Ulex europaeus agglutinin-I binding to dental primary afferent projections in the spinal trigeminal complex combined with double immunolabeling of substance P and GABA elements using peroxidase and colloidal gold.

Ulex europaeus agglutinin I (UEA-I) is a plant lectin with an affinity for L-fucosyl residues in the chains of lactoseries oligosaccharides associated with medium- and smaller-diameter dorsal root ganglion neurons and their axonal processes. These enter Lissauer's tract and terminate within the superficial laminae of the spinal cord overlapping projections known to have a nociceptive function. This implies that the surface coatings of neuronal membranes may have a relationship with functional modalities. The present investigation further examined this concept by studying a neuronal projection with a nociceptive function to determine whether fucosyl-lactoseries residues were incorporated in its primary afferent terminals. Transganglionic transport of horseradish peroxidase (HRP) following injection into tooth pulp chambers was employed to demonstrate dental pulp terminals in the trigeminal spinal complex, while peroxidase and fluorescent tags were used concomitantly to stain for UEA-I. Double immunolabeling for substance P (SP) and gamma-aminobutyric acid (GABA) using peroxidase and colloidal gold allowed a comparison of the distribution of a known excitatory nociceptive transmitter with that of UEA-I binding in specific subnuclei. Synaptic interrelationships between UEA-I positive dental pulp primary afferent inputs and specific inhibitory terminals were also examined. SP immunoreactivity occurred in laminae I and outer lamina II (IIo) of subnucleus caudalis (Vc) and in the ventrolateral and lateral marginal region of the caudal half of subnucleus interpolaris (Vi), including the periobex area in which Vi is slightly overlapped on its lateral aspect by cellular elements of Vc. The adjacent interstitial nucleus (IN) also showed an intense immunoreactivity for this peptide antibody. UEA-I binding displayed a similar distribution pattern in both Vc and Vi, but extended into lamina IIi and the superficial part of Lamina III in Vc. Dental pulp terminals were found to have a comparable distribution; however, many extended into the dorsal portion of the caudal half of Vi and the ventromedial quadrant of rostral Vi. Electron-microscopic analysis showed that transganglionically labeled dental pulp terminals contained ovoid, complex membrane-bound vacuoles laden with transported HRP. The preterminal axon and synaptic membranes of those dental pulp terminals located in zones of Vc and Vi displaying an affinity for UEA-I were usually characterized by a patchy, electron-dense coating of the peroxidase tag. SP was demonstrated ultrastructurally with Protein-A colloidal gold (3-nm particles), whereas GABA immunoreactivity was revealed by the avidin-biotin-peroxidase method.(ABSTRACT TRUNCATED AT 400 WORDS)

Golgi and immunocytochemical analysis of neurons in trigeminal subnucleus interpolaris: correlations with cellular localization of enkephalin.

Recent electrophysiological evidence shows that rostral levels of the trigeminal spinal complex are concerned with pain processing from receptive fields in the face and oral cavity. The ventrolateral quadrant of the subnucleus interpolaris contains concentrations of enkephalin, dynorphin, serotonin, substance P and GABA [Matthews M. A., Hernandez T. V. and Liles S. L. (1987) Synapse 1, 512-529; Matthews M. A., McDonald G. K. and Hernandez T. V. (1988) Somatosensory Res. 5, 205-217]. These transmitters have also been localized to the fusiform and stalked cells in Laminae I and II of the subnucleus caudalis [Basbaum A. I. and Fields H. L. (1984) A. Rev. Neurosci. 7, 309-338]. The present study compares Golgi impregnations of the subnucleus interpolaris with sections at the same levels immunoreacted against enkephalin to determine if comparable cells exist in the subnucleus interpolaris and if they occur predominantly in the ventrolateral quadrant of the subnucleus. Twelve, young adult cats were killed by perfusion, the brainstems removed and either processed for rapid Golgi impregnation or sectioned and immunoreacted for enkephalin using the avidin-biotin Vectastain method. Golgi impregnated tissue was sectioned in the coronal, transverse or sagittal plane to insure the most advantageous visualization of cells with a directional bias in their dendritic arbors. The subnucleus interpolaris contained several distinctive cell types. The predominant neuron throughout the subnucleus was the smooth pyramidal cell or multipolar cell, characterized by a large round soma (15-25 microns diameter) and a spherical dendritic arborization which allowed its identification in all planes of section. The second cell type was the fusiform cell which had a smaller ovoid soma (10-15 microns) with narrow, less ramified, dendritic arbors oriented dorsoventrally, thus giving a bipolar appearance. Fusiform cells were most concentrated along the lateral margin of the subnucleus interpolaris. Examination of sections at the same level reacted for enkephalin revealed cells with a bipolar appearance in these same locations. An additional cell population which tended to predominate in the lateral zone was the stalked cell. These displayed a rounded soma (12-20 microns) and were evident only in the transverse or sagittal plane. Two to four primary dendrites arose from the soma and extensively ramified into a dense spiny arbor directed into the body of the subnucleus interpolaris. Many examples contained enkephalin. Islet cells, characterized by a very small oval soma (6-12 microns) and dense, rostrocaudally oriented dendrites, were less common than stalked cells and were located deeper in the nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptic substrates for enkephalinergic and serotoninergic interactions with dental primary afferent terminals in trigeminal subnucleus interpolaris: an immunocytochemical study using peroxidase and colloidal gold.

Pain processing in the trigeminal complex has been thought to reside primarily in the spinal subnucleus caudalis (Vc). However, trigeminal tractotomies eliminating primary afferent input to Vc and severance of secondary trigemino-thalamic fibers from Vc do not disturb pain perception from the central face and oral cavity. Furthermore, large numbers of neurons that are highly responsive to noxious stimuli and suppressed by inputs from the periaqueductal gray and raphe complex have been identified in subnuclei interpolaris (Vi) and oralis (Vo). Therefore, the purpose of this study was to assess the distribution and spatial arrangements of nociceptive modulatory transmitters with nociceptive afferents and trigemino-thalamic relay cells in the rostral portion of the spinal trigeminal nuclear complex. The dental pulp contains predominantly nociceptors that project to all three subdivisions of the trigeminal spinal complex. These projections were visualized by anterograde transganglionic transport of horseradish peroxidase or by degeneration following administration of toxic ricin to the pulp chambers. The spatial arrangements of dental primary afferents with enkephalinergic (ENK) and serotoninergic (5HT) inputs was then assessed by employing avidin-biotin peroxidase and protein-A colloidal gold double-labeling immunocytochemistry. Trigemino-thalamic relay cells were also labeled by retrograde transport of HRP after stereotaxic injections into the ventrobasal thalamus. ENK and 5HT immunoreactivity was found in the ventrolateral quadrant and lateral margin of Vi, together with the adjacent interstitial nucleus (IN). This activity extended from the caudal pole of Vi and the periobex region, where it was most dense, rostrally to a position approximately 2.9 mm from the Obex. Neither ENK nor 5HT immunoreactivity was observed in Vo. Primary dental afferents projected into the ventromedial quadrant of rostral Vi and were found in the ventrolateral quadrant and dorsal aspect of the subnucleus farther caudally. They appeared as simple boutons with single contacts or as larger, sometimes scalloped terminals that formed multiple contacts. Postsynaptic elements were usually small dendritic profiles, although relay cell somata rarely received primary afferent inputs. Many primary afferents entered areas of synaptic clustering and contacted enkephalinergic dendrites, some of which were also postsynaptic to serotoninergic synapses. Alternatively, primary afferents contacted unlabeled processes that were also postsynaptic to the enkephalinergic element to form a triad arrangement. The least common occurrence was axo-axonic contacts in which enkephalinergic synapses were presynaptic to primary afferents. Both enkephalinergic and serotoninergic synaptic categories displayed round vesicles and generally formed asymmetric junctions.(ABSTRACT TRUNCATED AT 400 WORDS)

GABA distribution in a pain-modulating zone of trigeminal subnucleus interpolaris.

A recent model for control of spinal and medullary nociceptive neurons (Basbaum and Fields, 1984) incorporates a gamma-aminobutyric acid-ergic (GABA-ergic) cell into this circuitry and indicates that such elements could act as one substrate for presynaptic inhibition of primary afferents. This concept is supported by a variety of pharmacological and electrophysiological studies. We therefore examined the distribution of GABA-ergic activity in trigeminal subnucleus interpolaris (Vi) by focusing on the types of cells, together with dendritic and synaptic profiles, that are immunocytochemically labeled with an antiserum against glutamic acid decarboxylase (GAD). GAD occurred throughout Vi but was most concentrated in the ventrolateral quadrant and interstitial nucleus. It was localized to groups of small neurons with two to three primary dendrites, and within numerous punctate profiles suggestive of synaptic elements. Electron microscopy revealed labeled dendrites, some of which were postsynaptic to scalloped terminals of presumptive primary afferents. Other labeled dendritic elements, which were quite variable in size, engaged both GAD-labeled and unlabeled synapses. Most GAD synapses displayed clear round vesicles and formed contacts with unlabeled perikarya and a variety of dendritic processes. Numerous GAD-positive synapses were also incorporated into axoaxonic clusters, in which the GAD element was presynaptic to scalloped terminals. Others engaged in serial arrays with other unlabeled terminals, which, in turn, were presynaptic to dendrites. Occasionally, GAD synapses formed contacts with GAD-positive dendrites. These data show that GABA is localized to a variety of neuronal elements in ventrolateral Vi and the interstitial nucleus. These occur in spatial arrangements providing an anatomical substrate for postsynaptic modulation of activity in this area. GABA terminals also appear to be involved in a presynaptic inhibitory mechanism, which may, in some instances, affect transmission in primary afferents.

Immunocytochemistry of enkephalin and serotonin distribution in restricted zones of the rostral trigeminal spinal subnuclei: comparisons with subnucleus caudalis.

The spinal trigeminal subnucleus caudalis processes nociceptive input from the head. However, physiological and behavioral studies in monkeys and humans indicate that painful stimuli from the central face and oral cavity also project through trigeminal nuclei rostral to the spinal subnucleus caudalis. Both enkephalin (ENK) and serotonin (5-HT) are present in rostral trigeminal nuclei and these regions receive inputs from the raphe complex. Thus, it appears that elements of pain-modulating circuitry proposed by Basbaum and Fields (Annu. Rev. Neurosci., 7:309-338, 1984) for the spinal and medullary dorsal horn may also exist in this region. In order to begin an exploration of this circuitry, the present study combines the techniques of retrograde transport of HRP from the ventral posteromedial thalamic nucleus (VPM) of the cat's thalamus to label trigeminothalamic relay cells. Secondarily, immunocytochemical techniques are employed to define the distribution patterns of ENK and 5-HT cells and terminals in relationship to both labeled and nonlabeled neurons in each of the subnuclei of the spinal trigeminal nucleus. Trigeminothalamic relay cells were observed in laminae I and II, the magnocellular region, and the interstitial nucleus (IN) of subnucleus caudalis (Vc). ENK was found in axodendritic and axosomatic terminals, together with a population of small fusiform neurons in all these same areas except the magnocellular region. ENK axosomatic contacts innervated approximately 30% of labeled relay cells, chiefly in lamina I and the IN, or small unlabeled neurons in the same area. Serotonin activity occurred principally in lamina I and the IN and was confined almost exclusively to axodendritic terminals. Examination of subnucleus interpolaris (Vi) revealed relay cells distributed throughout the length of the nucleus and increasing in numbers at rostral levels. A rostral extension of the IN was found just ventrolateral to the main body of Vi and contained numerous labeled cells. The distribution of ENK activity was restricted to the ventral part of Vi and the IN and occurred in axodendritic and axosomatic terminals. These latter elements innervated 30-40% of labeled relay cells in Vi, particularly those located in the IN. Cells containing ENK generally resembled the fusiform cells found in Vc and were distributed in ventral Vi and the IN. Some ENK cells were larger, displayed several dendrites, and occurred only in the ventral Vi. Serotonin within Vi and Vc was confined principally to axodendritic terminals.(ABSTRACT TRUNCATED AT 400 WORDS)