Spinal Trigeminal Subnucleus Oralis Research Articles

Postsynaptic N-type or P/Q-type calcium channels mediate long-term potentiation by group I metabotropic glutamate receptors in the trigeminal oralis

AimsBoth N-type and P/Q-type voltage-gated Ca2+ channels (VGCCs) are involved in the induction of long-term potentiation (LTP), the long-lasting increase of synaptic strength, in the central nervous system. To provide further information on the roles of N-type and P/Q-type VGCCs in the induction of LTP at excitatory synapses of trigeminal primary afferents in the spinal trigeminal subnucleus oralis (Vo), we investigated whether they contribute to the induction of LTP by activation of group I metabotropic glutamate receptors (mGluRs). Main methods(S)-3,5-Dihydroxyphenylglycine (DHPG; 10μM for 5min), the group I mGluR agonist, was used to induce LTP of excitatory postsynaptic currents that were evoked in the Vo neurons by stimulating the trigeminal track. Key findingsWeak blockade of the N-type or P/Q-type VGCCs by ω-conotoxin GVIA or ω-agatoxin IVA, respectively, which inhibited only 20−40% of Ca2+ currents recorded in isolated trigeminal ganglion neurons but had no effect on the basal excitatory synaptic transmission, completely blocked the induction of LTP. In contrast, stronger blockade of the channels, which inhibited >50% of Ca2+ currents and about 30% of basal synaptic transmission, resulted in the development of long-term depression (LTD), the long-lasting decrease of synaptic strength. Interestingly, the postsynaptic mechanism of DHPG-induced LTP, which was determined by paired-pulse ratio, disappeared when LTP was blocked, or LTD occurred, while a presynaptic mechanism still remained. SignificanceOur data suggest that postsynaptic N-type and P/Q-type VGCCs mediate the DHPG-induced LTP at the trigeminal afferent synapses in the Vo.

Differential roles of signal transduction mechanisms in long-term potentiation of excitatory synaptic transmission induced by activation of group I mGluRs in the spinal trigeminal subnucleus oralis.

Group I metabotropic glutamate receptors (mGluR1 and 5) have been implicated in long-term potentiation (LTP), a persistent increase of synaptic efficiency, in the central nervous system including the spinal trigeminal nucleus (Vsp). In the ascending pathway from the caudalis (Vc) to the oralis (Vo) subnuclus in Vsp, it has been shown that the activation of group I mGluRs (mGluR1 and 5) with their agonist (S)-3,5-dihydroxyphenylglycine (DHPG) produces a delayed type of LTP of excitatory synaptic transmission and this LTP was mediated by mGluR1. Further, this study attempts to pharmacologically characterize essential signaling components for the expression of DHPG-induced LTP. As a result, it is found that the group I mGluRs essentially use G protein-mediated activation of the phospholipase C (PLC) pathway to express the LTP. However, recruited signaling molecules following the activation of PLC are differentially involved in the expression of LTP: i.e. IP3 receptor, intracellular Ca(2+) rise, CaMKII and ERK function as positive regulators, whereas PKC as a negative regulator. Furthermore, both L-type voltage-dependent Ca(2+) channel and canonical transient receptor potential channel positively contribute to the expression of LTP. Taken together, these results suggest that signaling molecules recruited by the activation of group I mGluRs collaboratively or oppositely control the optimal expression of synaptic plasticity at excitatory synapses in the Vo.

Long-term potentiation by activation of group I metabotropic glutamate receptors at excitatory synapses in the spinal trigeminal subnucleus oralis

In this study, I examined if activation of group I metabotropic glutamate receptors (mGluRs; mGluR1 and 5) induces long-term potentiation (LTP) at excitatory synapses in the ascending pathway from the spinal trigeminal subnuclei caudalis (Vc) to oralis (Vo), in which group I mGluRs are strongly expressed. As a result, the activation of group I mGluRs produced an initial short-lasting depression and subsequently a delayed type of long-term potentiation (LTP) of excitatory synaptic transmission. Analyses of paired pulse ratio and coefficient of variation indicated that the initial short-lasting depression was induced presynaptically, whereas LTP was expressed postsynaptically. In addition, the short-lasting depression and LTP were mostly mediated by mGluR1, and only partially by mGluR5 and the N-methyl-d-aspartate receptor. Thus, this study suggests that group I mGluRs play an important role in the expression of LTP in the Vc-to-Vo pathway.

NMDA receptor, PKC and ERK prevent fos expression induced by the activation of group I metabotropic glutamate receptors in the spinal trigeminal subnucleus oralis.

Fos, a protein product of immediate early gene c-fos, has been used as a marker for activation of nociceptive neurons in central nervous system including spinal trigeminal nucleus (Vsp). By noxious stimulation applied to orofacial area, the expression of Fos occurred in the Vsp pars oralis (Vo), the subnucleus receiving inputs from trigeminal primary afferents that predominantly innervate intraoral receptive fields. The present study demonstrates that the in vitro activation of group I metabotropic glutamate receptors (mGluRs; mGluR1 and 5) by bath-application of their well-known agonist (S)-3,5-dihydroxyphenylglycine (DHPG) increased the number of Fos-expressing neurons in the Vo area. In addition, bath application of DHPG caused inward currents, a parameter of neuronal excitation, in the Vo neurons held at -70 mV in voltage-clamp mode of whole-cell recordings. In further experiments characterizing two phenomena, the increased Fos expression in the Vo was mediated by an additive activation of both mGluR1 and mGluR5, which required the activation of N-methyl-D-aspartate (NMDA) receptors, protein kinase C (PKC) and extracellular signal-regulated kinase (ERK). In contrast, the inward currents were mediated only by mGluR1, but not by others. The data resulting from this in vitro study indicate that the DHPG-induced membrane depolarisation or neuronal excitation may be upstream to, or skip, the NMDA receptor, PKC and ERK pathways for the DHPG-induced Fos expression.

Organization of projections from the spinal trigeminal subnucleus oralis to the spinal cord in the rat: A neuroanatomical substrate for reciprocal orofacial–cervical interactions

The organization of efferent projections from the spinal trigeminal nucleus oralis (Sp5O) to the spinal cord in the rat was studied using the anterograde tracer Phaseolus vulgaris leucoagglutinin. Sp5O projections to the spinal cord are restricted to the cervical cord. No labeled terminal can be detected in the thoracic and lumbar cord. The organization of these projections happens to critically depend on the dorso-ventral location of the injection site. On the one hand, the dorsal part of the Sp5O projects to the medial part of the dorsal horn (laminae III–V) at the C1 level, on the ipsilateral side, and to the ventral horn, on both sides but mainly on the ipsilateral one. Ipsilateral labeled terminals are distributed throughout laminae VII to IX but tend to cluster around the dorso-medial motor nuclei, especially at C3–C5 levels. Within the contralateral ventral horn, label terminals are found particularly in the region of the ventro-medial motor nucleus. This projection extends as far caudally as C3 or C4 level. On the other hand, the ventral part of the Sp5O projects to the lateral part of the dorsal horn (laminae III–V) at the C1 level, on the ipsilateral side, and to the ventral horn, on both sides but mainly on the contralateral one. Contralateral labeled terminals are distributed within the region of the dorso- and ventro-medial motor nuclei at C1–C4 levels whereas they are restricted to the dorso-medial motor nucleus at C5–C8 levels. These findings suggest that Sp5O is involved in the coordination of neck movements and in the modulation of incoming sensory information at the cervical spinal cord.

Signal Transduction Mechanisms Underlying Group I Mglur-Mediated Increase in Frequency and Amplitude of Spontaneous EPSCs in the Spinal Trigeminal Subnucleus Oralis of the Rat

Group I mGluRs (mGluR1 and 5) pre- and/or postsynaptically regulate synaptic transmission at glutamatergic synapses. By recording spontaneous EPSCs (sEPSCs) in the spinal trigeminal subnucleus oralis (Vo), we here investigated the regulation of glutamatergic transmission through the activation of group I mGluRs. Bath-applied DHPG (10 μM/5 min), activating the group I mGluRs, increased sEPSCs both in frequency and amplitude; particularly, the increased amplitude was long-lasting. The DHPG-induced increases of sEPSC frequency and amplitude were not NMDA receptor-dependent. The DHPG-induced increase in the frequency of sEPSCs, the presynaptic effect being further confirmed by the DHPG effect on paired-pulse ratio of trigeminal tract-evoked EPSCs, an index of presynaptic modulation, was significantly but partially reduced by blockades of voltage-dependent sodium channel, mGluR1 or mGluR5. Interestingly, PKC inhibition markedly enhanced the DHPG-induced increase of sEPSC frequency, which was mainly accomplished through mGluR1, indicating an inhibitory role of PKC. In contrast, the DHPG-induced increase of sEPSC amplitude was not affected by mGluR1 or mGluR5 antagonists although the long-lasting property of the increase was disappeared; however, the increase was completely inhibited by blocking both mGluR1 and mGluR5. Further study of signal transduction mechanisms revealed that PLC and CaMKII mediated the increases of sEPSC in both frequency and amplitude by DHPG, while IP3 receptor, NO and ERK only that of amplitude during DHPG application. Altogether, these results indicate that the activation of group I mGluRs and their signal transduction pathways differentially regulate glutamate release and synaptic responses in Vo, thereby contributing to the processing of somatosensory signals from orofacial region.

Jaw-muscle spindle afferent feedback to the cervical spinal cord in the rat.

Putative synaptic contacts between masticatory-muscle spindle afferents and brainstem neurons which project to the cervical spinal cord were studied in rats by combining retrograde and intracellular neuronal labeling. Spinal cord projecting neurons were retrogradely labeled via injection of horseradish peroxidase unilaterally or bilaterally into cervical spinal cord segments C2 through C5. Twenty-four hours after the injection of horseradish peroxidase, one to five jaw-muscle spindle afferent axons were physiologically identified and intracellularly stained with biotinamide on each side of the brainstem. Horseradish-peroxidase-labeled neurons were found bilaterally in the supratrigeminal region, trigeminal principal sensory nucleus, parvicellular reticular nucleus including its alpha division, spinal trigeminal subnuclei oralis and interpolaris and the medullary reticular formation. Retrogradely labeled neurons were most numerous in the spinal trigeminal subnucleus oralis, parvicellular reticular formation and the ventral part of the spinal trigeminal subnucleus interpolaris. A small number of horseradish-peroxidase-labeled neurons were also present in the trigeminal mesencephalic nucleus and spinal trigeminal subnucleus caudalis. Appositions between jaw-muscle spindle afferent boutons and spinal projecting neurons were found in the supratrigeminal region, dorsomedial portions of the trigeminal principal sensory nucleus and spinal trigeminal subnuclei oralis and interpolaris, and the parvicellular reticular formation including its alpha division. Putative synaptic contacts were most frequent in the parvicellular reticular formation and the dorsomedial portion of the trigeminal subnucleus oralis. These results indicate that some orofacial proprioceptive feedback transmitted via the mesencephalic trigeminal nucleus reaches the cervical spinal cord directly and suggests that jaw-muscle spindle afferent feedback reaches the cervical spinal cord predominately via relays in the dorsomedial part of the spinal trigeminal subnucleus oralis and the parvicellular reticular formation. It is hypothesized that these pathways are primarily involved in the coordination of jaw and neck movement during mastication and biting.

Effects of lesions in the trigeminal oralis and caudalis subnuclei on different orofacial nociceptive responses in the rat

Behavioural responses to two different orofacial noxious stimulations were analysed following lesion of spinal trigeminal subnucleus oralis (Sp5O) in the rat. Lesions were obtained by intranuclear microinjections of quinolinic acid (0.4 μl of 60 nmol/μl solution). The control groups received microinjection of saline. Noxious stimulation was a subcutaneous injection of formalin into the upper lip or electrical stimulation of the tooth pulp. The measured behavioural responses were duration of rubbing induced by the formalin injection and thresholds of the jaw-opening reflex (JOR), head rotation (HR) and face rubbing (FR) evoked by the pulp stimulation. In addition, formalin injection was also performed in two groups of rats that had received intranuclear injection of quinolinic acid or saline into rostral subnucleus caudalis (Sp5C). Rubbing duration was not significantly modified by the lesion of Sp5O, whereas a significant decrease occurred after the lesion of rostral Sp5C. After the lesion of Sp5O, an increase in the threshold of JOR was observed whereas the thresholds of HR and FR were not significantly modified. These results suggest that Sp5O is not necessary for the processing and relay of nociceptive inputs triggered by intense stimulations of oral and perioral areas. However further experiments are needed to reconcile these results with the relevant data obtained from cell recording experiments which indicate the existence, in Sp5O, of neuronal activities related to the sensory discriminative aspect of intense nociception.

Central terminals of orofacial primary afferents and NADPH-diaphorase activity in the trigemino–solitary complex of rats

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and the central terminal fields of branches of the mandibular and chorda tympani nerves were visualized histochemically at the same time using transganglionic transport of wheat germ agglutinin conjugated with horseradish peroxidase. The blue NADPH-d-positive neurons comprised a sparse network in the dorsomedial spinal trigeminal subnucleus oralis and a dense one in the rostral lateral division of the nucleus of the solitary tract. In the subnucleus caudalis, most labeled neurons were in the superficial zone, and smaller numbers were in the magnocellular zone. The NADPH-d-positive neurons in the subnucleus oralis and the nucleus of the solitary tract overlapped mostly with the transganglionically labeled terminal field from the lingual nerve, partly with the terminal field from the inferior alveolar and chorda tympani nerves, and rarely with the terminal field from the mental nerve. The NADPH-d-positive neurons in the dorsomedial paratrigeminal nucleus and subnucleus caudalis overlapped mostly with the terminal field from the lingual nerve, partly with the terminal field from the inferior alveolar and mental nerves and never with the terminal field from the chorda tympani. A statistically significant reduction in the number of NADPH-d-positive neurons was seen bilaterally in subnucleus oralis and the nucleus of the solitary tract when the lingual nerve was transected. Inflammatory insults to the lingual nerve or tooth pulps significantly increased the number of NADPH-d-positive neurons in subnucleus oralis, the nucleus of the solitary tract, and subnucleus caudalis. These results show that the NO/cyclic GMP system in the trigeminal and solitary nuclei is differentially regulated trans-synaptically by trigeminal afferents depending on the nucleus and sensory modality.

Projection of jaw‐muscle spindle afferents to the caudal brainstem in rats demonstrated using intracellular biotinamide

Intracellular staining with biotinamide was used to study the axonal projection and synaptic morphology of rat jaw-muscle spindle afferents. Intracellular recordings in the mesencephalic trigeminal nucleus (Vme) were identified as spindle afferent responses by their increased firing during stretching of the jaw-elevator muscles. Biotinamide-stained axon collaterals with boutons were found in the trigeminal motor nucleus (Vmo), Vme, the region dorsal to Vmo including the supratrigeminal region, the dorsomedial portion of the trigeminal principal sensory nucleus, and the dorsomedial part of the rostral spinal trigeminal subnucleus oralis. Additional, previously undescribed projections of jaw-muscle spindle afferents were found to the dorsomedial portion of the caudal spinal trigeminal subnucleus oralis (Vodm), the dorsomedial part of the spinal trigeminal subnucleus interpolaris (Vidm), the caudal parvicellular reticular formation, laminae IV and V of the spinal trigeminal subnucleus caudalis (Vc), and the dorsal division of the medullary reticular field. Labeled spindle boutons in Vodm formed predominately axodendritic synapses. Some of these boutons received presynaptic inputs from unlabeled P-type boutons containing clear, spherical, or flattened vesicles. In Vidm, labeled collaterals and boutons were densely clustered into glomerular-like structures. Labeled boutons in Vidm made axodendritic, axosomatic, and axoaxonic synapses and received synaptic contacts from unlabeled boutons containing clear, spherical, or flat and pleomorphic vesicles. Unlabeled presynaptic boutons in Vidm occasionally contained dense core vesicles. Labeled boutons in Vc mainly formed synaptic contacts with large diameter dendrites. This projection of jaw-muscle spindle afferents to caudal brainstem regions may play a significant role in masticatory-muscle stretch reflexes and in the integration of trigeminal proprioceptive information and its transmission to higher centers.

Antinociceptive effect of morphine microinjections into the spinal trigeminal subnucleus oralis.

We analysed the effects of morphine microinjections (0.2 microliter) into the subnucleus oralis (SNO), i.e. the rostral division of the spinal trigeminal nucleus, on the formalin test adapted for orofacial pain. Duration of rubbing following an injection (50 microliters) of formalin solution (1.5%) in the right upper lip of the rat was measured. Compared to microinjections of saline (NaCl, 0.9%) used as control, 90 and 180 nmol microliter-1 of morphine solution in the SNO significantly curtailed rubbing. This effect was dose-dependent, reversed by naloxone, and also site-specific to the SNO since no effect was observed after microinjection of morphine into areas adjacent to the SNO. These results are further evidence for the involvement of the SNO in perioral nociceptive mechanisms.

Response characteristics of tooth pulp-driven postsynaptic neurons in the spinal trigeminal subnucleus oralis of the cat.

Neuronal activity in the spinal trigeminal subnucleus oralis in response to electrical tooth stimulation was recorded in the anaesthetized cat in order to compare the electrophysiological characteristics of the oralis neurons with those of subnucleus caudalis and interpolaris neurons recorded in previous studies. The most sensitive oralis neurons had lower thresholds and shorter latencies than the most sensitive caudalis and interpolaris neurons. The thresholds of the oralis neurons were lower and their strength-duration curves flatter than those depicting liminal dental pain in man but similar to those depicting liminal jaw reflexes in the cat. Noxious conditioning stimulus elevated the threshold of only 1 of 10 neurons tested. The converging input from the skin and oral mucosa was from low-threshold mechanoreceptors. The results indicate that the response properties of the subnucleus oralis neurons differ significantly from those of other spinal subnuclei. Human pain thresholds cannot be explained by the liminal response properties of oralis neurons. These neurons might be important in the mediation of liminal reflex events evoked by dental stimuli.

Bilateral and unilateral antinociceptive effects of rostral trigeminal nuclear complex lesions in rats

Behavioural responses to two different orofacial noxious stimulations were analysed following lesion of spinal trigeminal subnucleus oralis (Sp5O) in the rat. Lesions were obtained by intranuclear microinjections of quinolinic acid (0.4 μl of 60 nmol/μl solution). The control groups received microinjection of saline. Noxious stimulation was a subcutaneous injection of formalin into the upper lip or electrical stimulation of the tooth pulp. The measured behavioural responses were duration of rubbing induced by the formalin injection and thresholds of the jaw-opening reflex (JOR), head rotation (HR) and face rubbing (FR) evoked by the pulp stimulation. In addition, formalin injection was also performed in two groups of rats that had received intranuclear injection of quinolinic acid or saline into rostral subnucleus caudalis (Sp5C). Rubbing duration was not significantly modified by the lesion of Sp5O, whereas a significant decrease occurred after the lesion of rostral Sp5C. After the lesion of Sp5O, an increase in the threshold of JOR was observed whereas the thresholds of HR and FR were not significantly modified. These results suggest that Sp5O is not necessary for the processing and relay of nociceptive inputs triggered by intense stimulations of oral and perioral areas. However further experiments are needed to reconcile these results with the relevant data obtained from cell recording experiments which indicate the existence, in Sp5O, of neuronal activities related to the sensory discriminative aspect of intense nociception.