Yun-Qing Li

GABAergic and glycinergic neurons projecting to the trigeminal motor nucleus: A double labeling study in the rat

The distribution of GABAergic and glycinergic premotor neurons projecting to the trigeminal motor nucleus (Vm) was examined in the lower brainstem of the rat by a double labeling method combining retrograde axonal tracing with immunofluorescence histochemistry. After injection of the fluorescent retrograde tracer, tetramethylrhodamine dextran amine (TRDA), into the Vm unilaterally, neurons labeled with TRDA were seen ipsilaterally in the mesencephalic trigeminal nucleus, and bilaterally in the parabrachial region, the supratrigeminal and intertrigeminal regions, the reticular formation just medial to the Vm, the principal sensory and spinal trigeminal nuclei, the pontine and medullary reticular formation, especially the parvicellular part of the medullary reticular formation, the alpha part of the gigantocellular reticular nucleus, and the medullary raphe nuclei. Some of these neurons labeled with TRDA were found to display glutamic acid decarboxylase (the enzyme involved in GABA synthesis)‐like or glycine‐like immunoreactivity. Such double‐labeled neurons were seen mainly in the supratrigeminal region, the reticular region adjacent to the medial border of the Vm, and the dorsal part of the lateral reticular formation of the medulla oblongata; a number of them were further scattered in the intertrigeminal region, the alpha part of the gigantocellular reticular nucleus, the nucleus raphe magnus, the principal sensory trigeminal nucleus, and the interpolar subnucleus of the spinal trigeminal nucleus. These neurons were considered to be inhibitory (GABAergic or glycinergic) neurons sending their axons to motoneurons in the Vm, or to local interneurons within and around the Vm.

Identification of premotor interneurons which project bilaterally to the trigeminal motor, facial or hypoglossal nuclei: a fluorescent retrograde double-labeling study in the rat☆

With the aid of a fluorescent retrograde double-labeling technique, we examined in the rat the distribution of single neurons which project bilaterally to one of the orofacial motor nuclei (the trigeminal motor, facial and hypoglossal nuclei) by branching axons. The results suggested that premotor interneurons were distributed most frequently in the medial part of the parvicellular reticular nucleus; such neurons were further scattered in the other regions of the medullary reticular formation, in the regions around the trigeminal motor nucleus, in the parabrachial area, and in the mesencephalic reticular formation.

The sites of origin of serotoninergic afferent fibers in the trigeminal motor, facial, and hypoglossal nuclei in the rat.

The sites of origin of serotoninergic afferents in the trigeminal motor (Vm), facial (VII), and hypoglossal nuclei (XII) were studied in the rat by fluorescent retrograde labeling with Fluoro-Gold, in combination with immunofluorescence histochemistry for serotonin (5-HT). The results indicated: (1) The nucleus raphe magnus, nucleus raphe pallidus, and nucleus raphe obscurus contained 5-HT neurons projecting to the Vm, VII or XII. (2) The nucleus raphe dorsalis sends 5-HT fibres to the Vm and VII, but not to the XII. (3) The gigantocellular reticular nucleus pars alpha contained 5-HT neurons projecting to the VII.

Premotor neurons projecting simultaneously to two orofacial motor nuclei by sending their branched axons. A study with a fluorescent retrograde double-labeling technique in the rat

Premotor neurons innervating simultaneously two of the trigeminal motor (Vm), facial (VII) and hypoglossal nuclei (XII) by sending their branched axons were demonstrated in the lower brain stem of the rat by means of a fluorescent retrograde double-labeling method with Fast Blue (FB) and Diamidino Yellow (DY). After injections of FB and DY, respectively, into the Vm and VII, into the Vm and XII, or into the VII and XII, the majority of neuronal cell bodies labeled doubly with FB and DY were distributed in the lateral tegmental field, especially its medial part in the medulla oblongata.

Identification of periaqueductal gray and dorsal raphe nucleus neurons projecting to both the trigeminal sensory complex and forebrain structures: a fluorescent retrograde double-labeling study in the rat

The midbrain periaqueductal gray (PAG) including the dorsal raphe nucleus (DR) has been known to contain serotoninergic neurons projecting to many brain regions. Employing fluorescent retrograde double labeling combined with immunofluorescence histochemistry for serotonin (5-HT), we examined in the rat whether or not single PAG/DR neurons with 5-HT send their axons to both the trigeminal sensory complex and forebrain regions. Stereotaxic injections of Diamidino Yellow (DY) and Fast Blue (FB) were performed unilaterally; DY was injected into the caudal spinal trigeminal nucleus or principal sensory trigeminal nucleus, and FB into the ventrolateral orbital cortex, nucleus accumbens or amygdala. A small percentage of PAG/DR neurons were doubly labeled with DY and FB, and the majority of them showed 5-HT-like immunoreactivity (5-HT-LI). Most of these 5-HT-LI PAG/DR neurons that were indicated to send their axons simultaneously to both the trigeminal sensory complex and forebrain regions were distributed in the ventrolateral PAG subdivision and ventral aspects of the medial PAG subdivision at the middle and caudal PAG levels, bilaterally with a predominant distribution on the side ipsilateral to the injections. This indicates a possible role of these PAG/DR neurons in the limbic or affective-motivational aspect of the pain-related neural system.

Association of serotonin-like immunoreactive axons with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat.

Serotoninergic projections to the spinal dorsal horn are implicated in the modulation of nociceptive transmission. However, morphological evidence indicating that serotoninergic projection fibers make synapses on nociceptive neurons in the medullary dorsal horn is still meager. Thus, we examined whether axonal varicosities with serotonin (5‐HT)‐like immunoreactivity (5‐HT‐LI) might make synapses on nociceptive projection neurons in the caudal spinal trigeminal nucleus (Vc) of the rat. Projection neurons were retrogradely labeled with tetramethylrhodamine‐dextran amine (TMR‐DA) or wheat germ agglutinin‐horseradish peroxidase (WGA‐HRP) that was injected into the parabrachial or thalamic region. Vc neurons in which c‐fos protein‐like immunoreactivity (Fos‐LI) was induced by subcutaneous injection of formalin into the lip were considered nociceptive. Vc neurons in direct contact with axonal varicosities that bind isolectin I‐B4 were also considered nociceptive.

Serotonergic innervation of mesencephalic trigeminal nucleus neurons: a light and electron microscopic study in the rat

Neurons of the mesencephalic trigeminal nucleus (MTN) are considered to be homologous to mechanosensitive neurons in the sensory ganglia. The sites of origin of serotonin (5HT)-immunoreactive axons on neuronal cell bodies in the MTN were studied in the rat by combining immunofluorescence histochemical techniques with retrograde tracing of Fluoro-Gold (FG) and anterograde tracing of biotin-conjugated dextran amine (BDA). The tracing studies, which were combined with multiple-labeling immunohistochemistry and confocal microscopy, indicated that 5HT-immunoreactive axon terminals on the cell bodies of MTN neurons originated from the medullary raphe nuclei, such as the nucleus raphes magmus (RMg), alpha part of the nucleus reticularis gigantocellularis (GiA) and nucleus raphes obscurus (ROb), as well as from the mesopontine raphe nuclei, such as the nucleus raphes dorsalis (DR), nucleus raphes pontis (PnR) and nucleus raphes medianus (MnR); mainly from the RMg, GiA and DR, and additionally from the ROb, PnR and MnR. The cell bodies in close apposition to 5HT-immunoreactive axon terminals were found through the whole rostrocaudal extent of the MTN. Electron microscopically a number of axon terminals that were labeled with BDA injected into the raphe nuclei were confirmed to be in asymmetric synaptic contact with the cell bodies of MTN neurons. It was also indicated that substance P existed in some of the 5HT-containing axosomatic terminals arising from the ROb, RMg and GiA. The present results indicated that proprioceptive sensory signals from the muscle spindles or periodontal ligament might be modulated at the level of the primary afferent cell bodies in the MTN by 5HT-containing axons from the mesopontine and medullary raphe nuclei including the GiA.

Collateral projections of single neurons in the periaqueductal gray and dorsal raphe nucleus to both the trigeminal sensory complex and spinal cord in the rat.

After injecting Diamidino yellow (DY) and Fast blue (FB), respectively, into the trigeminal sensory complex and lumbar cord segments, neurons were doubly labeled retrogradely with both DY and FB in the periaqueductal gray (PAG) and dorsal raphe nucleus (DR). The fluorescent retrograde double-labeling method combined with serotonin (5HT) immunofluorescence histochemistry revealed that more than 70% of the doubly labeled PAG/DR neurons showed 5HT-like immunoreactivity (5HT-LI). These 5HT-LI PAG/DR neurons may modulate nociceptors in the trigeminal sensory complex and spinal cord by sending axon collaterals to these regions.

Collateral projections of single neurons in the nucleus raphe magnus to both the sensory trigeminal nuclei and spinal cord in the rat

After injecting Diamidino yellow and Fast blue respectively into the sensory trigeminal nuclei and spinal cord, we observed doubly labeled cells in the nucleus raphe magnus (NRM). Combining the fluorescent retrograde double labeling with serotonin (5-HT) immunofluorescence histochemistry, we further found that about 30% of the doubly labeled NRM neurons showed 5-HT-like immunoreactivity (5-HT-LI). Such 5-HT-LI NRM neurons may modulate nociceptive activities simultaneously in the sensory trigeminal nuclei and spinal cord by sending axon collaterals to these regions.

Collateral projections of trigeminal ganglion neurons to both the principal sensory trigeminal and the spinal trigeminal nuclei in the rat

Employing a combination of fluorescent retro grade double labelling and immunofluorescence histo chemistry for substance P (SP) and calcitonin gene-relat ed peptide (CGRP), we examined collateral projections from single neurons in the trigeminal ganglion (TG) of the rat to both the principal sensory trigeminal nucleus (Vp) and the oral, interpolar or caudal subnuclei of the spinal trigeminal nucleus (Vo, Vi or Vc). In the rats that were unilaterally injected with fast blue (FB) into the Vp and with diamidino yellow (DY) into the Vo, Vi or Vc, neurons labelled with FB and/or DY were observed in the TG ipsilateral to the injections. Of the labelled TG neurons, about 2% were double labelled with both trac ers in the rats that were injected with FB into the Vp and with DY into the Vo or Vi, and about 10% were double labelled in the rats that were injected with FB into the Vp and with DY into the Vc. The results indicate that TG neurons sending their axons to the Vp project, by way of axon collaterals, to the Vc more frequently than to the Vo or Vi.Some of the TG neurons double labelled with FB and DY exhibited SP-or CGRP-like immunoreactivity (LI): Of the TG neurons that were double labelled with FB injected into the Vp and with DY injected into the Vo, Vi or Vc, about 38%, 49% and 42%, respectively, displayed SP-LI, and about 54%, 58% and 59%, respectively, showed CGRP-LI. Some of the SP-or CGRP-LI TG neurons that were double labelled with FB and DY were assumed to mediate pain signals to both the Vp and the spinal trigeminal nucleus (Vo, Vi and/or Vc) by way of axon collaterals.