Masayuki Moritani

Trigeminal P2X3 receptor expression differs from dorsal root ganglion and is modulated by deep tissue inflammation

&NA; The distribution and modulation of the P2X3 receptor was studied in trigeminal ganglion neurons to provide insight into the role of ATP in craniofacial sensory mechanisms. Binding to the D‐galactose specific lectin IB4 was found in 73% of P2X3‐positive neurons while only 16% of IB4 neurons expressed P2X3. Neurons expressing P2X3 alone were significantly larger than IB4‐or IB4/P2X3‐positive neurons. Investigation of target‐specificity revealed that 22% of trigeminal ganglion muscle afferent neurons were positive for P2X3 versus 16% of cutaneous afferent neurons. Muscle P2X3 afferents were significantly smaller than the overall muscle afferent population while P2X3 cutaneous afferent neurons were not. Presumptive heteromeric (P2X2/3) muscle afferent neurons were also identified and comprised 77% of the P2X3 muscle afferent population. Muscle afferent neurons co‐expressed P2X3 with either calcitonin gene‐related peptide (15%) or substance P (4%). The number of P2X3‐positive muscle afferent neurons significantly increased one and four days following complete Freunds adjuvant‐induced masseter muscle inflammation, but significantly decreased after 12 days. These results indicate that within trigeminal ganglia: (1) the P2X3 receptor is expressed in both small and medium‐sized neurons; (2) the P2X3 receptor is not exclusively expressed in IB4 neurons; (3) P2X3 is co‐expressed with neuropeptides; (4) differences in the proportion of cutaneous versus muscle P2X3 afferents are not apparent. Trigeminal P2X3 neurons therefore differ markedly from dorsal root ganglion P2X3 afferents. This study also shows that deep tissue inflammation modulates expression of the P2X3 receptor and thus may warrant exploration as a target for therapeutic intervention.

Deep tissue inflammation upregulates neuropeptides and evokes nociceptive behaviors which are modulated by a neuropeptide antagonist.

Abstract Promising recent developments in the therapeutic value of neuropeptide antagonists have generated renewed importance in understanding the functional role of neuropeptides in nociception and inflammation. To explore this relationship we examined behavioral changes and primary afferent neuronal plasticity following deep tissue inflammation. One hour following craniofacial muscle inflammation ipsilateral as well as contralateral head withdrawal thresholds and ipsi‐ and contralateral hindpaw withdrawal thresholds were lowered and remained reduced for 28 days. Elevated levels of calcitonin gene‐related peptide (CGRP) within the trigeminal ganglion temporally correlated with this mechanical allodynia. Inflammation also induced an increase in the number of CGRP and substance P (SP)‐immunopositive trigeminal ganglion neurons innervating inflamed muscle but did not evoke a shift in the size distribution of peptidergic muscle afferent neurons. Trigeminal proprioceptive muscle afferent neurons situated within the brainstem in the mesencephalic trigeminal nucleus did not express CGRP or SP prior to or following inflammation. Intravenous administration of CGRP receptor antagonist (8‐37) two minutes prior to adjuvant injection blocked plasma extravasation and abolished both head and hindlimb mechanical allodynia. Local injection of CGRP antagonist directly into the masseter muscle prior to CFA produced similar, but less pronounced, effects. These findings indicate that unilateral craniofacial muscle inflammation produces mechanical allodynia at distant sites and upregulates CGRP and SP in primary afferent neurons innervating deep tissues. These data further implicate CGRP and SP in deep tissue nociceptive mechanisms and suggest that peptide antagonists may have therapeutic potential for musculoskeletal pain.

DISTRIBUTION PATTERN OF INHIBITORY AND EXCITATORY SYNAPSES IN THE DENDRITIC TREE OF SINGLE MASSETER ALPHA -MOTONEURONS IN THE CAT

Little is known about the differences in the distributions of inhibitory and excitatory synapses in the dendritic tree of single motoneurons in the brainstem and spinal cord. In this study, the distribution of γ‐aminobutyric acid (GABA)‐, glycine‐, and glutamate‐like immunoreactivity in axon terminals on dendrites of cat masseter α‐motoneurons, stained intracellularly with horseradish peroxidase, was examined by using postembedding immunogold histochemistry in serial ultrathin sections. The dendritic tree was divided into three segments: primary (Pd) and distal (Dd) dendrites and intermediate (Id) dendrites between the two segments. Quantitative analysis of 175, 279, and 105 boutons synapsing on 13 Pd, 54 Id, and 81 Dd, respectively, was performed. Fifty percent of the total number of studied boutons were immunopositive for GABA and/or glycine and 48% for glutamate. Among the former, 27% showed glycine immunoreactivity only and 14% were immunoreactive to both glycine and GABA. The remainder (9%) showed immunoreactivity for GABA only. As few as 3% of the boutons were immunonegative for the three amino acids. Most boutons immunoreactive to inhibitory amino acid(s) contained a mixture of spherical, oval, and flattened synaptic vesicles. Most boutons immunoreactive to excitatory amino acid contained clear, spherical, synaptic vesicles with a few dense‐cored vesicles. When comparisons of the inhibitory and excitatory boutons were made between the three dendritic segments, the proportion of the inhibitory to the excitatory boutons was high in the Pd (60% vs. 37%) but somewhat low in the Id (46% vs. 52%) and Dd (44% vs. 53%). The percentage of synaptic covering and packing density of the inhibitory synaptic boutons decreased in the order Pd, Id, and Dd, but this trend was not applicable to the excitatory boutons. The present study provides possible evidence that the spatial distribution patterns of inhibitory and excitatory synapses are different in the dendritic tree of jaw‐closing α‐motoneurons. J. Comp. Neurol. 414:454–468, 1999.

Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat

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.

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. J. Comp. Neurol. 383:94–111, 1997.

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.

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

Little is known about the organization of corticofugal projections controlling antagonistic jaw muscles. To address this issue, we employed retrograde (Fluorogold; FG) and anterograde (biotinylated dextran amine; BDA) tracing techniques in rats. Three groups of premotoneurons were identified by injecting FG into the jaw‐closing (JC) and ‐opening (JO) subdivisions of the trigeminal motor nucleus (Vmo). These were 1) the intertrigeminal region (Vint) and principal trigeminal sensory nucleus for JC nucleus; 2) the reticular region medial to JO nucleus (RmJO) for JO nucleus; and 3) the parabrachial (Pb) and supratrigeminal (Vsup) nuclei, reticular regions medial and ventral to JC nucleus, rostrodorsomedial oralis (Vor), and juxtatrigeminal region (Vjuxt) containing a mixture of premotoneurons to both the nuclei. Subsequently, FG was injected into the representative premotoneuron structures. The JC and JO premotoneurons received main afferents from the lateral and medial agranular fields of motor cortex (Agl and Agm), respectively, whereas afferents to the nuclei with both JC and JO premotoneurons arose from Agl also and from primary somatosensory cortex (S1). Finally, BDA was injected into each of the three cortical areas representing the premotoneuron structures to complement the FG data. The Agl and Agm projected to reticular regions around the Vmo, whereas the Pb, Vsup, Vor, and Vjuxt received input from Agl. The S1 projected to the trigeminal sensory nuclei as well as to the Pb, Vsup, and Vjuxt. These results suggest that corticofugal projections to Vmo via premotoneuron structures consist of multiple pathways, which influence distinct patterns of jaw movements. J. Comp. Neurol. 514:368–386, 2009.

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

Little is known about the differences of the terminations of group Ia and group II afferents within the brainstem or spinal cord. The present study was performed to classify cat jaw muscle spindle afferents by the use of succinylcholine (SCh) and to examine the morphological characteristics of the physiologically classified afferents at the light and electron microscopic levels through the use of the intra‐axonal horseradish peroxidase (HRP) injection technique. The effects of SCh on stretch responses of 119 jaw muscle spindle afferents from the masseter were examined. The SCh converted the single skew distribution of the values for dynamic index (DI) into a bimodal one. Fifty‐eight and 61 afferents were classified as group Ia and group II afferents, respectively. The central projections of 17 intra‐axonally stained afferents (10 group Ia and 7 group II afferents) were examined. The spindle afferents terminated mainly in the supratrigeminal nucleus (Vsup), region h, and the dorsolateral subdivision of trigeminal motor nucleus (Vmo.dl) but differed in the pattern of projections of group Ia and group II afferents. The proportion of group Ia afferent terminals was higher in Vmo.dl but lower in Vsup than that of group II afferents. In Vmo.dl, the proportion of group Ia afferent terminals was higher in the central region but lower in the more outer regions than that of group II afferents. The ultrastructure of serially sectioned afferent boutons (63 group Ia and 72 group II boutons) also was examined. The boutons from the two groups were distributed widely from the soma to small‐diameter dendrites, but the frequency of synaptic contacts on proximal dendrites was higher in group Ia than group II afferents. The present study provides evidence that the two groups of jaw muscle spindle afferents differ in their central projection and the spatial distribution of their synaptic contacts on Vmo.dl neurons. J. Comp. Neurol. 391:50–63, 1998.

Morphologic characteristics of physiologically defined neurons in the cat trigeminal nucleus principalis

Although the principalis nucleus (Vp) contains trigeminothalamic and internuclear tract cells, the functional and morphologic differences between the two kinds of neurons have remained unsettled. The present study was aimed to address these problems by using the intracellular horseradish peroxidase injection technique in the cat. Of 20 neurons stained, 7 and 13 were located in the dorsomedial subnucleus (Vpd) and ventrolateral subnucleus (Vpv) of Vp, respectively. The Vpd neurons received input from the intraoral structures only but the Vpv neurons from the intraoral or facial structures. Nineteen neurons could be divided as class I and class II, based on the branching pattern of their stem axons. Class I (eight neurons) had an ascending stem axon without branching. Class II was divided into two subclasses (IIa and IIb). Class IIa (eight neurons) had an ascending stem axon from which branches were given off. Their branches formed a local‐circuit restricted to the lower brainstem. Class IIb (three neurons) had a stem axon that formed the local‐circuit only. The dendritic morphology was indistinguishable between different classes of neurons and between the subdivisions. Although the dendritic arborization pattern was governed by the location of the somata, it was suggested to be also important elements for determining primary afferent arborizations. In the brainstem nuclei, the jaw‐closing motor nucleus received the highest density of projections from class II neurons with the receptive field involving the periodontal ligaments. The present study provides new findings that Vp neurons could be divided into three distinct populations and suggests that each population exerts a distinct function with respect to sensory discrimination, sensorimotor reflexes, or both. J. Comp. Neurol. 401:308–328, 1998.

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) and jaw‐opening (JO) α‐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) α‐motoneurons and an EPSP with an action potential in JO (mylohyoid) α‐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 α‐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. J. Comp. Neurol. 384:443–455, 1997.