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.     

Spinal topography of the projection of the auricular nerve in the rabbit: a transganglionic WGA-HRP study

The central projections of the nervous branches supplying the rabbit's concha auriculae have been determined by means of the transganglionic transport of WGA-HRP. Labeling appeared in trigeminal, C2 and C3 ganglion cells, but terminals were observed only at spinal levels (C2 and C3). There, they formed a wedge-shaped column of label centered mainly in lamina II; scattered terminals appeared in lamina III and I. These terminals constitute the afferent link of the pathway that accounts for the analgesia obtained after electrical stimulation of the concha auriculae.     

A comparative study of the transganglionic transport of cholera toxin-horseradish peroxidase (CT-HRP) and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the trigeminal system of the guinea pig.

A comparative study of cholera toxin (CT) and wheat germ agglutinin (WGA) conjugated with horseradish peroxidase (HRP) was made on trigeminal central projections of the lower incisor gingiva afferent neurons in the guinea pig. Considerably more CT-HRP-labeled endings were observed in the trigeminal sensory nuclear complex (TSNC) and in the cervical spinal cord (C1-C8). The substantia gelatinosa (lamina II) of both the caudal nucleus of the TSNC and C1-C2 was the only area where WGA-HRP labeled more terminals. CT-HRP-labeled fibers and endings were traced up to C7-C8, whereas with WGA-HRP were rare caudal to C5. A comparison of the two methods currently in use, i.e. the 2-step glutaraldehyde and sodium periodate, showed that the latter yields conjugates which are more sensitive as neuroanatomical tracers.     

Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary and spinal trigeminal tracts

The aim of this study was to map the viscerotopic representation of the upper alimentary tract in the sensory ganglia of the IXth and Xth cranial nerves and in the subnuclei of the solitary and spinal trigeminal tracts. Therefore, in 172 rats 0.5-65 microliters of horseradish peroxidase (HRP), wheat germ agglutinin-HRP, or cholera toxin-HRP were injected into the trunks and major branches of the IXth and Xth cranial nerves as well as into the musculature and mucosa of different levels of the upper alimentary and respiratory tracts. The results demonstrate that the sensory ganglia of the IXth and Xth nerves form a fused ganglionic mass with continuous bridges of cells connecting the proximal and distal portions of the ganglionic complex. Ganglionic perikarya were labeled in crude, overlapping topographical patterns after injections of tracers into nerves and different parts of the upper alimentary tract. After injections into the soft palate, pharynx, esophagus, and stomach, anterograde labeling was differentially distributed in distinct subnuclei in the nucleus of the tractus solitarius (NTS). Palatal and pharyngeal injections resulted primarily in labeling of the interstitial and intermediate subnuclei of the NTS and in the paratrigeminal islands (PTI) and spinal trigeminal complex. Esophageal and stomach wall injections resulted in labeling primarily of the subnucleus centralis and subnucleus gelatinosus, respectively. The distribution of upper alimentary tract vagal-glossopharyngeal afferents in the medulla oblongata has two primary groups of components, i.e., a viscerotopic distribution in the NTS involved in ingestive and respiratory reflexes and a distribution coextensive with fluoride-resistant acid-phosphatase-positive regions of the PTI and spinal trigeminal nucleus presumably involved in visceral reflexes mediated by nociceptive or chemosensitive C fibers.     

Cutaneous projections from the rat hindlimb foot to the substantia gelatinosa of the spinal cord studied by transganglionic transport of WGA-HRP conjugate

The somatotopic organization of cutaneous afferent fibers from the hindlimb foot in the substantia gelatinosa of the spinal cord was investigated in adult Sprague Dawley rats following intracutaneous injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). The different parts of the foot were found to project in a precise manner to the medial two-thirds of the substantia gelatinosa of the spinal cord segments L3-L5. The projections from the digits were arranged in a consecutive row rostrocaudally, with the most medial digit in caudal L3 and the most lateral digit in caudal L4 or rostral L5. The plantar skin was found to project both rostral and caudal to the projection of the digits. The dorsal foot skin projects lateral to the digits, neighboured by the medial edge of the foot rostrally and the lateral edge caudally. Within the projection of a particular digit, the plantar skin was found medially and the dorsal skin laterally. A certain degree of overlap for the projections from the different foot skin areas was found.     

Somatotopic organization of the dorsal column nuclei in the rat: transganglionic labelling with B-HRP and WGA-HRP.

To analyze the patterns of cutaneous primary afferent fibers projecting to the dorsal column nuclei in the rat, horseradish peroxidase (HRP)-based tracers were injected intracutaneously into a number of discrete regions of the forelimbs and hindlimbs. Three-4 days following the HRP injections, the rats were perfused transcardially; 60 microns transverse sections were cut, and the HRP was reacted using the tetramethyl benzidine method. Comparisons were made of projections following injections with choleragenoid-conjugated horseradish peroxidase (B-HRP) or with wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). B-HRP and WGA-HRP produced similar patterns of labelling, but B-HRP produced greater intensity of labelling and slightly larger projection areas. In the cuneate nucleus (CN), HRP labelling of primary afferents from small, delimited regions, e.g., from a portion of the skin of a single digit, appeared to be precisely restricted in rostrocaudally oriented columns, with little or no overlap (in the mediolateral and dorsoventral plane) into adjacent regions. With respect to rostrocaudal organization, a region in the CN containing a dense population of cutaneous primary afferent fibers appeared to be similar to the middle, or cluster, region in cats and in raccoons and the pars rotunda in primates. Projection patterns were very consistent from rat to rat, but their somatotopic organization differed from that suggested by electrophysiological studies: cutaneous afferents from forelimb digit 1 projected near the ventral border of the CN; those from digit 5 projected dorsomedially to those from digit 1; the projections from the remaining digits formed a crescent between the projections from digits 1 and 5. In the gracile nucleus, the organization of cutaneous afferent projections from hindlimb digits was more variable and complex than that found in the CN.     

Contributions from the upper cervical dorsal roots and trigeminal ganglia to the feline circle of Willis

To further define the sensory projections to the circle of Willis, we measured concentrations of immunoreactive substance P in pial arteries of cats following either bilateral removal of the C1-3 dorsal root ganglia (six cats) or bilateral removal of the trigeminal ganglia (three cats). Removal of the dorsal root ganglia decreased concentrations of the tachykinin substance P in the vertebral artery and the basilar artery and its branches by 72% and 50-66%, respectively. Bilateral removal of the trigeminal ganglia decreased substance P concentrations in all forebrain vessels including the rostral basilar artery, although only concentrations in the anterior cerebral artery were significantly lower than those in unilaterally lesioned cats (p less than 0.01). Hence, the vertebrobasilar artery and its tributaries are invested by substance P-containing fibers originating from the upper cervical dorsal root ganglia, and the anterior cerebral artery is innervated by both trigeminal ganglia. If a similar anatomy exists in humans, our data provide an explanation for the occipital localization of headaches arising from the vertebrobasilar arteries and for bilateral headaches following stimulation of the anterior cerebral artery.     

Calbindin-D28k in cerebrovascular extrinsic innervation system of the rat

Calbindin-D28k, one of the calcium-binding proteins, belongs to the EF hand family and is commonly found in neurons. It serves as a representative neuronal marker for neuroanatomical investigations. The authors' knowledge of its precise function, however, is yet very limited. In this study, we examined the existence of nerve fibers with calbindin-D28k immunoreactivity in the cerebral blood vessels and ganglia that innervate the cerebral blood vessels in the rat. Numerous nerve fibers with calbindin-D28k immunoreactivity were observed on the walls of the major extracerebral arteries forming the circle of Willis and its branches. Calbindin-D28k immunoreactivity was seen in many neurons of the trigeminal, dorsal root and jugular ganglia. A small number of neurons showed calbindin-D28k immunoreactivity in the otic and superior cervical ganglia. Calbindin-D28k immunoreactivity was not detected in the sphenopalatine or internal carotid ganglia. Pericellular basket-like formations of nerve terminals with calbindin-D28k immunoreactivity were observed in the sphenopalatine, otic, internal carotid and superior cervical ganglia. The present study demonstrated calbindin-D28k immunoreactivity in the cerebrovascular nerve fibers as well as in their origins--the cranial ganglia. These findings are significant in understanding the calcium-mediated mechanism of the neural control of the cerebral blood vessels.     

Stellate ganglion innervation of the vertebro-basilar arterial system demonstrated in the rat with anterograde and retrograde WGA-HRP tracing

Stellate ganglia projections to cerebral arteries have been investigated with wheatgerm-agglutinated horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into the stellate ganglia resulted in labelling of nerve fibres on the vertebral and basilar arteries, and their side branches. The innervation was bilateral, but with an ipsilateral predominance. After WGA-HRP application on the basilar artery, retrogradely labelled cells appeared in both stellate ganglia, but most numerously in the right ganglion (70-75%). Failure to detect stellate projections to cerebral arteries in 6-hydroxydopamine (6-OHDA)-pretreated animals indicates that these fibres are of noradrenergic sympathetic character. It is suggested that the stellate fibres follow the vertebral arteries towards the basilar artery and its branches.     

The central projection of muscle afferent fibres to the lower medulla and upper spinal cord: an anatomical study in the cat with the transganglionic transport method

The projection of muscle afferent fibres to the medulla oblongata and upper spinal cord was studied in the cat by using transganglionic transport of wheat germ agglutinin-horseradish peroxidase conjugate. The results demonstrate a precise, musculotopic termination pattern in the external cuneate nucleus; thus, fibres from the intrinsic muscles of the paw terminate medially; those from forearm, arm, and shoulder muscles terminate progressively more laterally; and those from neck and thoracic muscles terminate in the ventrolateral and dorsolateral parts, respectively. Muscle afferent fibres to the main cuneate nucleus terminate in the ventral "reticular" region of the nucleus, with a sparse projection also to the ventral part of the rostral and caudal regions, including the base of the dorsal horn. Fibres from the neck muscles terminate slightly more laterally in the ventral region than do those from the limb muscles, but otherwise, and thus contrary to the case in the external cuneate nucleus, no topographic organization was detected. In the spinal cord, projection was found to laminae I and V, and from the musculature of the back of the neck to the central cervical nucleus.     

Ultrastructure of transganglionic degeneration in brain stem trigeminal nuclei during normal primary tooth exfoliation and permanent tooth eruption in the cat

Electron microscopy is used to study changes in the axons and terminals in the cat brain stem trigeminal nuclei, main sensory, and partes interpolaris and caudalis, during the process of natural tooth shedding. Areas previously showing light optical argyrophilic degeneration products and adjacent areas lacking this degeneration are included. Various types of alteration occur early during tooth loss, including increased presumed glycogen, increased cytoplasmic density, flocculence, lucency, and neurofilamentous hyperplasia. By the stage of maximum exfoliation, terminals and axons of marked density become prominent in areas showing argyrophilia, whereas nondense forms occur elsewhere. By late eruption ages, all forms of degenerated terminals and axons are rare, but phagocytes are heavily laden with similar forms of debris. The sequence of ultrastructural events is discussed in light of recent studies of transganglionic degeneration, their correlation with light microscopic findings, and the potential implications for central plasticity in this system.     

Baclofen in trigeminal neuralgia: its effect on the spinal trigeminal nucleus: a pilot study

Experiments with cats showed that baclofen resembles carbamazepine and phenytoin sodium in its ability to depress excitatory synaptic transmission in the spinal trigeminal nucleus. Baclofen was, therefore, given to 14 patients with refractory trigeminal neuralgia. Ten patients were relieved of the paroxysms of tic douloureux while taking 60 to 80 mg/day of baclofen. A reduction in the dosage of baclofen in six of these patients resulted in a recurrence of painful paroxysms in five patients. Seven patients have been pain-free or almost pain-free on a regimen of baclofen for four to 12 months. Our results suggest that baclofen may be a useful drug in the treatment of trigeminal neuralgia and that our experimental model may successfully predict the efficacy of a drug in the treatment of this condition.     

Cholinergic innervation of canine thalamostriatal projection neurons: an ultrastructural study combining choline acetyltransferase immunocytochemistry and WGA-HRP retrograde labeling

Choline acetyltransferase (ChAT) immunocytochemistry and lectin-conjugated horseradish peroxidase (WGA-HRP) histochemistry were combined at the electron microscopic level to examine the morphology of cholinergic terminals in the canine centrum medianum-parafascicular complex (CM-Pf) and to localize cholinergic terminals making synaptic contact with retrogradely labeled CM-Pf thalamostriatal projection neurons. Following WGA-HRP injections into the caudate nucleus, CM-Pf neurons were heavily labeled with WGA-HRP reaction product. Examination with the electron microscope revealed retrogradely labeled neurons characterized by a large nucleus with deep infoldings of the nuclear envelope. ChAT-positive terminals were observed arising from small-diameter nonmyelinated axonal profiles. These terminals varied in size from 0.5 to 1.4 micron in long diameter. The smaller terminals (0.5-0.7 micron) were seen most frequently and established symmetrical or slightly asymmetrical synaptic contacts with small dendritic profiles. The larger ChAT-positive terminals (1.0-1.4 micron) were less frequently observed, contained several mitochondria and small clusters of pleomorphic vesicles, and contacted large dendritic shafts and cell somata. Some of the postsynaptic targets of both smaller and larger ChAT-positive terminals were identified as belonging to retrogradely HRP-labeled thalamostriatal neurons. These observations indicate that at least some thalamostriatal neurons within the CM-Pf complex are innervated by cholinergic terminals which probably arise from ChAT-positive cell bodies located within the pontomesencephalic tegmentum, particularly within the nucleus tegmenti pedunculopontinus and the laterodorsal tegmental nucleus. These findings provide evidence for direct influence by cholinergic brainstem nuclei over activities of thalamostriatal neurons.     

Development of the spinal projections of the nucleus paraventricularis hypothalami of the rat: an intra-uterine WGA-HRP study

The development of the spinal projections of the nucleus paraventricularis hypothalami (PVH) was studied by injecting wheatgerm-agglutinated horseradish peroxidase into the upper thoracic spinal cord of fetal rats at developmental ages ranging from embryonic day 16 (E16) to E21, and into the thoracic spinal cord of rat pups and adult rats. At E18 the first retrogradely labeled cells are seen in the PVH. These cells are located dorsally in the PVH, adjacent to the ventricular matrix. At E20 a more ventrally located group of retrogradely labeled cells appears. This ventral group joins caudally with the dorsal group of labeled cells. The dorsal group of labeled cells is closely packed, while the ventral group of labeled cells is intermingled with unlabeled cells. After E20 this spatial configuration remains essentially constant. Therefore descending projections from all spinal projecting cell groups in the PVH have reached lower cervical spinal cord levels at E20.     

Topographic representation of lower and upper teeth within the trigeminal sensory nuclei of adult cat as demonstrated by the transganglionic transport of horseradish peroxidase

Transganglionic transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) entrapped in hypoallergenic polyacrylamide gel was used to study the patterns of termination of primary afferents that innervate the lower and upper tooth pulps within the trigeminal sensory nuclear complex (TSNC). HRP injections were made into the inferior and superior alveolar nerves in order to compare the central projections of the whole nerve with those from tooth pulps. In addition, the relationship between the distribution of the trigeminothalamic tract cells and the projection sites of the tooth pulp afferents was investigated by injecting HRP into the posterior ventral thalamus. HRP-labeled tooth pulp afferent fibers innervating the lower and upper teeth projected to the subnucleus dorsalis (Vpd) of pars principalis, the rostrodorsomedial part (Vo.r) and nucleus dorsomedialis (Vo.dm) of pars oralis, the medial regions of pars interpolaris, and laminae I, II, and V of pars caudalis. Terminal fields of the lower tooth pulp afferents formed a rostrocaudally running, uninterrupted column from the midlevel of Vpd to the caudal tip of caudalis. In contrast, the column of termination of upper tooth pulp afferents was discontinuous at the Vpd/Vo.r transition, and ended at the more rostral level of the caudalis than that of the lower tooth pulp afferents. The representation of the lower and upper teeth in the TSNC was organized in a somatotopic fashion which varied from one subdivision to the next, although terminal zones of the inferior and superior alveolar nerves overlapped within the Vo.r, Vo.dm, and dorsomedial part of rostral pars interpolaris. The lower and upper teeth were represented in the Vpd, Vo.r, Vo.dm, medial region of pars interpolaris, and laminae I, II, and V, in a ventrodorsal or caudorostral, dorsoventral, lateromedial, dorsoventral, and mediolateral or dorsomedial-ventrolateral sequence, respectively. The smaller, more focal terminal areas of the teeth contrasted sharply with more extensive terminal fields of the alveolar nerves. The HRP injections within the thalamus indicated that neurons in Vpd, the caudal pars interpolaris, and laminae I/V of caudalis, which are subdivisions of TSNC that receive pulpal projections, sent their axons to the ipsilateral and contralateral posterior ventral thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensory innervation of the rat kidney and ureter as revealed by the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from dorsal root ganglia.

The sensory innervation of the rat kidney and ureter was investigated in wholemount preparations and sectioned materials by labeling the afferent nerve fibers with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) transported anterogradely from dorsal root ganglia. Labeled fibers were seen in large numbers in the ureter and in the lining of the renal pelvis, where they were located in the adventitia, smooth muscle, subepithelial connective tissue, and epithelium. Most of the fibers in the ureter and ureteropelvic junctional zone traveled parallel to the long axis of the organ. In contrast, fibers in the widest part of the funnel-shape renal pelvis were oriented predominantly in a circumferential fashion. Many of the pelvic afferents were extremely fine and appeared to terminate as free nerve endings. Modest networks of labeled axons were also observed around branches of the renal artery; the greatest innervation was supplied to the distal portions of the interlobar arteries and to the arcuate arteries. Only single axons were observed around the interlobular arteries, and very few fibers were seen around afferent arterioles or near glomeruli. In contrast to the arteries, branches of the renal vein were relatively sparsely innervated. Occasional labeled fibers entered the renal cortex and formed intimate associations with renal tubules; however, the vast majority of renal tubular elements were not contacted by labeled sensory fibers. Labeled fibers were never observed in the renal medulla or in the papilla. The present study represents the first time that the sensory innervation of the kidney and ureter has been investigated by using a highly specific anterograde nerve tracing technique. The pattern of innervation demonstrated here reveals an anatomical configuration of ureteral and renal pelvic sensory nerves consistent with a role in detection of ureteral and pelvic pressure and chemical changes and a renal vascular sensory innervation that may monitor changes in renal arterial and venous pressure and chemical content. Still other renal afferent nerve endings may signal renal pain.     

Peripheral patterns of calcitonin-gene-related peptide general somatic sensory innervation: cutaneous and deep terminations

The distribution of calcitonin-gene-related peptide (CGRP) immunoreactivity (IR) was studied in peripheral tissues of rats. The ganglionic origin, somatosensory nature, and anatomic relations of this thin-axon population were evaluated with particular emphasis on possible nociceptive roles. In animals untreated with colchicine, CGRP-IR is found in a vast proportion of small- and medium-diameter sensory ganglion cells that give rise to numerous thinly myelinated and unmyelinated axons that display CGRP-IR throughout the body. The integumentary innervation consists, in part, of an extensive subpapillary network largely traced to dermal blood vessels, sweat glands, and "free" nerve endings, some of which are found within regions containing only mast cells, fibroblasts, and collagen. Dermal papillae contain CGRP-IR axons surrounding each vascular loop; other papillary axons end freely or occasionally surround Meissner corpuscles. Intraepithelial axons enter glabrous epidermal pegs, branching and exhibiting terminals throughout the stratum spinosum. A similar pattern is found in hairy skin with additional innervation entering the base and surrounding the lower third of each hair follicle, but apparently not supplying sebaceous glands and arrector pili muscle. Axons innervating nonkeratinized oral epithelium are similar or greater in number and distribution compared to epidermis, often with more extensive branching. The high density of intraepithelial CGRP-IR innervation does not appear to correlate with the sensitive mechanoreceptor-based increase in spatial sensory discriminative capacities in the distal portions of the limb. In deep somatic tissues, CGRP-IR is principally related to vasculature and motor end plates of striated muscle, but there is an extensive network of thin axons within bone, principally in the periosteum, and focally in joint capsules, but not in relation to muscle spindles or tendon organs. These findings, together with the distribution in cranial tissues described in an accompanying paper (Silverman and Kruger: J. Comp. Neurol. 280:303-330, '89), are considered in the context of a "noceffector" concept incorporating the efferent role of these sensory axons in various tissues. It is suggested that involvement in tissue maintenance and renewal during normal function, as well as following injury, may predominate over the relatively infrequent nociceptive role of this peptidergic sensory system.     

Immunohistochemical study of the serotoninergic innervation of the basal ganglia in the squirrel monkey

A specific antibody raised against 5-hydroxytryptamine (5-HT) conjugated to bovine serum albumin was used to study the serotoninergic innervation of the basal ganglia in the squirrel monkey (Saimiri sciureus). At midbrain level, numerous fine 5-HT-immunoreactive axons were seen to arise from the immunopositive neurons of the dorsal raphe nucleus and less abundantly from those of the nucleus centralis superior. The bulk of these axons formed a rather loosely arranged bundle that arched ventrorostrally through the central portion of the midbrain tegmentum and ascended toward the ventral tegmental area. Several fascicles detached themselves from this bundle to reach the substantia nigra where they arborized into a multitude of heterogeneously distributed 5-HT terminals. The 5-HT innervation was particularly dense in the pars reticulata but much less so in the pars compacta of the substantia nigra. More rostrally other 5-HT fibers swept dorsolaterally and formed a remarkably dense network of varicose fibers within the subthalamic nucleus. A multitude of 5-HT axons continued their ascending course within the lateral hypothalamic area, and many of them swept laterally to invade the lenticular nucleus. At pallidal levels, the 5-HT axons arborized much less profusely in the external segment than in the internal segment, which contained numerous 5-HT varicose fibers and terminals arranged in a typical bandlike pattern. At striatal levels, the 5-HT terminals were particularly abundant in the ventral striatum, including the nucleus accumbens and deep layers of the olfactory tubercle. They also abounded in the ventrolateral region of the putamen and the ventromedial aspect of the caudate nucleus. Overall, the number of 5-HT fibers and terminals decreased progressively along the rostrocaudal axis of the striatum and several large and elongated zones rather devoid of 5-HT immunoreactivity were visualized, particularly in the caudate nucleus and the dorsal putamen. These zones of poor 5-HT immunoreactivity were in register with similar areas devoid of tyrosine hydroxylase immunoreactivity as seen on contiguous sections. These findings reveal that all the core structures of the basal ganglia in primates receive a significant serotoninergic input, but that the densities and patterns of innervation vary markedly from one structure to the other.