Trigeminal projections to hypoglossal and facial motor nuclei in the rat

This study was undertaken to identify the trigeminal nuclear regions connected to the hypoglossal (XII) and facial (VII) motor nuclei in rats. Anterogradely transported tracers (biotinylated dextran amine, biocytin) were injected into the various subdivisions of the sensory trigeminal complex, and labeled fibers and terminals were searched for in the XII and VII. In a second series of experiments, injections of retrogradely transported tracers (biotinylated dextran amine, gold-horseradish peroxidase complex, fluoro-red, fluoro-green) were made into the XII and the VII, and labeled cells were searched for in the principal sensory trigeminal nucleus, and in the pars oralis, interpolaris, and caudalis of the spinal trigeminal nucleus. Trigeminohypoglossal projections were distributed throughout the ventral and dorsal region of the XII. Neurons projecting to the XII were found in all subdivisions of the sensory trigeminal complex with the greatest concentration in the dorsal part of each spinal subnucleus and exclusively in the dorsal part of the principal nucleus. Trigeminofacial projections reached all subdivisions of the VII, with a gradual decreasing density from lateral to medial cell groups. They mainly originated from the ventral part of the principal nucleus. In the spinal nucleus, most of the neurons projecting to the VII were in the dorsal part of the nucleus, but some were also found in its central and ventral parts. By using retrograde double labeling after injections of different tracers in the XII and VII on the same side, we examined whether neurons in the trigeminal complex project to both motor nuclei. These experiments demonstrate that in the spinal trigeminal nucleus, neurons located in the pars caudalis and pars interpolaris project by axon collaterals to XII and VII. J. Comp. Neurol. 415:91–104, 1999. © 1999 Wiley-Liss, Inc.     

Projections from the superior colliculus to the trigeminal system and facial nucleus in the rat

To determine the influence of the superior colliculus (SC) in orienting behaviors, we examined SC projections to the sensory trigeminal complex, the juxtatrigeminal region, and the facial motor nucleus in rats. Anterograde tracer experiments in the SC demonstrated predominantly contralateral colliculotrigeminal projections. Microinjections in the deep layers of the lateral portion showed labeled nerve fibers and terminals in the ventromedial parts of the caudal principal nucleus and of the rostral oral subnucleus and in the medial part of the interpolar subnucleus. Some terminals were also observed in the juxtatrigeminal region and in the dorsolateral part of the facial motor nucleus contralaterally, overlying the orbicularis oculi motoneuronal region. Verification by retrograde tracer injections into the trigeminal target regions showed labeled SC neurons mostly in lateral portions of layers 4-7. When the juxtatrigeminal region was involved, a remarkable increase of labeled neurons was observed, having a patch-like arrangement with a decreasing gradient from lateral to medial SC portions. Retrograde tracer injections in the dorsolateral VII nucleus showed bilateral labeled neurons mainly in the deep lateral SC portion. Retrograde BDA microinjections into the same trigeminal or juxtatrigeminal regions, followed by gold-HRP into the dorsolateral VII nucleus, demonstrated a significant number of SC neurons in deep layers 6-7 projecting to both structures by axon collaterals. These neurons are mediolaterally grouped in patches along the rostrocaudal SC extent; a subset of them are immunoreactive for glutamic acid decarboxylase (GAD). They could be involved in the coordination of facial movements. Simultaneous anterograde and retrograde tracer injections into the lateral SC portion and the VII nucleus respectively localized trigeminofacial neurons receiving collicular input in the trigeminal principal nucleus and pars oralis. Therefore the SC should play a crucial role in regulating motor programs of both eye and eyelid movements.     

Trigeminovestibular and trigeminospinal pathways in rats: retrograde tracing compared with glutamic acid decarboxylase and glutamate immunohistochemistry

This study identified neurons in the sensory trigeminal complex with connections to the medial (MVN), inferior (IVN), lateral (LVN), and superior (SVN) vestibular nuclei or the spinal cord. Trigeminovestibular and trigeminospinal neurons were localized by injection of retrograde tracers. Immunohistochemical processing revealed gamma-aminobutyric acid (GABA)- and glutamate-containing neurons in these two populations. Trigeminovestibular neurons projecting to the MVN and the IVN were in the caudal principal nucleus (5P), pars oralis (5o), interpolaris (5i), and caudalis (5c) and scattered throughout the rostral 5P. Projections were bilateral to the IVN, with an ipsilateral dominance to the MVN, except from the rostral 5P, which was contralateral. Neurons projecting to the LVN were numerous in the ventral caudal 5P and the 5o and less abundant in the rostral 5P, 5i, and 5c. Our results suggested that only 5P and 5o project to the dorsal LVN. Neurons projecting to the SVN were in the dorsal 5P, 5o, and 5i but not in 5c. Trigeminospinal neurons were mainly in the ventral 5o and 5i and in the lateral 5c, rarely or never in 5P. Among trigeminovestibular neurons, most of the somas were immunoreactive for glutamate, but some reacted for GABA. Among trigeminospinal neurons, the number of somas immunoreactive for each of the two amino acids was similar. Trigeminal terminals were observed in contact with vestibulospinal neurons in the IVN and LVN, giving evidence of a trigeminovestibulospinal pathway. Therefore, inhibitory and excitatory facial inputs may contribute through trigeminospinal or trigeminovestibulospinal pathways to the control of head/neck movements.     

Organisation of reciprocal connections between trigeminal and vestibular nuclei in the rat.

In order to study the connection patterns between the sensory trigeminal and the vestibular nuclei (VN), injections of anterogradely and/or retrogradely transported neuronal tracers were made in the rat. Trigeminal injections resulted in anterogradely labelled fibres, with an ipsilateral preponderance, within the VN: in the ventrolateral part of the inferior nucleus (IVN), in the lateral part of the medial nucleus (MVN), in the lateral nucleus (LVN) with a higher density in its ventral half, and in the superior nucleus (SVN), more in the periphery than in the central part. Moderate trigeminal projections were observed in the small vestibular groups f, x and y/l and in the nucleus prepositus hypoglossi. Additional retrogradely labelled neurones were seen in the IVN, MVN, and LVN, in the same regions as those receiving trigeminal afferents. Morphological analysis of vestibular neurones demonstrated that vestibulo-trigeminal neurones are relatively small and belong to a different population than those receiving projections from the trigeminal nuclei. The trigeminovestibular and vestibulo-trigeminal relationships were confirmed by tracer injections in the VN. The results show that, in the VN, there is sensory information from facial receptors in addition to those reported from the neck and body. These facial afferents complement those from the neck and lower spinal levels in supplying important somatosensory information from the face and eye muscles. The oculomotor connections of the respective zones of the VN receiving trigeminal afferents suggest that sensory inputs from the face, including extraocular proprioception, may, through this pathway, influence the vestibular control of eye and head movements.     

Hypertensive effect of yohimbine following selective injection into the nucleus tractus solitarii of normotensive rats

We administered yohimbine (0.5 nmol in 0.2 microliter) directly into the nucleus tractus solitarii (NTS) of urethane-anesthetized normotensive male rats. We noted a prompt rise in blood pressure which lasted 30 min. These data suggest that the NTS is a central site in which the hypertensive effects of yohimbine can originate.     

Central afferents to the nucleus of the solitary tract in rats and mice

The nucleus of the solitary tract (NTS) regulates life-sustaining functions ranging from appetite and digestion to heart rate and breathing. It is also the brain's primary sensory nucleus for visceral sensations relevant to symptoms in medical and psychiatric disorders. To better understand which neurons may exert top-down control over the NTS, here we provide a brain-wide map of all neurons that project axons directly to the caudal, viscerosensory NTS, focusing on a medial subregion with aldosterone-sensitive HSD2 neurons. Injecting an axonal tracer (cholera toxin b) into the NTS produces a similar pattern of retrograde labeling in rats and mice. The paraventricular hypothalamic nucleus (PVH), lateral hypothalamic area, and central nucleus of the amygdala (CeA) contain the densest concentrations of NTS-projecting neurons. PVH afferents are glutamatergic (express Slc17a6/Vglut2) and are distinct from neuroendocrine PVH neurons. CeA afferents are GABAergic (express Slc32a1/Vgat) and are distributed largely in the medial CeA subdivision. Other retrogradely labeled neurons are located in a variety of brain regions, including the cerebral cortex (insular and infralimbic areas), bed nucleus of the stria terminalis, periaqueductal gray, Barrington's nucleus, Kölliker-Fuse nucleus, hindbrain reticular formation, and rostral NTS. Similar patterns of retrograde labeling result from tracer injections into different NTS subdivisions, with dual retrograde tracing revealing that many afferent neurons project axon collaterals to both the lateral and medial NTS subdivisions. This information provides a roadmap for studying descending axonal projections that may influence visceromotor systems and visceral “mind–body” symptoms.     

Quantitative analysis of the bilateral brainstem projections from the whisker and forepaw regions in rat primary motor cortex

The whisker region in rat primary motor (MI) cortex projects to several brainstem regions, but the relative strength of these projections has not been characterized. We recently quantified the MI projections to bilateral targets in the forebrain (Alloway et al. [2009] J Comp Neurol 515:548-564), and the present study extends those findings by quantifying the MI projections to bilateral targets in the brainstem. We found that both the whisker and forepaw regions in MI project most strongly to the basal pons and superior colliculus. While the MI forepaw region projects mainly to the ipsilateral basilar pons, the MI whisker region has significantly more connections with the contralateral side. This bilateral difference suggests that corticopontine projections from the MI whisker region may have a role in coordinating bilateral whisker movements. Anterograde tracer injections in MI did not reveal any direct projections to the facial nucleus, but retrograde tracer injections in the facial nucleus revealed some labeled neurons in MI cortex. The number of retrogradely labeled neurons in MI, however, was dwarfed by a much larger number of labeled neurons in the superior colliculus and other brainstem regions. Together, our anterograde and retrograde tracing results indicate that the superior colliculus provides the most effective route for transmitting information from MI to the facial nucleus.     

Neurons containing calcitonin gene-related peptide in the parabrachial nucleus project to the central nucleus of the amygdala

The source, distribution, and morphology of axons displaying calcitonin gene-related peptide (CGRP) immunoreactivity in the central amygdaloid nucleus of the adult rat were investigated with immunohistochemical techniques, both alone and in combination with retrograde transport of fluorescent tracers. An extremely dense plexus of CGRP-immunoreactive axons is differentially concentrated within the lateral capsular and lateral central subdivisions of the central nucleus, and much lighter concentrations of labeled fibers are present in the rostral part of the medial subdivision. No immunoreactive neurons were observed in the central nucleus in any of the experimental animals. The immunoreactive axons characteristically form prominent pericellular terminal arborizations surrounding unlabeled neurons. The number of cells receiving this dense input increases at caudal levels of the central nucleus. Retrograde label of central nucleus neurons by dye transport from injections into the pontine parabrachial nucleus and the nucleus of the tractus solitarius combined with CGRP immunohistochemistry established that many neurons in the central nucleus which receive dense pericellular innervation from CGRP-immunoreactive axons are projecting caudally to the parabrachial nucleus or, to a lesser extent, to the nucleus tractus solitarii. Central amygdaloid injections of rhodamine-labeled microspheres or fluorogold followed by immunohistochemical localization of cellular CGRP immunoreactivity revealed that the central amygdaloid CGRP fiber plexus originates bilaterally from the parabrachial nucleus. These multipolar CGRP-containing neurons are preferentially concentrated in the external medial and external lateral subnuclei, in the ventral aspect of the parabrachial nucleus. These results relating central amygdaloid CGRP to ascending and descending brainstem pathways, taken together with the extreme density of the fiber plexus, strongly suggest the relevance of the CGRP input to central nucleus function in cardiovascular and other autonomic regulation.     

Organization of trigeminocollicular connections and their relations to the sensory innervation of the eyelids in the rat

Relationships between the trigeminal component of blinking and the superior colliculus (SC) were studied in rats. To localize primary afferent eyelid projections in the sensory trigeminal complex, neuronal tracing experiments were performed as well as analysis of c-Fos protein expression after supraorbital (SO) nerve stimulation. Labelled nerve fibers were found to enter ventrally within the ipsilateral sensory trigeminal complex. Labelled boutons were observed at the junction of the principal nucleus (5P) and the pars oralis (5o) and in the pars caudalis (5c). The c-Fos immunoreactivity was observed in neurons located in the ipsilateral ventral parts of 5P, 5o, and the pars interpolaris (5i) and bilaterally in 5c. Injections in 5P, 5o, 5i, and 5c resulted in anterogradely labelled fibers, with a contralateral preponderance, within the intermediate and deeper SC layers. Injections in 5P or 5o showed anterogradely labelled nerve fibers, profusely terminating in small patches in the medial and central portions of SC layer 4. Subsequently, dense labelling was found in the lateral portion of SC layers 4-7, without patch-like organization. Injections in SC showed retrogradely labelled neurons predominantly within the contralateral part of the sensory trigeminal complex (28% in 5P, 20% in 5o, 50% in 5i, and 2% in 5c). Colocalization of the retrograde tracer after SC injections and c-Fos immunoreactivity in neurons demonstrated that some 5P, 5o, and 5i neurons receive SO nerve inputs and project to SC. This implies that intermediate and deeper SC layers receive sensory information from the eyelids and may be directly involved in the regulation of eye-eyelid coordination.     

Trigeminal projections to the nucleus submedius of the thalamus in the rat.

Methods involving the anterograde and retrograde transport of wheat-germ agglutinin conjugated horseradish peroxidase and the retrograde transport of Fluoro-Gold were used in rats to examine the distribution within the spinal trigeminal nucleus of trigeminal neurons projecting to the nucleus submedius (Sm) of the thalamus, as well as the distribution of axon terminals within the Sm. Following injections into the trigeminal nucleus, axon terminals were seen in the dorsal part of the anterior Sm; the terminals occurred bilaterally but had an obvious contralateral dominance. To help determine the precise location of the Sm-petal neurons, the border between trigeminal subnuclei interpolaris and caudalis was examined by the use of immunohistochemical procedures for calcitonin gene-related peptide (CGRP). The Sm-petal neurons that were labeled retrogradely occurred only at the caudal interpolaris and rostral caudalis levels; the number of labeled neurons on the contralateral side was approximately six times that on the ipsilateral side. Most of these neurons were located in the ventral part of the caudal interpolaris and rostral caudalis and spinal trigeminal tract; in caudalis, the neurons were almost exclusively localized to its superficial layers. There were approximately three times more labeled neurons in interpolaris than in caudalis. In the experiments combined with immunohistochemistry for CGRP, many neurons (34%) were seen in proximity to CGRP-like immunopositive fibers. These results suggest that the Sm of the rat receives its orofacial afferent inputs from brainstem neurons that are localized to the caudal interpolaris and rostral caudalis. In view of previous studies that have implicated these three structures in somatosensory function, and in particular nociception, our data point to a role for this direct projection from interpolaris and caudalis to Sm in the central processing of pain.     

Vasopressin-induced pressor responses in rats to bilateral electrolytic lesioning of the caudal portion of the nucleus tractus solitarii

Bilateral electrolytic lesioning of the nucleus tractus solitarii elicited a pressor response in rats with spinal transection. This response was abolished by a vasopressin pressor antagonist and plasma vasopressin was increased during the response. This evidence suggests that the nucleus tractus solitarii is involved in inhibiting vasopressin release and that an impaired function would lead to an enhancement of vasopressin release, to the extent of eliciting a pressor response.     

Adenosine A<Subscript>1</Subscript> receptor distribution in the nucleus tractus solitarii of normotensive and spontaneously hypertensive rats

Summary. Adenosine acts at many sites to modulate neuronal activity. The nucleus tractus solitarii (NTS) is a major brain site in cardiovascular control. The present study was undertaken for a detailed analysis of the distribution of A₁ adenosine receptor (A₁R) in the NTS of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY), using in vitro autoradiography with [³H]DPCPX. The density of [³H]DPCPX in the whole NTS decreased according to the rostral-caudal levels. This high level of [³H]DPCPX binding at rostral sites is due to an specific label of the dorsomedial/dorsolateral subnuclei. On the other hand, analysis of subpostremal subnucleus, showed opposite results. The density of [³H]DPCPX binding in the subpostremal NTS increased according to the rostral-caudal levels. Furthermore, it was observed an increased [³H]DPCPX binding in the SHR compared with WKY. The results show a complex pattern of A₁R distribution in the NTS, which highlight the powerful modulatory actions mediated by adenosine in the NTS barosensitive neurons.     

The ultrastructural localization of enkephalin and substance P immunoreactivities in the nucleus tractus solitarii of the cat

In the medial and commissural subdivisions of the nucleus tractus solitarii enkephalin and substance P immunoreactivities were localized within synaptic terminals, unmyelinated axons, and neuronal cell bodies. Both enkephalin and substance P immunoreactivities were contained within synaptic terminals which had a mixture of small clear vesicles and dense core vesicles. The presence of dense core vesicles within both the enkephalin- and substance P-immunoreactive terminals was a consistent feature, although they were not associated with the actual synaptic junction. While enkephalin- and substance P-immunoreactive terminals shared a similar morphology, their respective distributions along the dendritic tree were quite distinct. Enkephalin-immunoreactive terminals contacted mainly the cell body and proximal portions of the dendritic tree. In contrast, substance P-immunoreactive terminals synapsed predominantly with spines and shafts of small to medium-sized dendrites. Few substance P-immunoreactive terminals contacted proximal dendrites and they were never presynaptic to the neuronal cell body. This apparent segregation of synaptic terminals on neurons suggests that enkephalin synapses have a more pronounced effect than substance P terminals.     

Autoradiographic localization of thyrotropin-releasing hormone and substance P receptors in the rat dorsal vagal complex

We utilized quantitative autoradiography to localize receptors for thyrotropin-releasing hormone (TRH) and substance P in individual subnuclei of the rat nucleus tractus solitarii (NTS) and the dorsal vagal complex. Within the NTS, TRH receptor concentrations were highest within the gelatinosus and centralis subnuclei and the medial subnucleus rostral to the area postrema, moderate within the intermediate subnucleus and the medial subnucleus adjacent to the area postrema, and low within the ventrolateral and commissural subnuclei and the medial subnucleus caudal to the area postrema. In contrast, substance P receptor concentrations were high throughout the medial subnucleus, moderate in all other subnuclei medial to the tractus solitarius, and relatively low in subnuclei lateral to the tractus solitarius. The dorsal motor nucleus of the vagus contained high concentrations of both TRH and substance P receptors, whereas we observed low TRH and moderate substance P receptors in the area postrema. High TRH and moderate substance P receptors were observed in the adjacent hypoglossal nucleus. In addition, we compared the concentrations of TRH receptors between chloroform-defatted and nondefatted tissue sections, and noted little effect of white matter tritium quench upon the observed TRH receptor concentrations. These results suggest that neurotransmitter receptors within the rat dorsal vagal complex are organized in a manner consistent with previous cytoarchitectural and hodological partitioning of the NTS and that the distribution of an individual neurotransmitter receptor in the NTS may correspond to the role of that transmitter in modulating autonomic function.     

Axonal projections of medullary swallowing neurons in guinea pigs

A central pattern generator (CPG) for swallowing in the medulla oblongata generates spatially and temporally coordinated movements of the upper airway and alimentary tract. To reveal the medullary neuronal network of the swallowing CPG, we examined the cytoarchitecture of the swallowing CPG and axonal projections of its individual neurons by extracellular recording and juxtacellular labeling of swallowing-related neurons (SRNs) in the medulla in urethane-anesthetized and paralyzed guinea pigs. Three major types of neuronal discharge patterns were identified during fictive swallowing induced by stimulation of the superior laryngeal nerve: early (burst-like activation during the pharyngeal stage), late (activation after the pharyngeal stage), and inhibited (inhibition during the pharyngeal stage) types. Sixteen neurons were successfully labeled in the nucleus tractus solitarii (NTS) and in the medullary reticular formation (RF). No motoneuron was labeled. The SRNs in the NTS had axons projecting to the NTS, RF, nucleus ambiguus, nucleus hypoglossus, and dorsal motor nucleus of the vagus on the ipsilateral side. Some NTS SRNs projected only within the NTS. The axons of SRNs in the RF projected also to the NTS, RF, motor nuclei on the ipsilateral side, and to the other side RF. These findings show anatomic substrates for the neuronal network of the CPG for swallowing, which consists of complex neuronal connections among SRNs in the NTS, RF, and motor nuclei.     

Localization of trigeminal, spinal, and reticular neurons involved in the rat blink reflex

Electrical stimulation of the supraorbital nerve (SO) induces eyelid closure by activation of orbicularis oculi muscle motoneurons located in the facial motor nucleus (VII). Neurons involved in brainstem central pathways implicated in rat blink reflex were localized by analyzing c-Fos protein expression after SO stimulation in conjunction with tracing experiments. A retrograde tracer (gold-horseradish peroxidase [HRP]) was injected into the VII. The distribution patterns of activated c-Fos-immunoreactive neurons and of neurons exhibiting both c-Fos immunoreactivity and gold-HRP labeling were determined in the sensory trigeminal complex (STC), the cervical spinal cord (C1), and the pontomedullary reticular formation. Within the STC, c-Fos immunoreactivity labeled neurons in the ipsilateral ventral part of the principal nucleus, the pars oralis and interpolaris, and bilaterally in the pars caudalis. Colocalization of gold-HRP and c-Fos immunoreactivity was observed in neurons of ventral pars caudalis layers I-IV and ventral pars interpolaris. In C1, SO stimulation revealed c-Fos neurons in laminae I-V. After additional injections in VII, the double-labeled c-Fos/gold-HRP neurons were concentrated in laminae IV and V. Although c-Fos neurons were found throughout the pontomedullary reticular formation, most appeared rostrally around the motor trigeminal nucleus and in the ventral parvocellular reticular nucleus medial to the fiber bundles of the seventh nerve. Caudally, c-Fos neurons were in the lateral portion of the dorsal medullary reticular field. In addition, these reticular areas contained double-labeled neurons in electrically stimulated rats that had received gold-HRP injections in the VII. The presence of double-labeled neurons in the STC, C1, and the reticular formation implies that these neurons receive sensory information from eyelids and project to the VII. These double-labeled neurons could then be involved in di- or trisynaptic pathways contributing to the blink reflex.     

Anatomical organization of the limb premotor network in the turtle (Chrysemys picta) revealed by in vitro transport of biocytin and neurobiotin

The in vitro turtle brainstem-cerebellum preparation has been a valuable tool in the study of central motor programs. In the present study, we investigate the anatomical organization of the turtle rubrocerebellar limb premotor network and its sensory connections in vitro by combining the rapid anterograde and retrograde transport of neurobiotin and biocytin with the extended viability of the isolated turtle brainstem-cerebellum. These compounds retrogradely labeled soma, dendrites, and axons, and orthogradely labeled axons and, to a lesser extent, terminals. The chelonian red nucleus receives a dense input form the contralateral lateral cerebellar nucleus and projects heavily to the contralateral spinal cord. Rubral axons sparsley innervate the lateral cerebellar nucleus and project heavily to the lateral reticular nucleus. Lateral reticular axons heavily innervate the lateral cerebellar nucleus before terminating in the pars laterlalis of the cerebellar cortex as mossy fibers. These prominent, recurrent loops among the lateral cerebellar nucleus, red nucleus, and lateral reticular nucleus constitute the turtle rubrocerebellar limb premotor network. Sensory inputs to the red nucleus orginate in the contralateral dorsal column nuclei, the principle trigeminal nucleus, and the spinothalamic system. These sites project bilaterally to the lateral reticular nucleus. The lateral cerebellar nucleus receives a contralateral input from the dorsal column nuclei. The red nucleus projects sparsely to the dorsal column nuclei. The red nucleus also receives an ipsilateral descending projection from the suprapeduncular nucleus, located in the diencephalon, and an ascending input from the rostral rhombencephalic reticular formation. An ipsilateral descending pathway originating in the red nucleus is likely to be the rubro-olivary tract. © 1994 Wiley-Liss, Inc.