Specializations of gastrin‐releasing peptide cells of the mouse suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus regulates daily rhythms in physiology and behavior. It is composed of a heterogeneous population of cells that together form the circuits underlying its master clock function. Numerous studies suggest the existence of two regions that have been termed core and shell. At a gross level, differences between these regions map to distinct functional differences, although the specific role(s) of various peptidergic cellular phenotypes remains unknown. In mouse, gastrin‐releasing peptide (GRP) cells lie in the core, are directly retinorecipient, and lack detectable rhythmicity in clock gene expression, raising interest in their role in the SCN. Here, we provide evidence that calbindin‐expressing cells of perinatal mouse SCN express GRP, identified by a green fluorescent protein (GFP+), but lack detectable calbindin later in development. To explore the intra‐SCN network in which GRP neurons participate, individual GFP+ cells were filled with tracer and their morphological characteristics, processes, and connections, as well as those of their non‐GFP‐containing immediate neighbors, were compared. The results show that GFP+ neurons form a dense network of local circuits within the core, revealed by appositions on other GFP+ cells and by the presence of dye‐coupled cells. Dendrites and axons of GFP+ cells make appositions on arginine vasopressin neurons, whereas non‐GFP cells have a less extensive fiber network, largely confined to the region of GFP+ cells. The results point to specialized circuitry within the SCN, presumably supporting synchronization of neural activity and reciprocal communication between core and shell regions. J. Comp. Neurol. 518:1249–1263, 2010. © 2009 Wiley‐Liss, Inc.     

Central terminations of low-threshold mechanoreceptive afferents in the trigeminal nuclei interpolaris and caudalis of the cat

Previous studies indicate that vibrissa, nonvibrissa, guard hair, hairy skin, and periodontal ligament afferents give rise to morphologically distinct terminal arbors in the trigeminal nuclei principalis (Vp) and oralis (Vo) in the cat. The present study describes the extent to which morphological and functional relationships exist in the nuclei interpolaris (Vi) and caudalis (Vc). Twenty-two fibers were physiologically characterized and stained by intra-axonal HRP injection techniques. The fast adapting (FA) vibrissa (VF) afferents gave rise to compact and circumscribed arbors in Vj and Vc. These tended to be larger in Vc than in Vi. The slowly adapting (SA) vibrissa (VS) afferents in Vi and Vc had more widespread and larger arbors than those of the VF afferents. The VS arbors in Vc tended to be larger and less circular than those in Vi. Guard hair (GH) afferents gave rise to circumscribed arbors in both nuclei, but those in Vc tended to have larger and more circular arbors than those in Vi. Down hair (DH) afferents gave rise to small, circumscribed arbors or a few distinct patches of boutons within a small area in Vi; arbors in Vc were less extensive and “stringy.” Unlike other afferents, DH arbors were larger in Vi than in Vc, but smaller than those of GH afferents in either nuclei. The SA hairy skin (SS) afferents had arbors that were widespread with a few distinct patches of boutons; the arbors in Vc were larger than those in Vi. The arbors of SS afferents were smaller than those of VS and GH afferents in both nuclei. Like GH afferents, FA periodontal ligament (PF) afferents gave rise to large and circumscribed arbors in Vi, although the arbors in Vc were smaller and less dense. The present study demonstrated significant functional and morphological relationships for primary afferents in Vi and Vc, thus suggesting that sensory information from each of the distinct fiber or functional classes is processed in a characteristic manner in the individual nuclei. © Wiley-Liss, Inc.     

Osteopontin-immunoreactive primary sensory neurons in the rat spinal and trigeminal nervous systems

1200 micrometer(2) and 9% of those in the range 600-1200 micrometer(2) showed the immunoreactivity (ir). DRG neurons <600 micrometer(2)800 micrometer(2) showed the ir and 21% of those in the range 400-800 micrometer(2) were immunoreactive for this protein. TG neurons <400 micrometer(2) were mostly devoid of OPN-ir (2%). Virtually all (99%) Mes5 primary sensory neurons exhibited the ir. Muscle spindles in the soleus and masseter muscles contained OPN-ir spiral axon terminals. In the hard palate and incisor periodontal ligament, unencapsulated corpuscular endings exhibited the ir. The co-expression of OPN with parvalbumin and calcitonin gene-related peptide (CGRP) was also examined in the DRG and TG. In the DRG, virtually all (97%) OPN-ir neurons exhibited parvalbumin-ir. Conversely, 66% of parvalbumin-ir DRG neurons co-expressed OPN-ir. In the TG, 81% of OPN-ir neurons exhibited parvalbumin-ir and 69% of parvalbumin-ir ones showed OPN-ir. Virtually all OPN-ir DRG and TG neurons were devoid of CGRP-ir. The present study indicates that OPN-ir primary sensory neurons in the DRG and Mes5 are spinal and trigeminal proprioceptors. OPN-ir TG neurons appear to include low-threshold mechanoreceptors.     

Studies of the principal sensory and spinal trigeminal nuclei of the rat: Projections to the superior colliculus,inferior olive,and cerebellum

We have analyzed the connections between the sensory trigeminal nuclei and two major sensorimotor areas (i.e., the superior colliculus and crura I and II of the cerebellar cortex) in which tactile input from peri-oral and other facial regions is a prominent feature. Following injections of horseradish peroxidase into the superior colliculus, retrogradely labeled cells occupy the ventral one chird of the contralateral principal sensory and spinal trigeminal nucleus; trigeminocollicular neurons are especially numerous within, the sub-nucleus interpolaris (Svi). Injections of either 3H-proline or horseradish per-oxidase (HRP) into the Svi reveal that trigeminocollicular axons reach the rostral two-thirds to three-quarters of the contralateral superior colliculus, where they distribute in a nonuniform, patchy manner within layers IV-VI. In addition to demonstrating the trigeminocollicular projection, antero-grade and retrograde transport studies of the Svi also reveal a trigeminooli-vary projection which terminates primarily within the contralateral rostral dorsal accessory (DAO) and adjacent principal (PO) olives; some of the Svi neurons innervate both the superior colliculus and the DAO-PO via axon collaterals. Data from a final set of retrograde tracing experiments show that the trigeminorecipient zone of the DAO-PO contains neurons which project to crura I and/or II of the cerebellar cortex. Of the various submodalities conveyed by the trigeminal system, it is likely that the trigeminal connections we have demonstrated are carrying tactile information. This is indicated by the fact that responses to tactile stimulation of the face have been reported for cells in (1) the deeper collicular layers, (2) the trigeminorecipient zone of the DAO-PO, and (3) cerebellar targets of this zone, crura I and II. All data are discussed in the context of the anatomical and physiological literature.     

Organization of HRP-labeled trigeminal mandibular primary afferent neurons in the rat

Horseradish peroxidase (HRP) applied to the transected mandibular division of the trigeminal (V) ganglion was transported anterogradely to pri-mary afferent terminal zones in the dorsal and dorsomedial trigeminal brain-stem nuclear complex (TBNC). Primary V afferents of ganglionic origin were also visible in the ipsilateral cerebellar cortex (crus I and II, paraflocculus) and the dentate, cuneate, solitary, supratrigeminal, and dorsal motor vagal nuclei, parvicellular reticular formation, area postrema and C1–C6 dorsal horn, laminae I–V. Contralateral subnucleus caudalis and C1–C2 dorsal horn were also innervated by primary afferents which crossed in the spinal gray to terminate medially, primarily in laminae I, II, and V. Almost all of these projections were also labeled in various combinations when HRP was applied to individual sensory branches of the mandibular nerve: lingual, infe-rior alveolar, mylohyoid, and auriculotemporal. Transganglionic transport of HRP in the latter four cases revealed strong evidence for mtradivisional somatotopy among the four branches in both the ganglion and TBNC. Cell bodies innervating posterior and/or lateral portions of the head and face (i.e., auriculotemporal and mylohyoid) were found with greater frequency in dor-sal mandibular ganglion regions, while somata supplying more rostral oral-perioral regions (i.e., lingual and inferior alveolar) were predominant ventrally. Components of the mandibular projection to the TBNC were organized topographically in at least some portion of all of its three dimen-sions. Subnuclear preferences were not clear-cut; all four nerves innervated at least some portion of principalis, oralis, interpolaris, and caudalis, save for mylohyoid, which did not project to caudalis. Lingual fibers were most prominent in principalis and oralis, occupied medial portions of the mandib-ular projection to the TBNC, and descended only to rostral caudalis, most notably laminae I-III. Inferior alveolar afferents were ubiquitous in the mandibular component of the TBNC and C1–C2, save for its far lateral bor-der. Mylohyoid terminals were sparse, most prominent in interpolaris, and occupied only dorsolateral TBNC regions and laminae III and IV of C1–C3. The auriculotemporal innervation of the mandibular TBNC was heaviest in interpolaris and was restricted to mostly ventrolateral regions. Its primary focus, however, was laminae III and IV of C1–C4. The clinical implications of this topographical organization are discussed, particularly with respect to the rostrocaudal intradivisional lamination in caudalis and the cervical dorsal horn.     

Oral and facial representation in the trigeminal principal and rostral spinal nuclei of the cat

Transganglionic transport of horseradish peroxidase (HRP) was used to study the patterns of termination of somatic afferent fibers innervating oral and facial structures within the principal nucleus (Vp), nucleus oralis (Vo), and nucleus interpolaris (Vi). The primary trigeminal afferent fibers that innervate the oral cavity supplied by the pterygopalatine, superior alveolar, lingual, buccal, and inferior alveolar branches, as well as the facial skin supplied by the frontal, corneal, zygomatic, infraorbital, auriculotemporal, mylohyoid, and mental branches, were traced in this experiment. The results show that trigeminal afferent nerves that innervate the oral cavity project mainly to the principal nucleus, the rostrodorsomedial part (Vo.r) and dorsomedial division (Vo.dm) of pars oralis, and the dorsomedial region of pars interpolaris, while an extensive overlap of projections is found in the Vo.r, Vo.dm, and rostral Vi. The central processes of fibers innervating the anterior face (i.e., mental, infraorbital, and frontal nerves) terminate in the ventral division of principalis (Vpv), caudal region pars oralis (Vo.c), and ventrolateral Vi, with the largest numbers of terminals being found in the Vpv and Vi. In contrast, the central projection patterns of the corneal, zygomatic, mylohyoid, and auriculotemporal afferents are different from those of other afferent nerves examined, and present a discrete projection to the trigeminal sensory nuclear complex (TSNC). The corneal, mylohyoid, and auriculotemporal afferents mainly project to the restricted regions of principalis and caudal Vi, while zygomatic afferent nerve fibers project to the caudal third of pars interpolaris. The typical somatotopic organization with the face of the mouth open inverted is represented in the rostrocaudal midlevels of the Vpv and caudal pars interpolaris. The Vpd receives topographical projection from primary afferent nerves that innervate the oral structure only, while this projection was organized in a complicated manner. The relationship between the functional segregation and the cytoarchitectonic differentiation of the TSNC is discussed, particularly with respect to this somatotopic organization, combined with the characteristics of projecting cells in the TSNC.     

Site‐specific effects of gastrin‐releasing peptide in the suprachiasmatic nucleus

The effects of gastrin‐releasing peptide (GRP) on the circadian clock in the suprachiasmatic nucleus (SCN) are dependent on the activation of N‐methyl‐d ‐aspartate (NMDA) receptors in the SCN. In this study, the interaction between GRP, glutamate and serotonin in the regulation of circadian phase in Syrian hamsters was evaluated. Microinjection of GRP into the third ventricle induced c‐fos and p‐ERK expression throughout the SCN. Coadministration of an NMDA antagonist or 8‐hydroxy‐2‐di‐n‐propylamino‐tetralin [a serotonin (5‐HT)_1A,7 agonist, DPAT] with GRP limited c‐fos expression in the SCN to a region dorsal to GRP cell bodies. Similar to the effects of NMDA antagonists, DPAT attenuated GRP‐induced phase shifts in the early night, suggesting that the actions of serotonin on the photic phase shifting mechanism occur downstream from retinorecipient cells. c‐fos and p‐ERK immunoreactivity in the supraoptic (SON) and paraventricular hypothalamic nuclei also increased following ventricular microinjection of GRP. Because of this finding, a second set of experiments was designed to test a potential role for the SON in the regulation of clock function. Syrian hamsters were given microinjections of GRP into the peri‐SON during the early night. GRP‐induced c‐fos activity in the SCN was similar to that following ventricular administration of GRP. GRP or bicuculline (a γ‐aminobutyric acid_A antagonist) administered near the SON during the early night elicited phase delays of circadian activity rhythms. These data suggest that GRP‐induced phase‐resetting is dependent on levels of glutamatergic and serotonergic neurotransmission in the SCN and implicate activity in the SON as a potential regulator of photic signaling in the SCN.     

Trigeminal ganglion neurons which project by way of axon collaterals to both the caudal spinal trigeminal and the principal sensory trigeminal nuclei

Employing a combination of fluorescent retrograde double labeling and immunofluorescence histochemistry, we found that some single neurons in the trigeminal ganglion of the rat projected by way of axon collaterals both to the caudal spinal trigeminal nucleus and to the principal sensory trigeminal nucleus, and that about 40% or 57% of these neurons showed respectively substance P- or calcitonin gene-related peptide-like immunoreactivity.     

Osteopontin-immunoreactivity in the rat trigeminal ganglion and trigeminal sensory nuclei

Osteopontin-immunoreactivity (OPN-ir) was examined in the oro-facial tissues and trigeminal sensory nuclei (principal sensory nucleus and spinal trigeminal nucleus) to ascertain the peripheral ending and central projection of OPN-containing primary sensory neurons in the trigeminal ganglion (TG). No staining was observed using mouse monoclonal anti-OPN antibody preabsorbed with recombinant mature OPN. OPN-immunoreactive (ir) peripheral endings were classified into two types: encapsulated and unencapsulated types. Unencapsulated endings were subdivided into two types: simple and complex types. Simple endings were characterized by the thin neurite that was usually devoid of ramification. These endings were seen in the hard plate and gingiva. The complex type was characterized by the thick ramified neurite, and observed in the vibrissa, hard palate, and molar periodontal ligament. Encapsulated endings were found only in the hard palate. The trigeminal sensory nuclei contained OPN-ir cell bodies and neuropil. The neuropil was devoid of ir in laminae I and II of the medullary dorsal horn (MDH), and had various staining intensities in other regions of the trigeminal sensory nuclei. Transection of the infraorbital and inferior alveolar nerves caused an increase of OPN-ir intensity in ipsilateral TG neurons. The staining intensity of the neuropil also increased in the trigeminal sensory nuclei ipsilateral to the neurotomy excepting laminae I and II of the MDH. The present study indicates that OPN-ir primary sensory neurons in the TG innervate encapsulated and unencapsulated corpuscular endings. Such neurons probably project their central terminals to the trigeminal sensory nuclei except for the superficial laminae of the MDH.     

The organization of the neonatal rat's brainstem trigeminal complex and its role in the formation of central trigeminal patterns

The present study delimits the relationship of primary trigeminal afferents to their targets, the brainstem trigeminal nuclei of the neonatal rat. Previously, the brainstem trigeminal complex of the rat has been subdivided on the basis of either cytoarchitectonics or patterns of succinic dehydrogenase activity into the principal sensory nucleus and the three subnuclei of the spinal trigeminal nucleus, oralis, interpolaris, and caudalis. In this paper, we demonstrate that each of these subdivisions can also be identified by its pattern of primary trigeminal afferents. In addition, we demonstrate that the terminations of these afferents are distributed in a punctate fashion which correlates with vibrissae-related patterns of histochemical staining. Further, vibrissae removal in the neonatal rat at any age studied results in a corresponding deafferentation of both the principal sensory nucleus and all subnuclei of the spinal trigeminal nucleus. This same procedure has a graded, age-dependent effect on the vibrissae-related pattern of cytochrome oxidase staining in somatosensory cortex. On this basis, we conclude that vibrissae-related pattern formation in the central trigeminal system can be best understood in terms of a single "sensitive" period for the entire system. We hypothesize that this is the period during which an interaction normally occurs between primary trigeminal afferents and target neurons of the principal sensory nucleus.     

Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye

The occurrence, distribution, and origin of immunoreactive calcitonin gene-related peptide (CGRP) in nerves of rat, guinea pig, cat, and monkey eyes were investigated by immunocytochemistry, radioimmunoassay, and chromatography. A rich network of CGRP-immunoreactive nerve fibres was noted in the anterior uvea, which was widely distributed in both dilator and constrictor pupillae muscles and extended to the ciliary body and uveal blood vessels. Numerous CGRP-immunoreactive neuronal cells were present in the trigeminal ganglion. The extractable CGRP was 8.6 +/- 1.8 pmoles/gm of tissue in the iris and 44.0 +/- 8.1 pmoles/gm in the trigeminal ganglion. Following damage to the Gasserian ganglion a marked decrease of CGRP immunoreactivity was observed in the anterior uvea (control 11.3 +/- 1.6 pmoles/gm; operated 1.4 +/- 0.1 pmoles/gm) confirming the origin of the immunoreactive fibres from trigeminal primary sensory neurons. The sensory nature of the CGRP-immunoreactive fibres was substantiated by the depletion of CGRP immunoreactivity observed after treatment with capsaicin, which is known to cause selective degeneration of sensory neurons. Comparative studies on the distribution and colocalisation of CGRP and the putative sensory neurotransmitter substance P revealed a closely parallel distribution of the two peptides in certain regions of the uvea and their coexistence in a subpopulation of trigeminal primary sensory neurons. This study suggests that the sensory nervous system in the eye is more heterogeneous in terms of its putative neurotransmitters than previously indicated.     

Somatotopic organization of the primary sensory trigeminal neurons in the hagfish, Eptatretus burgeri

Primary sensory trigeminal projections were investigated in the hagfish following application of horseradish peroxidase (HRP) to the sensory branches. In our control preparations we were able to distinguish five sensory ganglia and their respective nerves. HRP application confirmed the almost exclusive relation of each of these nerves to their respective ganglia, with very little overlap. In normal frontal sections of the medulla oblongata, five columns of fibers surrounded by neuronal cell bodies could be clearly distinguished, but the number is probably fortuitous, for there was no one-on-one relationship with the five trigeminal ganglia. From their peripheral connections, we surmised that columns 1 and 3 handle general cutaneous sensation, columns 2, 4, and 5 handle taste sensation, and column 5 handles general mucous cutaneous sensation conveyed by utricular ganglion cells. Dorsally located columns received projections from nerves with dorsal peripheral connections, and more ventrally located columns received projections from nerves with ventral peripheral connections. This relation is the reverse of that seen in other vertebrates.     

Local anesthetics inhibit veratridine-induced secretion of calcitonin gene-related peptide (CGRP) from cultured rat trigeminal ganglion cells

Secretion of calcitonin gene-related peptide (CGRP) was studied with the model system of dispersed adult rat trigeminal ganglion cells. Veratridine stimulated secretion of CGRP immunoreactivity. Tetrodotoxin and local anesthetics inhibited veratridine-stimulated peptide secretion. These observations implicate sodium channels in CGRP secretion and are consistent with a role for the peptide as an extracellular neuromodulator in the sensory nervous system.     

c-Jun expression after axotomy of corneal trigeminal ganglion neurons is dependent on the site of injury

The proto-oncogene c-Jun has been implicated in the control of neuronal responses to injury and in axonal growth during regenerative processes. We have investigated the expression of c-Jun during normal terminal remodelling in trigeminal ganglion neurons innervating the cornea and after acute injury of epithelial nerve terminals or parent axons. Remodelling and rearrangement, or damage limited to corneal epithelium endings, was not a trigger for activation of c-Jun expression. However, injury of parent axons in the stroma or in the orbital ciliary nerves induced c-Jun expression in 50% of the population of corneal neurons, which included all of the large myelinated and 20% of the small neuropeptide-containing corneal neurons. This suggests that c-Jun expression in trigeminal ganglion neurons is not associated with normal remodelling or regeneration of peripheral nerve terminals, and that it takes place only when parent axons are injured. A substantial number of damaged neurons do not express c-Jun, indicating that in primary sensory neurons, injury and regeneration may not always be coupled to the expression of this proto-oncogene.     

The central projection of masticatory afferent fibers to the trigeminal sensory nuclear complex and upper cervical spinal cord

Retrograde and anterograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) conjugate was used to study the organization of primary afferent neurons innervating the masticatory muscles. HRP applied to the nerves of jaw-closing muscles--the deep temporal (DT), masseter (Ma), and medial pterygoid (MP)--labeled cells in the trigeminal ganglion and the mesencephalic trigeminal nucleus (Vmes), whereas HRP applied to nerves of the jaw-opening muscles--anterior digastric (AD) and mylohyoid (My)--labeled cells only in the trigeminal ganglion. Cell bodies innervating the jaw-closing muscles were found with greater frequency in the intermediate region of the mandibular subdivision, while somata supplying the jaw-opening muscles were predominant posterolaterally. The distribution of their somatic sizes was unimodal and limited to a subpopulation of smaller cells. Projections of the muscle afferents of ganglionic origin to the trigeminal sensory nuclear complex (TSNC) were confined primarily to the caudal half of pars interpolaris (Vi), and the medullary and upper cervical dorsal horns. In the Vi, Ma, MP, AD, and My nerves terminated in the lateral-most part of the nucleus with an extensive overlap in projections, save for the DT nerve, which projected to the interstitial nucleus or paratrigeminal nucleus. In the medullary and upper cervical dorsal horns, the main terminal fields of individual branches were confined to laminae I/V, but the density of the terminals in lamina V was very sparse. The rostrocaudal extent of the terminal field in lamina I differed among the muscle afferents of origin, whereas in the mediolateral or dorsoventral axis, a remarkable overlap in projections was noted between or among muscle afferents. The terminals of DT afferents were most broadly extended from the rostral level of the pars caudalis to the C3 segment, whereas the MP nerve showed limited projection to the middle one-third of the pars caudalis. Terminal fields of the Ma, AD, and My nerves appeared in the caudal two-thirds of the pars caudalis including the first two cervical segments, the caudal half of the pars caudalis and the C1 segment, and in the caudal part of the pars caudalis including the rostral C1 segment, respectively. This rostrocaudal arrangement in the projections of muscle nerves, which corresponds to the anteroposterior length of the muscles and their positions, indicates that representation of the masticatory muscles in lamina I reflects an onion-skin organization. These results suggest that primary muscle afferent neurons of ganglionic origin primarily mediate muscle pain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cerebral arterial innervation by nerve fibers containing calcitonin gene-related peptide (CGRP): I. Distribution and origin of CGRP perivascular innervation in the rat

The origin, density and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in cerebral perivascular nerves and the trigeminal ganglion of rats were examined in this study. CGRP immunoreactive axons were abundant on the walls of the rostral circulation of the major cerebral arteries in the circle of Willis. The fibers form a grid- or meshwork of longitudinal and circumferential axons studded with numerous varicose swellings. The density of CGRP fibers was particularly high at the bifurcation of major arteries. A few CGRP fibers cross the midline to innervate arteries on the contralateral side of the arterial tree. The arteries of the caudal circulation were sparsely innervated by CGRP fibers. In the trigeminal ganglion, about 30% of the ganglion cells had CGRP immunoreactivity. The cell size of most (75%) of CGRP neurons was less than 30 micron in diameter. There was no significant difference in staining density between small and large CGRP neurons. Unilateral transection of the maxillary and mandibular divisions of the trigeminal nerve caused a substantial decrease of CGRP immunoreactivity in the ipsilateral dorsal two-thirds of the trigeminal nucleus and cervical spinal cord but did not noticeably change the diameter of the vascular lumen or the densities of CGRP fibers in the walls of the cerebral arteries. In contrast, unilateral transection that included the ophthalmic division eliminated CGRP fibers on the ipsilateral cerebral arteries and eliminated CGRP immunoreactivity throughout the trigeminal nucleus in the brainstem and rostral cervical cord. In addition, these lesions caused a significant reduction in the diameter of the denervated arteries. The present study demonstrates that CGRP, a putative neurotransmitter/neuromodulator, is especially abundant in the rostral cerebral circulation and is derived from the ipsilateral ophthalmic division of the trigeminal nerve. In addition, the loss of CGRP perivascular nerves is associated with a reduction of the arterial lumen. This suggests that CGRP is a strong candidate as a nerve-derived trophic factor at trigeminal terminals and provides additional evidence that CGRP is a component in the trigeminovascular system influencing vascular diameter.     

The central projections of tooth pulp afferent neurons in the rat as determined by the transganglionic transport of horseradish peroxidase

Transganglionic transport of horseradish peroxidase (HRP) or horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) was used to map in detail the central projections of trigeminal primary afferent neurons that innervate the dental pulp organ of the rat. In each of ten animals, 0.5-2.0 microliters of enzyme solution was injected into the pulp chamber of the first maxillary molar tooth. Postmortem examination of the decalcified teeth in all cases showed that the HRP/HRP-WGA remained confined to the pulp chamber and pulp roots, with no spread of enzyme into periapical tissues. HRP-labeled tooth pulp afferent fibers projected to all four rostrocaudal subdivisions of the ipsilateral trigeminal brainstem nuclear complex (TBNC) and to the upper cervical spinal cord. The labeled terminal fields formed a column that stretched relatively uninterrupted from just caudal to the rostromedial tip of the trigeminal principal sensory nucleus to at least the C2 segment of the spinal cord. The density of the afferent projection varied markedly from one rostrocaudal level of the TBNC to the next but was heaviest in an area encompassing the caudal one-half of the principal sensory nucleus and the rostral two-thirds of pars oralis. Fibers projected only lightly to pars caudalis, where they terminated preferentially in laminae I, IIa, and the junctional zone between laminae IV and V. HRP-labeled terminals in C1 and C2 were located almost exclusively in laminae I. In the dorsoventral axis, the terminal fields in the TBNC were located in a surprisingly dorsal part of the complex, well within what has been shown by others to be largely an area of termination for mandibular division fibers. Most fibers ended in medial parts of the TBNC, with the exception of two modestly labeled terminal fields located in the lateral aspects of rostral pars oralis and rostral pars caudalis. No labeled fibers terminated in the contralateral TBNC or contralateral cervical spinal cord.     

The effect of sympathectomy on calcitonin gene-related peptide levels in the rat trigeminovascular system

The effect of sympathectomy on the calcitonin gene-related peptide (CGRP) level in the rat primary trigeminal sensory neurone was investigated. Six weeks after bilateral removal of the superior cervical ganglion there was a 70% rise in the CGRP content of the iris and the pial arteries, a 34% rise in the concentration in the trigeminal ganglion but no change in the brainstem. The CGRP rise in both end organs suggests that this phenomenon may be common to all peripheral organs receiving combined sensory and sympathetic innervations. The lack of any rise in the brainstem CGRP content raises the possibility that this process spares central terminations. In contrast, the level of neuropeptide Y, a peptide mainly contained in sympathetic terminals, fell to 35% of control values in the iris and pial arteries whilst the trigeminal ganglion and brainstem concentrations remained unchanged. The possible relevance of these observations to the clinical syndrome of postsympathectomy pain (sympathalgia) is discussed. There are similarities between the delayed onset of the human pain state and the delayed rise in sensory peptides after sympathectomy.     

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.