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

Activation of Brainstem Neurons by Underwater Diving in the Rat

The mammalian diving response is a powerful autonomic adjustment to underwater submersion greatly affecting heart rate, arterial blood pressure, and ventilation. The bradycardia is mediated by the parasympathetic nervous system, arterial blood pressure is mediated via the sympathetic system and still other circuits mediate the respiratory changes. In the present study we investigate the cardiorespiratory responses and the brainstem neurons activated by voluntary diving of trained rats, and, compare them to control and swimming animals which did not dive. We show that the bradycardia and increase in arterial blood pressure induced by diving were significantly different than that induced by swimming. Neuronal activation was calculated after immunohistochemical processing of brainstem sections for Fos protein. Labeled neurons were counted in the caudal pressor area, the medullary dorsal horn, subnuclei of the nucleus tractus solitarii (NTS), the nucleus raphe pallidus (RPa), the rostroventrolateral medulla, the A5 area, the nucleus locus coeruleus, the Kölliker–Fuse area, and the external lateral and superior lateral subnuclei of the parabrachial nucleus. All these areas showed significant increases in Fos labeling when data from voluntary diving rats were compared to control rats and all but the commissural subnucleus of the NTS, A5 area, and RPa were significantly different from swimming rats. These data provide a substrate for more precise experiments to determine the role of these nuclei in the reflex circuits driving the diving response

The caudalmost ventrolateral medulla exhibits numerous peptides associated with pain pathways.

<p>Photomicrographs through the cmVLM showing immunoreactivity to multiple peptides (white oval encircles the CPA). All these peptides have been implicated in the processing of painful information. See text for discussion and abbreviations.</p

Neurons in the caudalmost ventrolateral medulla and lateral reticular formation are suspected relays of the trigeminoreticulothalamic tract.

<p>Photomicrographs of immunolabeling of neurons in the cmVLM to c-Fos after temporalis injections of capsaicin (A; CPA encircled), after BDA injections into the LRF (E; dashed outline) and FG into the PBil injections (G; CPA encircled). The medullary LRF was labeled with Fos immunoreactivity after temporalis injections of capsaicin (B, C; dashed white outline) which mimicked the distribution labeled neurons after the PBil injection of FG (H, I; dashed white outline). The injection of BDA into the LRF also labeled rostral facial motoneurons and the superior salivatory nucleus (F). Abbreviations: Li, linear nucleus of the medulla; SSN, superior salivary nucleus. See text and previous figures for other abbreviations.</p

Sparse c-Fos labeling was seen in control rats.

<p>Line drawings of brainstem sections showing nuclei immunoreactive to c-Fos after an injection of saline into the temporalis muscle of a rat. Note the minimal c-Fos label in dorsomedial parts of the MDH (A, B, C), the CPA in the cmVLM (A, B, C), lateral medulla (D, E), RVM (F), A5 area (H), and the external lateral, dorsal lateral and internal lateral subnuclei of the parabrachial complex (I, J) from this control animal. Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024499#pone-0024499-g003" target="_blank">figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024499#pone-0024499-g004" target="_blank">4</a>. Abbreviations: SpVe, spinal vestibular nucleus; SuVe, superior vestibular nucleus; ml, medial lemniscus. See text and previous figures for other abbreviations.</p

Pontine neurons implicated in pain pathways are labeled with c-Fos, BDA and FG in their respective experiments.

<p>Photomicrographs and line drawings through the rostral medulla and pons showing immunoreactivity to c-Fos after capsaicin injection into the temporalis muscle (A–D), fibers and varicosities after an injection of BDA into the LRF (E–F), and retrogradely-labeled neurons after an injection of FG into the PBil (I–L). Note that the PBil is labeled both with c- Fos (B, arrow; D, encircled) and BDA (F, arrow; H, encircled). Labeling with each marker was in the A5 area (A, C, E, G, I, K), while dorsomedial parts of subnucleus oralis were labeled after PBil injections of FG (I, K). Abbreviations: LVe, lateral vestibular nucleus; Me5, mesencephalic trigeminal nucleus; Mo5, motor trigeminal nucleus; MVe, medial vestibular nucleus; PN, pontine nuclei; PnR, pontine raphe nucleus; RtTg, reticulotegmental nucleus; SO, superior olivary nucleus; Tz, trapezoid nucleus; VC, ventral cochlear nucleus; VLL, ventral nucleus of the lateral lemniscus; bc, brachium conjunctivum; g7, genu of facial nerve; mcp, middle cerebellar peduncle; 7n, facial nerve root. See text and previous figures for other abbreviations.</p

Other areas implicated in pain were labeled after capsaicin injections into the temporalis muscle.

<p>Numerous nuclei were immunohistochemically labeled with Fos in lamina I (B; yellow arrowheads) and lamina V of the TCC near the spinomedullary junction (A, B). Similar labeling was seen in the dorsomedial MDH near levels of the calamus scriptorius, but also included neurons in lamina II and III (C; D, arrow). Nuclei were labeled in dorsal subnucleus interpolaris (E, arrow; F, encircled), but their function is unknown. C-Fos profiles were found in the RVM bilaterally (G, H, arrow) dorsal and lateral to the pyramidal tract. The superior salivatory nucleus also was labeled with FOS after temporalis injections bilaterally (I), as were neurons in the subcoeruleus area (J) and A7 area (K). Abbreviations: RVM, rostral ventromedial medulla; SubC, subcoeruleus nucleus. See text and previous figures for other abbreviations.</p

The lateral medulla is implicated in a pathway from the TCC through the caudalmost ventrolateral medulla.

<p>Line drawings showing immunoreactivity in the caudal brainstem to c-Fos after a 20 µl injection of 0.05% capsaicin into the temporalis muscle (A, B, C), projections from the injection of BDA into the lateral medulla (D, E, F), and the location of retrogradely-labeled neurons after injections of FG into the PBil (G, H, I). Dorsomedial parts of the MDH were labeled with Fos after temporalis injections of capsaicin (A), as was the CPA in the cmVLM (A, arrow). Since numerous neurons in the LRF were labeled after temporalis injections of capsaicin (B, C; arrows), injections of BDA were made here (E; arrow). Labeled fibers with varicosities spread to the cmVLM caudally (D; arrow) and more rostrally in the LRT (F). Numerous retrogradely-labeled neurons were found in the LRF (H, I) and CPA in the cmVLM (G) after injections of FG into the PBil (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024499#pone-0024499-g005" target="_blank">Fig. 5L</a>). Abbreviations: ECu, external cuneate nucleus; Gr, gracile nucleus; Sol, nucleus of the solitary tract; Sp5I, nucleus of the spinal tract of the trigeminal nerve, interpolar part; Sp5O, nucleus of the spinal tract of the trigeminal nerve, oral part; 7, facial motor nucleus; icp, inferior cerebellar peduncle. See text and previous figures for other abbreviations.</p

A trigeminoreticular pathway as a route for ascending noxious information.

<p>Summary diagram illustrating the presumptive pain pathway from the TCC to the cmVLM and continuing through the LRF and PBil. We propose this is the initial part of a paleo-reticulo-thalamic pathway of the trigeminal system and suggest it may be important for transmitting deep pain from head and neck regions.</p