-endings. These results suggest that pulpal afferent information is regulated in a unique manner in the each trigeminal sensory 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.