Subnucleus Interpolaris Research Articles

Nitric Oxide Synthase Immunoreactivity in the Rat Spinal Trigeminal Nucleus

The spinal trigeminal nucleus (SpV) is involved in the transmission of nociceptive information from the orofacial region. This study investigates nitric oxide synthase (NOS) immunohistochemical staining and conducts statistical data analysis within the three subnuclei of the SpV in rats. Our immunohistochemistry study revealed that the SpV has a high expression of nNOS, with immunostaining mostly seen in the perikarya and nerve processes of some neurons. Furthermore, we found that glial cells in the nucleus surrounding blood vessels display a significant nNOS immunohistochemical staining. NOS-immunopositive neurons had small cell bodies. Strong staining is also evident in their processes. NOS-immunoreactive neuronal processes encircled a significant amount of blood vessels. There were statistically significant variations in the nNOS expression between the SpV subnuclei. In particular, the caudal subnucleus exhibited the greatest expression, followed by the interpolar subnucleus, and the oral subnucleus showed the lowest expression. Remarkably, there was no discernible variation in the expression of nNOS in the interpolar and oral regions of the SpV. We suggest that NO can act as a neurotransmitter in increasing nociceptive stimuli in the SpV.

Orthodontic pain: c-Fos expression in rat brain nuclei after rapid maxillary expansion

The aim of this in vivo study was to quantitatively evaluate pain after rapid maxillary expansion (RME) in young rats by analyzing the activation of nociception-related structures, that is, the caudalis, interpolaris, and oralis subnuclei, according to the Fos expression. A total of 65 Wistar rats were assigned to three groups: control group (n=15) with no treatment, positive control group (n=25), and experimental group (n=25) with RME. The experimental animals were euthanized at 6, 12, 24, 48, and 72 hours after RME, and the brain was later carefully collected. Coronal sections through the spinal trigeminal caudalis, spinal trigeminal interpolaris, and spinal trigeminal oralis were cut (thickness of 40 µm) on a cryostat and processed for Fos immunohistochemistry. Images from the sections were captured under light microscopy, and ImageJ software was used to count Fos-like immunoreactive neurons. The Analysis of variance (ANOVA) and Tukey test were used for statistical analysis, and the significance level was set at 5%. RME induced incisor distalization and opening of the midpalatal suture, as well as neuronal activation of the spinal trigeminal nucleus. The experimental group demonstrated significantly more Fos-positive neurons in subnuclei caudalis and subnuclei interpolaris 6 hours after the maxillary expansion. The Fos immunoreactivity significantly decreased at 12 hours and increased again at 24 and 48 hours (P < 0.001). The RME increases the neural activation of brain regions involved in the nociception region, as determined by the Fos expression. The most intense Fos-like immunoreactive expression was detected in the brain 6 hours after the start of the palatal expansion.

Role of transient receptor potential vanilloid type 1 in the trigeminal ganglion and brain stem following dental pulp inflammation.

To verify whether experimentally induced pulpitis activates the expression of transient receptor potential vanilloid type 1 (TRPV1) and c-Fos, both peripherally and centrally. Acute pulpitis was induced in Sprague-Dawley rats via pulp exposure and application of complete Freund's adjuvant (CFA; n=13). Saline-treated (n=13) rats and rats that did not undergo tooth preparation (n=13) served as control groups. Three days post-CFA or post-saline application, face grooming activity was recorded, and the rats were then euthanized to allow for immunohistochemical analysis of the trigeminal ganglion (TG) and spinal trigeminal nucleus. anova with Student's t-test for post-hoc analysis was used to quantify the differences in behavioural tests and immunohistochemical labelling (c-Fos and TRPV1) in TG amongst groups. Kruskal-Wallis test with Dunnett's test for post-hoc analysis was used to compare immunohistochemical labelling (c-Fos and TRPV1) in the brainstem amongst groups. Histological evidence of severe pulp inflammation was found, and there was a significant increase in pain-like behaviour (P<0.05) in CFA-treated animals. C-Fos labelling and TRPV1 immunoreactivity in the TG were significantly higher (both P<0.05) in the CFA group than in the control groups. In the spinal trigeminal nucleus, the immunoreactivity for c-Fos was absent in the intermediate region (trigeminal subnucleus interpolaris) in all animals, with comparable expression of TRPV1 amongst all groups. In contrast, neurons in the trigeminal subnucleus caudalis (TSC) exhibited significant c-Fos immunoreactivity in the CFA group (P=0.0063). The expression of TRPV1 did not differ amongst the three groups, but the superficial laminae of the TSC exhibited significantly greater expression of TRPV1 than did the deep layers (P=0.0014). Following acute pulp inflammation, the TRPV1 channel was significantly involved in nociceptive signal processing in the peripheral nervous system, but not in the CNS. Because pulpitis induced some neuronal activation at the brainstem levels, further studies are needed to identify additional transducers that mediate signal transmission from pulpal afferents to their central targets.

Somatotopy in the Medullary Dorsal Horn As a Basis for Orofacial Reflex Behavior.

The somatotopy of the trigeminocervical complex of the rat was defined as a basis for describing circuitry for reflex behaviors directed through the facial motor nucleus. Thus, transganglionic transport of horseradish peroxidase conjugates applied to individual nerves/peripheral receptive fields showed that nerves innervating oropharyngeal structures projected most rostrally, followed by nerves innervating snout, periocular, and then periauricular receptive fields most caudally. Nerves innervating mucosae or glabrous receptive fields terminated densely in laminae I, II, and V of the trigeminocervical complex, while those innervating hairy skin terminated in laminae I–V. Projections to lamina II exhibited the most focused somatotopy when individual cases were compared. Retrograde transport of FluoroGold (FG) deposited into the facial motor nucleus resulted in labeled neurons almost solely in lamina V of the trigeminocervical complex. The distribution of these labeled neurons paralleled the somatotopy of primary afferent fibers, e.g., those labeled after FG injections into a functional group of motoneurons innervating lip musculature were found most rostrally while those labeled after injections into motoneurons innervating snout, periocular and preauricular muscles, respectively, were found at progressively more caudal levels. Anterograde transport of injections of biotinylated dextran amine into lamina V at different rostrocaudal levels of the trigeminocervical complex confirmed the notion that the somatotopy of orofacial sensory fields parallels the musculotopy of facial motor neurons. These data suggest that neurons in lamina V are important interneurons in a simple orofacial reflex circuit consisting of a sensory neuron, interneuron and motor neuron. Moreover, the somatotopy of primary afferent fibers from the head and neck confirms the “onion skin hypothesis” and suggests rostral cervical dermatomes blend seamlessly with “cranial dermatomes.” The transition area between subnucleus interpolaris and subnucleus caudalis is addressed while the paratrigeminal nucleus is discussed as an interface between the somatic and visceral nervous systems.

The sensory trigeminal complex and the organization of its primary afferents in the zebra finch (Taeniopygia guttata).

Our knowledge of the avian sensory trigeminal system has been largely restricted to the principal trigeminal nucleus (PrV) and its ascending projections to the forebrain. Studies addressing the cytoarchitecture and organization of afferent input to the sensory trigeminal complex, which includes both the PrV and the nuclei of the descending trigeminal tract (nTTD), have only been performed in pigeons and ducks. Here we extend such an analysis to a songbird, the zebra finch (Taeniopygia guttata). We describe the cytoarchitecture of the sensory trigeminal complex, the patterns of calbindin-like and substance P-like immunoreactivity, and the organization of afferents from the three branches of the trigeminal nerve and from the lingual branch of the hypoglossal nerve. On the basis of cytoarchitecture and immunohistochemistry, the sensory trigeminal column can be subdivided from caudal to rostral, as in other species, into cervical dorsal horn, subnucleus caudalis, subnucleus interpolaris, subnucleus oralis, and nucleus principalis. The relative positions of the terminal fields of the three trigeminal branches move from medial to lateral in the dorsal horn to dorsomedial to ventrolateral in nTTD, whereas in PrV there is considerable overlap of mandibular and ophthalmic terminal fields, with only a small maxillary input ventrally. The hypoglossal afferents, which terminate medially in the dorsal horn and dorsolaterally in nTTD, terminate in specific cell groups in the dorsolateral nTTDo and in PrV. This work sets the grounds for further analyses of the ascending connections of the nTTD and the afferents from the syrinx to the trigeminal sensory column.

UNDERSTANDING THE REGIONAL SPECIFICITY OF NEURONS IN THE TRIGEMINAL SUBNUCLEUS CAUDALIS AND SPINAL DORSAL HORN

Neurons in the trigeminal subnucleus caudalis (Vc) and spinal cord dorsal horn (SDH) play important roles in modulating and relaying pain signals to the higher centers of the central nervous system (CNS). Morphologically, many aspects including a laminated structure, cytoarchitecture and cellular elements in these two central regions are very similar [1,2,3,4] . Most nociceptive afferents with a small-diameter terminate in the superficial laminae of Vc and SDH. Functionally, sensory neurons in Vc and SDH respectively receive nociceptive inputs from the orofacial and other somatic regions, and convey the inputs to higher brain centers [1,2,3,4,5] . According to the mechanoreceptive properties, sensory neurons in both the Vc and SDH are classified into three types. They are low-threshold mechanoreceptive (LTM), wide-dynamic range (WDR) and nociceptive-specific neurons (NS) [1,2,3,4,5] . Because of these similarities Vc has been considered as analogous to SDH [4,6] . However, some differences between Vc and SDH have also been reported. For example, the distribution pattern of substance P (SP) and calcitonin gene related peptide (CGRP) in Vc of adult animals is different from that in SDH. In SDH there is no dual representation of peripheral regions, which can be found in rostral and caudal Vc. A transitional zone found between Vc and trigeminal subnucleus interpolaris plays a special role in sensorimotor function. However, no such region is found in the spinal system [5,7,8] . CNS neurons are regionalized. They are organized as groups through their circuitry. Although it is known that differences in functions among these groups in the adult CNS are predetermined during the development of the neuroepithelium [9,10,11,12,13] , understanding how the regional specificity is developed in the CNS is still a big challenge in neuroscience research. It has recently been found that ninhydrin-reacting small molecules released locally are involved in the region-specific regulation of neuronal development in cultured Vc and SDH neurons [14,15] . In this review we focused on present knowledge about the region-specific regulation of neuronal development in these two CNS regions associated with the transmission of nociceptive signals.

SCN2B in the Rat Trigeminal Ganglion and Trigeminal Sensory Nuclei.

The beta-2 subunit of the mammalian brain voltage-gated sodium channel (SCN2B) was examined in the rat trigeminal ganglion (TG) and trigeminal sensory nuclei. In the TG, 42.6% of sensory neurons were immunoreactive (IR) for SCN2B. These neurons had various cell body sizes. In facial skins and oral mucosae, corpuscular nerve endings contained SCN2B-immunoreactivity. SCN2B-IR nerve fibers formed nerve plexuses beneath taste buds in the tongue and incisive papilla. However, SCN2B-IR free nerve endings were rare in cutaneous and mucosal epithelia. Tooth pulps, muscle spindles and major salivary glands were also innervated by SCN2B-IR nerve fibers. A double immunofluorescence method revealed that about 40% of SCN2B-IR neurons exhibited calcitonin gene-related peptide (CGRP)-immunoreactivity. However, distributions of SCN2B- and CGRP-IR nerve fibers were mostly different in facial, oral and cranial structures. By retrograde tracing method, 60.4 and 85.3% of TG neurons innervating the facial skin and tooth pulp, respectively, showed SCN2B-immunoreactivity. CGRP-immunoreactivity was co-localized by about 40% of SCN2B-IR cutaneous and tooth pulp TG neurons. In trigeminal sensory nuclei of the brainstem, SCN2B-IR neuronal cell bodies were common in deep laminae of the subnucleus caudalis, and the subnuclei interpolaris and oralis. In the mesencephalic trigeminal tract nucleus, primary sensory neurons also exhibited SCN2B-immunoreactivity. In other regions of trigeminal sensory nuclei, SCN2B-IR cells were very infrequent. SCN2B-IR neuropil was detected in deep laminae of the subnucleus caudalis as well as in the subnuclei interpolaris, oralis and principalis. These findings suggest that SCN2B is expressed by various types of sensory neurons in the TG. There appears to be SCN2B-containing pathway in the TG and trigeminal sensory nuclei.

Essential roles of mGluR1 and inhibitory synaptic transmission in NMDA-independent long-term potentiation in the spinal trigeminal interpolaris.

Patterns of synaptic activity determine synaptic strengthening or weakening that is typically represented as long-term potentiation (LTP) and long-term depression (LTD), respectively. In the present study, we aim to test whether a conditioning stimulation of the spinal trigeminal subnucleus caudalis (Vc) induces LTP at excitatory synapses in the subnucleus interpolaris (Vi) and to characterize the LTP. Generally, a presynaptic high-frequency stimulation (HFS) protocol can induce LTP at excitatory synapses in the brain, including the spinal cord. Therefore, LTP in the Vi was induced by the HFS (3 tetani at 100 Hz) of Vc in the horizontal brainstem slices. By pretreating slices with antagonists for NMDA receptors, metabotropic glutamate receptor subtype 1 or 5 (mGluR1 or 5), GABAA receptors, glycine receptors and Ca(2+) chelator, the LTP was characterized. The HFS reliably but slowly induced LTP of excitatory synaptic transmission in the Vi. This LTP was not dependent on NMDA receptor activation; however, it did require the activation of mGluR1, but not mGluR5, and an intracellular Ca(2+) rise. Interestingly, this LTP induction required inhibitory synaptic transmission mediated by GABAA and glycine receptors, and coincided with the slow development of LTD at GABAergic synapses. The GABAergic LTD was mediated by mGluR1 and the intracellular Ca(2+) rise. These data suggest that the modulation of GABAergic synaptic transmission by conditioning synaptic activity contributes to the induction and expression of LTP at excitatory synapses in the Vi.

Whisker-related circuitry in the trigeminal nucleus principalis: Topographic precision

Single whiskers are topographically represented in the trigeminal (V) nucleus principalis (PrV) by a set of cylindrical aggregates of primary afferent terminals and somata (barrelettes). This isomorphic pattern is transmitted to the thalamus and barrel cortex. However, it is not known if terminals in PrV from neighboring whiskers interdigitate so as to violate rules of spatial parcellation predicted by barrelette borders; nor is it known the extent to which higher order inputs are topographic. The existence of inter-whisker arbor overlap or diffuse higher order inputs would demand additional theoretical principles to account for single whisker dominance in PrV cell responses. In adult rats, first, primary afferent pairs responding to the same or neighboring whiskers and injected with Neurobiotin or horseradish peroxidase were rendered brown or black to color-code their terminal boutons. When collaterals from both fibers appeared in the same topographic plane through PrV, the percentage of the summed area of the two arbor envelopes that overlapped was computed. For same-whisker pairs, overlap was 5 ± 6% (mean ± SD). For within-row neighbors, overlap was 2 ± 5%. For between-row neighbors, overlap was 1 ± 4%. Second, the areas of whisker primary afferent arbors and their corresponding barrelettes in the PrV were compared. In the transverse plane, arbor envelopes significantly exceeded the areas of cytochrome oxidase-stained barrelettes; arbors often extended into neighboring barrelettes. Third, bulk tracing of the projections from the spinal V subnucleus interpolaris (SpVi) to the PrV revealed strict topography such that they connect same-whisker barrelettes in the SpVi and PrV. Thus, whisker primary afferents do not exclusively project to their corresponding PrV barrelette, whereas higher order SpVi inputs to the PrV are precisely topographic.

Theta-burst stimulation induces LTP at excitatory and inhibitory synapses in the spinal trigeminal subnucleus interpolaris

Long-lasting synaptic modifications of excitatory and inhibitory synaptic transmissions induced by theta-burst stimulation (TBS) were examined in the spinal trigeminal subnucleus interpolaris (Vi). We found that conditioning afferents of another subnucleus caudalis (Vc) to the Vi with TBS produced long-term depression (LTD). However, when GABAA and glycine receptors were blocked, the same stimulation paradigm produced long-term potentiation (LTP). The induction of LTP involved neither NMDA receptors nor a presynaptic change. The expression of LTP was obviously suppressed by the activation of group I mGluRs because its magnitude increased in the presence of antagonists for group I mGluRs. Besides the LTP at excitatory synapses, TBS also induced LTP at inhibitory GABAergic synapses, which required the activation of NMDA receptors and NO-cGMP signaling but was not involved in the increase of postsynaptic Ca2+ concentration. Therefore, this study shows, for the first time, an activity-dependent plasticity at excitatory and inhibitory synapses in the Vi by the same conditioning stimulation.

Organization of the spinal trigeminal nucleus in star‐nosed moles

Somatosensory inputs from the face project to multiple regions of the trigeminal nuclear complex in the brainstem. In mice and rats, three subdivisions contain visible representations of the mystacial vibrissae, the principal sensory nucleus, spinal trigeminal subnucleus interpolaris, and subnucleus caudalis. These regions are considered important for touch with high spatial acuity, active touch, and pain and temperature sensation, respectively. Like mice and rats, the star-nosed mole (Condylura cristata) is a somatosensory specialist. Given the visible star pattern in preparations of the star-nosed mole cortex and the principal sensory nucleus, we hypothesized there were star patterns in the spinal trigeminal nucleus subnuclei interpolaris and caudalis. In sections processed for cytochrome oxidase, we found star-like segmentation consisting of lightly stained septa separating darkly stained patches in subnucleus interpolaris (juvenile tissue) and subnucleus caudalis (juvenile and adult tissue). Subnucleus caudalis represented the face in a three-dimensional map, with the most anterior part of the face represented more rostrally than posterior parts of the face. Multiunit electrophysiological mapping was used to map the ipsilateral face. Ray-specific receptive fields in adults matched the CO segmentation. The mean areas of multiunit receptive fields in subnucleus interpolaris and caudalis were larger than previously mapped receptive fields in the mole's principal sensory nucleus. The proportion of tissue devoted to each ray's representation differed between the subnucleus interpolaris and the principal sensory nucleus. Our finding that different trigeminal brainstem maps can exaggerate different parts of the face could provide new insights for the roles of these different somatosensory stations.

Null mutations of NT-3 and Bax affect trigeminal ganglion cell number but not brainstem barrelette pattern formation

Trigeminal ganglion (TG) neurons innervate the grid-like array of whisker follicles on the face of the mouse. Central TG axons project to the trigeminal (V) brainstem nuclear complex, including the nucleus principalis (PrV) and the spinal subnucleus interpolaris (SpVi), where they innervate barrelettes that are organized in a pattern that recapitulates the whisker pattern on the face. Neurotrophin-3 (NT-3) supports a population of TG cells that supply slowly adapting mechanoreceptors in the whisker pad. We examined mice at embryonic day 17 (E17) and on the day of birth (P0) with null mutations of NT-3, Bax, a proapoptotic gene associated with naturally occurring cell death, and Bax/NT-3 double knockout (KO) mutants to determine if: (1) the number of TG cells would be reduced; (2) eliminating the Bax gene would rescue the NT-3-dependent neurons; and (3) the central projections of the rescued axons in the Bax/NT-3 double KO mice would fail to develop the barrelette patterns in the PrV and SpVi subnuclei. In mice at E17, NT-3−/− mutants had 65% fewer TG neurons than found in age-matched wild-type (WT) mice, and at P0, the number was reduced by 55% (p < 0.001 for both). Bax null mutant mice at E17 had 132% of the WT number of TG cells (p < 0.001), although the numbers returned to WT levels by P0. Bax/NT-3 double KO mice at E17 had TG cell numbers equal to those seen in WT, but the double KO failed to retain WT TG neuron numbers in P0 mice (39% fewer cells; p < 0.001). In all cases of reduced experimental neuron numbers, and in the E17 Bax−/− mice with supernumerary cells, the barrelette patterns in the PrV and SpVi were normal. Only a slight qualitative reduction in overall barrelette field area and clarity of barrelettes were seen. These results suggest that NT-3 is not necessary for barrelette pattern formation in the brainstem.

Corneal dry-responsive neurons in the spinal trigeminal nucleus respond to innocuous cooling in the rat

Corneal primary afferent neurons that respond to drying of the ocular surface have been previously characterized and found to respond to innocuous cooling, menthol, and hyperosmotic stimuli. The purpose of the present study was to examine the receptive field properties of second-order neurons in the trigeminal nucleus that respond to drying of the ocular surface. Single-unit electrophysiological recordings were performed in anesthetized rats, and dry-responsive corneal units were isolated in the brain stem at the transition zone between the spinal trigeminal subnucleus caudalis and subnucleus interpolaris. Corneal units were characterized according to their responses to changes in temperature (cooling and heating), hyperosmotic artificial tears, menthol, and low pH. All dry-responsive neurons (n = 18) responded to cooling of the ocular surface. In addition, these neurons responded to hyperosmotic stimuli and menthol application to the cornea. One-half of the neurons were activated by low pH, and these acid-sensitive neurons were also activated by noxious heat. Furthermore, neurons that were activated by low pH had a significantly lower response to cooling and menthol. These results indicate that dry-responsive neurons recorded in the trigeminal nucleus receive input from cold, sensitive primary afferent neurons, with a subset of these neurons receiving input from corneal primary afferent neurons sensitive to acid and noxious heat. It is proposed that acid-insensitive corneal neurons represent a labeled line for lacrimation in response to evaporation of tears from the ocular surface, whereas acid-sensitive neurons are involved in tearing, elicited by damaging or potentially damaging stimuli.

Ginsenosides Have a Suppressive Effect on c-Fos Expression in Brain and Reduce Cardiovascular Responses Increased by Noxious Stimulation to the Rat Tooth

The purpose of this study is to investigate the antinociceptive effects of ginsenosides on toothache. c-Fos immunoreactive (IR) neurons were examined after noxious intrapulpal stimulation (NS) by intrapulpal injection of 2 M KCl into upper and lower incisor pulps exposed by bone cutter in Sprague Dawley rats. The number of Fos-IR neurons was increased in the trigeminal subnucleus caudalis (Vc) and the transitional region between Vc and subnucleus interpolaris (Vi) by NS to tooth. The intradental NS raised arterial blood pressure (BP) and heart rate (HR). The number of Fos-IR neurons was also enhanced in thalamic ventral posteromedial nucleus (VPMN) and centrolateral nucleus (CLN) by NS to tooth. The intradental NS increased the number of Fos-IR neurons in the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN), central cardiovascular regulation centers. Ginsenosides reduced the number of c-Fos-IR increased by NS to tooth in the trigeminal Vc and thalamic VPMN and CLN. Naloxone, an opioid antagonist, did not block the effect of ginsenoside on the number of Fos-IR neurons enhanced by NS to tooth in the trigeminal Vc and thalamic VPMN and CLN. Ginsenosides ameliorated arterial BP and HR raised by NS to tooth and reduced the number of Fos-IR neurons increased by NS to tooth in the NTS, RVLM, hypothalamic SON, and PVN. These results suggest that ginsenosides have an antinociceptive effect on toothache through non-opioid system and attenuates BP and HR increased by NS to tooth.

ERK is Involved in Tooth-pressure-induced Fos Expression in Vc Neurons

Discomfort and pain encountered during orthodontic treatment are major problems for patients, but the details of the underlying neural processes and molecular mechanisms are not well-understood. Here we show that noxious tooth mechanical pressure induced by orthodontic elastics resulted in a rapid and transient activation of extracellular signal-regulated protein kinase (ERK) in the trigeminal spinal subnucleus interpolaris and caudalis transition zone (Vi/Vc), trigeminal spinal subnucleus caudalis (Vc), and upper cervical spinal cord (Vc/C2). The phosphorylated ERK (pERK) was observed in neurons but not in astroglia and microglia. Single-plane scanning analysis indicated that the pERK was localized to the nucleus of Vc neurons. In addition, the tooth mechanical pressure led to Fos expression in the pERK-positive Vc neurons that would be suppressed by intrathecal administration of an MEK1/2 inhibitor (PD98059). Taken together, these findings suggest that activation of the ERK signaling cascade following noxious mechanical pressure on the teeth regulates Fos expression in Vc neurons and may thereby contribute to pain associated with orthodontic treatment.

Somatotopic direct projections from orofacial areas of secondary somatosensory cortex to trigeminal sensory nuclear complex in rats

Little is known about the projections from the orofacial areas of the secondary somatosensory cortex (S2) to the pons and medulla including the second-order somatosensory neuron pools. To address this in rats, we first examined the distribution of S2 neurons projecting to the trigeminal principal nucleus (Vp) or oral subnucleus (Vo) of the trigeminal sensory nuclear complex (TSNC) after injections of a retrograde tracer, Fluorogold (FG), into five regions in the Vp/Vo which were responsive to stimulation of trigeminal nerves innervating the orofacial tissues. A large number of FG-labeled neurons were found with a somatotopic arrangement in the dorsal areas of S2 (orofacial S2 area). This somatotopic arrangement in the orofacial S2 area was shown to closely match that of the orofacial afferent inputs by recording cortical surface potentials evoked by stimulation of the trigeminal nerves. We then examined the morphology of descending projections from these electrophysiologically defined areas of the orofacial S2 to the pons and medulla after injections of an anterograde tracer, biotinylated dextranamine (BDA), into the areas. A large number of BDA-labeled axon fibers and terminals were seen only in some of the second-order somatosensory neuron pools, most notably in the contralateral TSNC, although the labeled terminals were not seen in certain rostrocaudal levels of the contralateral TSNC including the rostrocaudal middle level of the trigeminal interpolar subnucleus. The projections to the TSNC showed somatotopic arrangements in dorsoventral, superficial-deep and rostrocaudal directions. The somatotopic arrangements in the Vp/Vo closely matched those of the electrophysiologically defined central projection sites of the orofacial trigeminal afferents in the TSNC. The present results suggest that the orofacial S2 projects selectively to certain rostrocaudal levels of the contralateral TSNC, and the projections may allow the orofacial S2 to accurately modulate orofacial somatosensory transmission to higher brain centers including the orofacial S2 itself.

Peptide 19-containing neurons in the medullary dorsal horn, subnuclei interpolaris and oralis, and nucleus principalis of the rat

Peptide 19 (PEP 19) is a 7.6 kDa polypeptide which can bind to calmodulin and inhibit calcium-calmodulin signaling. In this study, PEP 19-immunoreactivity (ir) was examined in the rat trigeminal sensory nuclei. Numerous PEP 19-immunoreactive (ir) neurons were detected in the medullary dorsal horn (MDH) and rostral parts of the trigeminal sensory nuclei (subnuclei interpolaris and oralis, and nucleus principalis). The mean numbers ± S.D. per section of PEP 19-ir neurons were 104.2 ± 30.4 in the MDH, 137.8 ± 39.5 in the subnucleus interpolaris, 129.2 ± 46.9 in the subnucleus oralis and 157.2 ± 34.1 in the nucleus principalis. In the MDH, small to medium-sized PEP 19-ir neurons were abundant within superficial laminae. PEP 19-ir neurons with various cell body sizes were also distributed in the rostral parts of the trigeminal sensory nuclei. A double immunofluorescence analysis also demonstrated that many PEP 19-ir neurons co-expressed parvalbumin (PV)-ir in the MDH (9.0%), subnucleus oralis (7.7%) and nucleus principalis (19.7%). In the subnucleus interpolaris, such neurons were relatively rare (1.7%). PEP 19-ir neurons were mostly devoid of calbindin D-28k. In addition, a retrograde tracing method revealed that a substantial number of PEP 19-ir neurons projected to the thalamus. PV-ir was common in thalamus-projecting PEP 19-ir neurons. These findings suggest that PEP 19-ir neurons in the MDH may have a function in modulation of nociceptive and thermo-receptive signaling. It is also likely that PEP 19-ir neurons in rostral parts of the trigeminal sensory nuclei are related to transduction of mechano-receptive information from facial regions to the thalamus.

Differential ascending projections of temporomandibular joint-responsive brainstem neurons to periaqueductal gray and posterior thalamus of male and female rats

Several craniofacial pain conditions, including temporomandibular joint disorders (TMJDs), are more prevalent in women than men. The basis for sex differences in deep craniofacial pain is not known. The present study compared the magnitude of ascending projections from temporomandibular joint (TMJ)-responsive neurons in trigeminal brainstem with the ventrolateral periaqueductal gray (vlPAG) or posterior nucleus of the thalamus (Po) in males and female rats. Fluorogold (FG) was injected into vlPAG or Po, and TMJ-responsive neurons were identified by Fos-like immunoreactivity (Fos-LI) after mustard oil injection. TMJ-evoked Fos-LI was similar in males and females; however, significant differences in cell counts were seen for FG single-labeled and Fos/FG double-labeled neurons in trigeminal brainstem. After vlPAG injections, the number of FG-labeled neurons in trigeminal subnucleus interpolaris (Vi), ventral interpolaris/caudalis transition (vl-Vi/Vc), and dorsal paratrigeminal region (dPa5) was greater in females than males. The percentage of Fos/FG double-labeled neurons in vl-Vi/Vc and dPa5 after vlPAG injection also was greater in females than males. In contrast, after Po injections, males displayed a greater number of FG-labeled neurons in superficial laminae (Lam I/II) of trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1–2) and deeper laminae (Lam III/V) at C1–2 than females. The percentage of Fos/FG double-labeled neurons in Lam I/II of Vc after Po injection also was greater in males than females. These data revealed significant sex differences in ascending projections from TMJ-responsive neurons in trigeminal brainstem. Such differences may influence the ability of males and females to recruit autonomic reflexes and endogenous pain control circuits relevant for TMJ nociception.

A trigeminoreticular pathway: implications in pain.

Neurons in the caudalmost ventrolateral medulla (cmVLM) respond to noxious stimulation. We previously have shown most efferent projections from this locus project to areas implicated either in the processing or modulation of pain. Here we show the cmVLM of the rat receives projections from superficial laminae of the medullary dorsal horn (MDH) and has neurons activated with capsaicin injections into the temporalis muscle. Injections of either biotinylated dextran amine (BDA) into the MDH or fluorogold (FG)/fluorescent microbeads into the cmVLM showed projections from lamina I and II of the MDH to the cmVLM. Morphometric analysis showed the retrogradely-labeled neurons were small (area 88.7 µm2±3.4) and mostly fusiform in shape. Injections (20–50 µl) of 0.5% capsaicin into the temporalis muscle and subsequent immunohistochemistry for c-Fos showed nuclei labeled in the dorsomedial trigeminocervical complex (TCC), the cmVLM, the lateral medulla, and the internal lateral subnucleus of the parabrachial complex (PBil). Additional labeling with c-Fos was seen in the subnucleus interpolaris of the spinal trigeminal nucleus, the rostral ventrolateral medulla, the superior salivatory nucleus, the rostral ventromedial medulla, and the A1, A5, A7 and subcoeruleus catecholamine areas. Injections of FG into the PBil produced robust label in the lateral medulla and cmVLM while injections of BDA into the lateral medulla showed projections to the PBil. Immunohistochemical experiments to antibodies against substance P, the substance P receptor (NK1), calcitonin gene regulating peptide, leucine enkephalin, VRL1 (TPRV2) receptors and neuropeptide Y showed that these peptides/receptors densely stained the cmVLM. We suggest the MDH- cmVLM projection is important for pain from head and neck areas. We offer a potential new pathway for regulating deep pain via the neurons of the TCC, the cmVLM, the lateral medulla, and the PBil and propose these areas compose a trigeminoreticular pathway, possibly the trigeminal homologue of the spinoreticulothalamic pathway.

&lt;b&gt;The study of migraine model rat with photophobia &lt;/b&gt;&lt;b&gt;in the upper cervical spinal cord &lt;/b&gt;

Background and purposes: Photophobia is characteristic and important sign of the migraine headache. Recently, we found that the response of dura-sensitive neuron to photic stimulation was enhanced after dura inflammation. Some previous papers also have reported that neurons located in the dorsal horn of the C2 spinal cord (C2 neurons) receive photic inputs suggesting that C2 neurons somehow involved in photophobia. However, how the C2 neurons are involved in photophobia associated with migraine is unknown. Therefore, the change in neuronal excitability of C2 nociceptive neurons was analyzed in rat model with dural inflammation. Methods: The Sprague-Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and the small hole was made in the frontal bone, and 10% mustard oil (MO) was applied to the dura surface around the transverse sinus (migraine rat). Photic stimulation was applied to the eye in migraine and vehicle-treated rats, and c-Fos like immunoreactive (LI) cells and pERK-immunoreactive (IR) cells in C2 were precisely analyzed. To identify trigeminal ganglion (TG) neurons innervating the retina, the fluorogold (FG) solution (4% in distilled water, 10 µl) was injected to the retina using a 31-gauge needle. Substance P (SP), calcitonin generelated peptide (CGRP), TRPV1 immunoreactivities were also detected in the ratina. Results: Following MO application to the dura, a large number of c-Fos-LI cells was expressed in the transition zone between subnuclei interpolaris and caudalis (Vi/Vc) zone bilaterally and was also in the ipsilateral C2. Photic stimulation to the eye in migraine rat caused an expression of pERK-IR cells in C2, and many of them also showed c-Fos immunoreactivity. FG-labeled trigeminal ganglion cells were observed in the first branch-innervated area of the TG following FG injection into the retina. Many SP, CGRP and TRPV1 immunoreactivities were detected in nerve fibers in the retina. The present findings suggested that C2 neurons are involved in photophobia associated with migraine, and also suggested that the unmyelinated trigeminal afferents innervating the retina might be one of the sources of photic information in photophobia.