Chemical phenotypes of muscle and cutaneous afferent neurons in the rat trigeminal ganglion

Retrograde labeling was combined with cytochemistry to investigate phenotypic differences in primary afferent neurons relaying sensory information from deep and superficial craniofacial tissues. Calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM) immunoreactivity and isolectin IB4, and cholera toxin B (ChTB) binding were examined for trigeminal masticatory muscle and cutaneous afferent neurons. Somata labeled from muscle were larger than cutaneous afferent neurons. Muscle afferent neurons exhibited positive staining as follows: 22% CGRP, 5% SP, 0% SOM; 18% ChTB, 5% IB4. The somata of CGRP- and SP-positive muscle afferent neurons were smaller than that of the overall muscle afferent population. Size differences were not detected between IB4- or ChTB-binding muscle afferent neurons and the total muscle afferent population. The following distribution was found for cutaneous afferent neurons: 26% CGRP, 7% SP, 1% SOM, 26% ChTB, 44% IB4. Cutaneous afferent neurons positive for SP were smaller, while ChTB-binding cutaneous afferents were larger than the overall cutaneous afferent population. No size differences were found between cutaneous CGRP-, SOM-, or IB4-positive neurons and the total cutaneous afferent population. Target-specific differences exist for SOM and IB4. The percentage of cutaneous afferent neurons positive for SOM and IB4 exceeds that for SOM- or IB4-positive muscle afferents. The number of retrogradely labeled neurons never differed between sexes. The percentage of retrogradely labeled muscle afferent neurons that were CGRP-positive was greater in males than females. These data indicate the presence of phenotypic, target, and sex differences in trigeminal ganglion primary afferent neurons.     

Enrichment of a vasoactive neuropeptide (calcitonin gene related peptide) in the trigeminal sensory projection to the intracranial arteries

Trigeminal sensory innervation of cerebral vessels and the surrounding dura is responsible for most intracranial head pain. Small-diameter fibers containing substance P (Sub P) have been observed in the periadventitia around feline cerebral blood vessels, and it has been suggested that these fibers are the trigeminal substrate for vascular pain associated with cluster and migraine headaches. Calcitonin gene related peptide (CGRP) coexists with Sub P in some of these fibers and with some Sub P containing neurons in the trigeminal ganglion. In addition, a population of trigeminal neurons containing CGRP but not Sub P has been observed. We now report that the population of trigeminal ganglion cells projecting to the cerebral vasculature is enriched in CGRP-containing neurons, and especially in the population of neurons containing CGRP and not Sub P. Using retrograde tracing of fluorescent tracers combined with immunocytochemistry after explant culture, we found approximately 32% of trigeminal ganglion cells projecting to the cerebral vasculature contained CGRP. Approximately 18 and 17% of these cells contained Sub P and cholecystokinin (CCK), respectively. The 32% of ganglion cells projecting to the cerebral vasculature that contain CGRP stands in contrast to the 12% CGRP positive seen in the population of ganglion cells projecting out to another target (the forehead), and the 21 and 23% CGRP positive observed in the mandibular branch and entire ganglion, respectively. Sub P and CCK are not enriched in the trigeminal innervation of the vasculature compared with their presence in cells throughout the ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peripheral and central distribution of TRPV1, substance P and CGRP of rat corneal neurons

The rat corneal neurons expressing vanilloid receptor TRPV1, substance P (SP) and calcitonin-gene-related peptide (CGRP) were examined. In the cornea, some TRPV1-immunoreactive nerve fibers displayed either SP- or CGRP immunoreactivity also. For observing corneal neuronal elements in the trigeminal ganglion (TG) and in the medulla oblongata, retrograde and anterograde cholera toxin subunit B (CTB) tracing methods combining with triple immunofluorescence technique were performed. The corneal neuronal somata were located in the ophthalmic division of the TG; 37% of them were immunoreactive for TRPV1. One third and three quarters of the corneal TRPV1-immunoreactive neurons co-expressed SP and CGRP, respectively. All of SP-immunoreactive corneal neurons exhibited TRPV1 immunoreactivity. They were predominantly medium-sized (mean +/- SE = 638.2 +/- 49.5 microm(2)) and significantly larger than SP-immunoreactive and TRPV1-immunonegative neurons in the ophthalmic division of the TG. The central projection fibers of corneal neurons co-expressing TRPV1 with SP and CGRP were observed at the subnucleus interpolaris/caudalis transition within trigeminal nucleus. The present study suggests that TRPV1 of the corneal neurons works in close relation to SP and CGRP both in the cornea and CNS for healing and nociceptive transduction.     

Location,morphology, and central projections of mesencephalic trigeminal neurons innervating rat masticatory muscles studied by axonal transport of choleragenoid-horseradish peroxidase

Retrograde and transganglionic transport of horseradish peroxidase conjugated to the B-fragment of cholera toxin (B-HRP) was used to study the location, morphology, and central projections of mesencephalic trigeminal (Me5) neurons innervating rat masticatory muscles. Labeled Me5 cell bodies were found throughout the Me5 nucleus from a level slightly caudal to the trigeminal motor nucleus to the level of the superior colliculus 5 mm further rostrally. Occasionally, labeled Me5 cells were observed in the anterior medullary velum, in the cerebellum, and in the brainstem contralateral to the B-HRP injection. The vast majority of the labeled Me5 cells were pseudounipolar, but multipolar cells were also found. Extensive central projections from labeled Me5 cells could be seen extending from the nucleus of Darkschewitsch rostrally to the C2 segment caudally. Small but consistent projections from Me5 neurons were observed in nuclear islands among the incoming Me5 root fibers. Trigeminal and hypoglossal motor nuclei received direct projections from Me5 cells, but not the facial motor nucleus. The most prominent Me5 projections appeared in the brainstem reticular formation, including the supratrigeminal nucleus. Smaller projections also extended into the main sensory trigeminal nucleus, trigeminal subnucleus oralis, and the nucleus of the solitary tract. © 1993 Wiley-Liss, Inc.     

Selective distribution and function of primary afferent nociceptive inputs from deep muscle tissue to the brainstem trigeminal transition zone

Orofacial injury activates two distinct regions in the spinal trigeminal complex, the subnuclei interpolaris/caudalis (Vi/Vc) transition zone and the laminated Vc, or medullary dorsal horn (MDH). Studies suggest that the Vi/Vc transition zone plays an important role in processing orofacial deep input. To test this hypothesis, we employed a double-tracing strategy to compare central projections of primary afferent neurons that innervate the masseter muscle and the overlying skin. Different tracers were injected either centrally (Fluoro-Gold: ventral Vi/Vc, or MDH) or peripherally (wheat germ agglutinin-conjugated horseradish peroxidase or cholera toxin B: masseter or overlying skin) in the same rat. Trigeminal ganglion tissue sections were processed for single or double immunohistochemistry. The double labeling of ganglion neurons indicates their site of peripheral and central innervations. A population of small to medium-sized neurons was doubly labeled after injections of the tracers into the masseter-Vi/Vc, masseter-MDH, or the skin-MDH. However, only a few double-labeled neurons were occasionally observed after injections of the tracers into the skin-Vi/Vc. Injection of an N-methyl-D-aspartate receptor antagonist, AP-5, into the Vi/Vc and MDH attenuated masseter inflammatory hyperalgesia. In contrast, hyperalgesia after inflammation of the skin overlying the masseter was attenuated by injection of AP-5 into the MDH but not Vi/Vc. These results indicate that while both masseter and cutaneous inputs project to the MDH, masseter afferents provide an additional input to the Vi/Vc. These findings provide further evidence to support a role of the trigeminal transition zone in response to orofacial deep injury.     

Somatotopic organization of the trigeminal ganglion cells in a cichlid fish, Oreochromis (Tilapia) niloticus

Somatotopic organization of the trigeminal ganglion is known in some vertebrates. The precise pattern of somatotopy, however, seems to vary in different vertebrate groups. Furthermore, the somatotopic organization remains to be studied in teleosts. From an evolutionary point of view, the morphology and somatotopic organization of the trigeminal ganglion of a percomorph teleost, Tilapia, were investigated by means of the tract-tracing method using biocytin and three-dimensional reconstruction models with a computer. The trigeminal ganglion was one cell aggregate elongated in the dorsoventral direction, which was separate from the facial and anterior lateral line ganglia. Biocytin applications to the trigeminal nerve root labeled ordinary ganglion cells in the trigeminal ganglion and a few displaced trigeminal ganglion cells in the facial ganglion. Biocytin applications to three primary branches (the ophthalmic, maxillary, and mandibular nerves) revealed that trigeminal ganglion cells were somatotopically distributed in the ganglion reflecting the dorsoventral order of the three branches. Ganglion cells of the ophthalmic nerve were distributed in the dorsal part of the trigeminal ganglion, those of the mandibular nerve in the ventral part, and those of the maxillary nerve in the intermediate part. Some of maxillary and mandibular ganglion cells appear to overlap in their boundary region, whereas ophthalmic ganglion cells did not intermingle with ganglion cells of other branches. Labeled-primary fibers terminated in the sensory trigeminal nucleus, descending trigeminal nucleus, medial funicular nucleus, a ventral part of the facial lobe, reticular formation, and trigeminal motor nucleus. Labeled cells were observed in the mesencephalic trigeminal nucleus and the trigeminal motor nucleus. The results suggest that the morphology and somatotopic organization of the trigeminal ganglion of tilapia are similar to those of mammals, except that the axis of the somatotopic organization of the ganglion in mammals is a mediolateral direction reflecting the mediolateral order of the ophthalmic, maxillary, and mandibular nerves.     

Immunocytochemical study on cholera toxin binding sites by monoclonal anti-cholera toxin antibody in neuronal tissue culture

An indirect method of immunocytochemistry showed that cholera toxin and its B-subunit served as specific neuronal surface markers in conjunction with monoclonal anti-cholera toxin and FITC labeled anti-mouse Fab. The cell types which get specifically stained in culture were peripheral neurons from dorsal root ganglion cells, superior cervical ganglion cells, and cerebral neurons, all of which were rat tissue, and NGF-treated PC12 cells. Non-neuronal cells, i.e. Schwann cells, fibroblasts and glia cells, were not stained. This method can, therefore, be used to distinguish neurons from non-neuronal cells in neuronal tissue cultures, as in the case of tetanus toxin described in the literature^{5,10,23,25,28,29}.     

Immunohistochemical identification of trigeminal ganglion neurons that innervate the mouse cornea: Relevance to intercellular spread of herpes simplex virus

Inoculation of the scarified cornea with herpes simplex virus (type 1) leads to herpetic infection of trigeminal ganglion cells. A recent study of the susceptibility of ganglion cells revealed that there may be at least four populations of trigeminal ganglion cells that are infectable by herpes. Two classes were identified by their neuropeptide content: Substance P or calcitonin gene-related peptide. One class was identified by its affinity for a monoclonal antibody, SSEA-3. The fourth class was recognized by its common affinity for both the monoclonal antibody LD2 and for the lectin Bandeiraea simplicifolia isolectin. However, there has been no direct evidence of which types are infected directly as a result of retrograde transport from the corneal site and which may be infected by cell-to-cell spread. The aim of this study was to determine which classes of neurons, which are known to become infected with HSV after ocular inoculation, supply corneal innervation. We have identified four classes of trigeminal ganglion neurons that supply axons to the central cornea of the mouse, on the basis of their ability to transport Fluoro-Gold retrograde from axons in the central corneal epithelium and stroma. About 40% of the neurons that innervate the cornea contain Substance P or calcitonin gene-related peptide; about 60% of the neurons that innervate the cornea react with the monoclonal antibody SSEA-3. About 36% of all neurons in the whole ophthalmic division react with the LD2 or Bandeiraea simplicifolia isolectin, and Fluoro-Gold labels only 2% of them. Thus, the population of LD2/Bandeiraea Simplicifolia Isolectin neurons that innervates the cornea represents less than 1% of the total neurons in the ophthalmic division, although it constitutes about 20% of the infected cells in that division at three days after viral inoculation. We conclude that most of the LD2/Bandeiraea simplicifolia isolectin-positive ganglion cells that are infected with herpes at 3 days are infected as a result of HSV spread from nearby cells in the ganglion or proximal trigeminal root. Neurons labeled with antibodies to Substance P, calcitonin gene-related peptide, or α-SSEA-3 that are primarily infected may be sources of the cell-to-cell spread of virus. © 1993 Wiley-Liss, Inc.     

Sensory innervation of the guinea pig extraocular muscles: A 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate tracing and calcitonin gene-related peptide immunohistochemical study

The sensory apparatus of the extraocular muscles attains special interest because of the great variation among different species with respect to the proprioceptors. The sensory innervation of the guinea pig extraocular muscles, lacking both muscle spindles and tendon organs, was investigated with a fluorescence double-labelling method. Primary sensory perikarya were assessed by postmortem application of 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (Di-I) to the extraocular muscle nerves. Traced neurons were found in the ipsilateral ophthalmic part of the trigeminal ganglion. This is in line with findings in other species. Calcitonin gene-related peptide (CGRP) was detected immunohistochemically within the trigeminal ganglion. No somatotopic organization was observed for CGRP-like immunoreactive perikarya. Small (maximal diameter below 30 μm), medium (maximal diameter between 30 and 50 μm), and large (maximal diameter larger than 50 μm) trigeminal ganglion cells were found among the primary afferent perikarya from extraocular muscles. Among CGRP-like immunoreactive cells, only small and medium cells were observed. Double-labelling experiments indicated the CGRP content of primary afferents of the guinea pig extraocular muscles. The relationship to former morphological categories of ganglion cells is discussed. Primary afferent neurons with CGRP-like immunoreactivity might have efferent functions and might also be involved in inflammatory processes of extraocular muscles. J. Comp. Neuol. 380:16–22, 1997. © 1997 Wiley-Liss, Inc.     

Primary sensory ganglion cells projecting to the principal trigeminal nucleus in the mallard, Anas platyrhynchos

The trigeminal and glossopharyngeal ganglia of the adult mallard were studied following HRP injections into the principal trigeminal nucleus (PrV). The PrV consists of the principal trigeminal nucleus proper (prV) and the principal glossopharyngeal nucleus (prIX). After an injection into the prV, the labeled cells were found in the ipsilateral trigeminal ganglion. After an injection into the prIX, labeled cells were found in the ipsilateral distal glossopharyngeal ganglion, but not in the proximal ganglion of the IX and X cranial nerve (pGIX + X). In Nissl preparations, two types of ganglion cells in the trigeminal ganglion, pGIX + X, and distal ganglion of N IX could be distinguished: larger light cells and smaller dark cells. We could not determine whether the HRP-labeled cells belonged to both types or to one of them; but because all the labeled cells were over 20 microns, we concluded that the smallest cells (10-19 microns) in the trigeminal ganglion and distal ganglion of N IX did not project to the PrV. The labeling of the cells in the distal ganglion of N IX (average 34.5 microns) was uniformly moderate. In the trigeminal ganglion there were two types of labeled cells: heavily labeled cells (average 29.1 microns) and moderately labeled cells (average 35.1 l microns). These two types of labeling (moderate and heavy) may reflect two types of primary sensory neurons: cells with ascending, nonbifurcating axons, and cells with bifurcating axons. We speculate that the former are proprioceptive neurons and the latter tactile neurons. Labeled bifurcating axons in the sensory trigeminal complex gave off collaterals to all parts of the descending trigeminal nucleus except to the caudalmost laminated spinal part.     

Demonstration of GM1-ganglioside in nervous system in generalized GM1-gangliosidosis using cholera toxin B subunit

Summary By using cholera toxin B subunit and its antibody, the deposition of GM₁-ganglioside in the cerebral cortex and peripheral nerves including Meissner and Auerbach's plexuses in the intestine and other visceral nerves of generalized GM₁-gangliosidosis was demonstrated. The GM₁-ganglioside was found in the swollen neurons of cerebral cortex and ganglion cells of the peripheral nerves. Electron microscopically, parts of membranous cytoplasmic bodies, and amorphous substances among them, revealed a positive reaction for the cholera toxin staining.     

Identification of signal substances in synapses made between primary afferents and their associated axon terminals in the rat trigeminal sensory nuclei

The relationships between primary afferent terminals (PATs) and their associated presynaptic terminals in the rat trigeminal sensory nuclear complex (TSNC) were examined with special reference to amino acid transmitters glutamate (Glu) and gamma-aminobutyric acid (GABA). Primary afferent terminals anterogradely labeled from the trigeminal ganglion with the B subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) were sectioned for electron microscopy. Serial sections from the principal nucleus (Vp), dorsomedial parts of the oral and interpolar nuclei (Vdm), and lamina III/IV of caudal nucleus (Vc) were immunostained for Glu and GABA by using a postembedding immunogold technique. The tracer, CTB-HRP to the trigeminal ganglion, preferentially labeled myelinated primary afferents. Sections immunostained with Glu antiserum showed that most labeled PATs were enriched with immunoreactivity (IR) for Glu. The Glu-IR PATs contained clear, round, synaptic vesicles and formed asymmetric synaptic contacts with somata or dendrites. They were frequently postsynaptic to, unlabeled axon terminals filled with a mixture of clear, round, oval, and flattened vesicles (p-endings), with symmetric synaptic junctions. The frequency of synapses onto somata or primary dendrites per Glu-IR PAT was higher in the Vdm than in either the Vp or Vc lamina III/IV. The frequency of contacts of the p-endings per Glu-IR PAT was higher in the Vp than in the Vdm and Vc lamina III/IV. Sections immunostained with GABA antiserum showed that most axon terminals presynaptic to PATs were enriched with GABA in the three nuclei. The GABA-IR axon terminals and their postsynaptic PATs had a similar ultrastructural character to p-endings and their postsynaptic Glu-IR PATs, respectively. The present study suggests that primary afferent neurons with large-caliber fibers use glutamate as a neurotransmitter and are subject to presynaptic modulation by GABAergic fibers.     

Infrared sensory neurons in the trigeminal ganglia of crotaline snakes: transganglionic HRP transport

Trigeminal neurons were labeled by inserting HRP into holes cut in the pit receptor membranes of a crotaline snake, Agkistrodon blomhoffi brevicaudus. Neurons were labeled in the ophthalmic ganglion and the maxillary division of the maxillo-mandibular ganglion, and the HRP was further transported across the ganglia and through the lateral descending trigeminal tract (dlv) to label axon terminals exclusively in the dlv nucleus (DLV). In 6 successful preparations, 7.1-19.3% of totals of 5568-5986 cells in the maxillary division of the ganglion were labeled, but none at all were labeled in the mandibular division. Only a few or none at all were labeled in the ophthalmic ganglion. Cells in the two ganglia ranged in size from 10 to 55 micrometers, but large cells (greater than or equal to 40 micrometers) were scarce (4.9% of the total population). All HRP-labeled neurons fell in the median range of 20-39 micrometers. We concluded that these ganglion cells were infrared neurons, and were therefore the origin of the A delta fibers in the pit membrane. There were no HRP-labeled neurons above or below this range, in spite of the fact that smaller cells (less than or equal to 19 micrometers) made up 35.8% of the total population. In normal Nissl preparations we found both light- and dark-staining cells, but the size range of neither corresponded to the size range of infrared neurons.     

Neurogenesis in the trigeminal ganglion of the albino rat: A quantitative autoradiographic study

The time of neuron origin in the trigeminal ganglion was examined in autoradiograms of 60-day-old rats that were exposed to a single pulse of ³H-thymidine on day 11, 12, 13, 14, or 15 of gestation. Heavily labeled neurons, representing cells in or near their last mitotic division at the time of the pulse label, were present in animals injected between embryonic days 11 and 13 with a peak on day 12. Within this time period, larger neurons were generated prior to smaller neurons with a peak for larger cells on day 12 and for smaller cells on day 13. Thus, the majority of trigeminal ganglion neurons are generated over a three-day period just after the midpoint of gestation. Neuron number, size, type, and cytoarchitectural organization were also examined in the ganglion. The mean neuron count per ganglion was 52,372. The size distribution of these cells ranged continuously from 7–61 μm (mean diameter) with no evidence for clearly defined subpopulations. The staining intensity and distribution patterns of the Nissl substance varied greatly from cell to cell precluding the classification of cells as light or dark. Little correspondence between these Nissl features and cell size was found. Among the clusters and rows of neurons in the ganglion, we did not see consistent cytoarchitectonic patterns which might reflect specific sensory receptive fields.     

Retrograde analysis of the cerebellar projections to the posteroventral part of the ventral lateral thalamic nucleus in the macaque monkey

The organization of cerebellothalamic projections was investigated in macaque monkeys using injections of retrograde tracers (cholera toxin B and fluorescent dextrans) in the posteroventral part of the ventrolateral thalamic nucleus (VLpv), the main source of thalamic inputs to the primary motor cortex. Injections that filled all of VLpv labeled abundant neurons that were inhomogeneously distributed among many unlabeled cells in the deep cerebellar nuclei (DCbN). Single large pressure injections made in face-, forelimb-, or hindlimb-related portions of VLpv using physiological guidance labeled numerous neurons that were broadly dispersed within a coarse somatotopographic anteroposterior (foot to face) gradient in the dentate and interposed nuclei. Small iontophoretic injections labeled fewer neurons with the same somatotopographic gradient, but strikingly, the labeled neurons in these cases were as broadly dispersed as in cases with large injections. Simultaneous injections of multiple tracers in VLpv (one tracer per somatic region with no overlap between injections) confirmed the general somatotopography but also demonstrated clearly the overlapping distributions and the close intermingling of neurons labeled with different tracers. Significantly, very few neurons (<2%) were double-labeled. This organizational pattern contrasts with the concept of a segregated "point-to-point" somatotopy and instead resembles the complex patterns that have been observed throughout the motor pathway. These data support the idea that muscle synergies are represented anatomically in the DCbN by a general somatotopography in which intermingled neurons and dispersed but selective connections provide the basis for plastic, adaptable movement coordination of different parts of the body. Indexing terms:     

Innervation of the pancreas by neurons in the gut

Experiments were done in order to test the hypothesis that neurons in the bowel send axonal projections to the pancreas and can modify pancreatic activity. pancreatic injections of the retrograde tracer, Fluoro-Gold, labeled neurons in the myenteric plexus of the antrum of the stomach and in the first 6 cm of the duodenum. this labeling was not due to the diffusion of Fluoro-Gold from the pancreas, because the injections did not label longitudinal muscle cells overlying labeled ganglia in the bowel or neurons in the phrenic nerve nucleus or nucleus ambiguous; nor were enteric neurons labeled if insufficient time was allotted for retrograde transport. More Fluoro-Gold labeled neurons were found in the stomach (9.2 +/- 0.9/ganglion) than in the duodenum (3.8 +/- 0.3/ganglion; p less than 0.001). Neurons were found in myenteric ganglia of both duodenum and stomach that were doubly labeled by retrograde transport of Fluoro-Gold and anti-serotonin (5-HT) sera. In addition, thick bundles of 5-HT immunoreactive nerve trunks were found to run between the duodenum and the pancreas. Most 5-HT immunoreactive axons in the pancreas terminated in ganglia, although some fibers were also observed near acini, ducts, vessels, and islet cells. The B subunit of cholera toxin (B-CT) was microinjected into single myenteric ganglia in order to determine if axon terminals in the pancreas would become labeled by anterograde transport in the pancreas. B-CT labeled bundles of axons in the pancreatic stroma. Branches of these bundles entered the pancreatic parenchyma and varicose B-CT labeled terminal axons were found in pancreatic ganglia and in proximity to acinar and insulin immunoreactive cells. The intercalating fluorochrome 1, 1', dioctadecyl-3,3,3',3'-tetramethylcarbocyanine perchlorate (Dil), which moves by lateral diffusion to outline entire cells, was introduced by microinjection into individual myenteric ganglia of fixed preparations. Fluorescence was seen in sequential observations to move away from the injected ganglion along connectives of the myenteric plexus. After about a month, neurons in ganglia at some distance from the injection site displayed Dil fluorescence as did nerve bundles that exited from the myenteric plexus and pierced the longitudinal muscle in the direction of the pancreas. Varicose Dil fluorescent terminal varicosities were also observed int he pancreas. These observations indicate that there is an extensive entero-pancreatic innervation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Visualization of efferent retinal projections by immunohistochemical identification of cholera toxin subunit B.

The present study describes the use of cholera toxin subunit B as an anterograde and retrograde neuronal tracer for studying retinal projections of the rat, mouse, gerbil, and hamster. The tracer was pressure injected in the posterior chamber of the eye and the labeled neurons were identified using an avidin-biotin immunoperoxidase technique using diaminobenzidine as chromagen. Doses of 3-8 microliters (30-80 micrograms) cholera toxin subunit B and a survival for 24 h resulted in an optimal transport of the tracer in all rodent species investigated. The cholera toxin subunit B-containing retinal efferents were effectively stained and yielded the presence of axons with delicate boutons on passage and nerve endings. Smooth and thick fibers were also observed, indicating a distinction between passing and terminating axons, respectively. Immunoreactive axons were observed in the hypothalamus, thalamus, ad mesencephalon, and the precise distribution of positive nerves could be identified in counterstained sections, some of them as delicate endings in apposition to neuronal surfaces. Labeled cell bodies were observed in the oculomotor nucleus and the pretectum, indicating that the tracer is transported retrogradely as well. Because the tracer is identified immunohistochemically, the retinofugal and retinopetal pathways can be mapped more precisely, perhaps in combination with immunohistochemical detection of other antigens.     

Trigeminal collaterals in the nasal epithelium and olfactory bulb: a potential route for direct modulation of olfactory information by trigeminal stimuli

The nasal epithelium is richly invested with peptidergic (substance P and calcitonin gene-related peptide [CGRP]) trigeminal polymodal nociceptors, which respond to numerous odorants as well as irritants. Peptidergic trigeminal sensory fibers also enter the glomerular layer of the olfactory bulb. To test whether the trigeminal fibers in the olfactory bulb are collaterals of the epithelial trigeminal fibers, we utilized dual retrograde labeling techniques in rats to identify the trigeminal ganglion cells innervating each of these territories. Nuclear Yellow was injected into the dorsal nasal epithelium, and True Blue was injected into the olfactory bulb of the same side. Following a survival period of 3-7 days, the trigeminal ganglion contained double-labeled, small (11.8 x 8.0 microm), ellipsoid ganglion cells within the ethmoid nerve region of the ganglion. Tracer injections into the spinal trigeminal complex established that these branched trigeminal ganglion cells also extended an axon into the brainstem. These results indicate that some trigeminal ganglion cells with sensory endings in the nasal epithelium also have branches reaching directly into both the olfactory bulb and the spinal trigeminal complex. These trigeminal ganglion cells are unique among primary sensory neurons in having two branches entering the central nervous system at widely distant points. Furthermore, the collateral innervation of the epithelium and bulb may provide an avenue whereby nasal irritants could affect processing of coincident olfactory stimuli.     

Parvalbumin- and calretinin-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

Calcium-binding proteins and neuropeptides were examined in trigeminal neuronal cell bodies retrogradely labeled with Fast blue (FB) from the maxillary molar tooth pulp of the rat. FB-labeled cells were located in the maxillary division of the trigeminal ganglion. 30 and 50% of the labeled cells were immunoreactive for parvalbumin and calcitonin gene-related peptide (CGRP), respectively. The coexpression of these substances was observed in 9.5% of FB-labeled cells. On the other hand, 2.4% of FB-labeled cells exhibited calretinin-immunoreactivity (CR-ir) and 20% tachykinin (TK)-ir. The coexpression of CR and TK was observed in 1.9% of FB-labeled cells, i.e., most of CR-ir FB-labeled neurons coexpressed TK-ir. An immuno-EM method revealed that all parvalbumin-ir nerve fibers in the root pulp were myelinated and that CGRP-ir nerve fibers were both myelinated (15%) and unmyelinated (85%). The present study indicated that primary nociceptors innervating the rat molar both pulp contained parvalbumin and CR and coexpressed these calcium-binding proteins and neuropeptides. It was suggested that peripheral axons of parvalbumin-ir tooth pulp primary neurons are all myelinated. Most peripheral CR-ir axons are probably unmyelinated because TK-ir myelinated axons have never been demonstrated in any peripheral organ.     

Neuroanatomical evidence that vagal afferent nerves do not possess a high affinity uptake system for glutamate.

The ability of vagal and glossopharyngeal afferent neurons to retrogradely transport 3H-D-aspartate from the nucleus tractus solitarius to the nodose and petrosal ganglia was examined. Injections of 3H-D-aspartate centered in the medial NTS at the rostral-caudal level of the area postrema failed to consistently label cells in the nodose and petrosal ganglia. In 5 of the 10 rats studied no retrogradely labeled neurons were observed in these ganglia ipsilateral to the injection site, while in the other 5 rats a small number of cells (less than 3%) were labeled. Injections of 3H-D-aspartate into the NTS consistently produced retrograde labeling of neurons in the ipsilateral paratrigeminal area. In addition, many heavily labeled neurons were observed in the injected as well as the contralateral NTS. Injections of 3H-D-asparate into the spinal trigeminal nucleus consistently labeled neurons in the trigeminal ganglion. Since the uptake and retrograde transport of 3H-D-aspartate appears to be characteristic of neurons that use glutamate or aspartate as a neurotransmitter, these results suggest that vagal and glossopharyngeal afferents are not glutamatergic or aspartatergic.