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Eduardo Osinaga - One of the best experts on this subject based on the ideXlab platform.
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Monoclonal antibodies against the Tn-specific Isolectin B4 from Vicia villosa seeds: characterization of the epitope of the blocking antibody VV34.
Hybridoma and Hybridomics, 2004Co-Authors: Andrea Medeiros, Nora Berois, Henia Balter, Ana Robles, Enrique Pérez-payá, Ana Gimenez, Juan J. Calvete, Eduardo OsinagaAbstract:Vicia villosa Isolectin B4 (VVLB4) recognizes the Tn antigen (GalNAc-O-Ser/Thr) exposed in certain human carcinomas. We have produced anti-VVLB4 monoclonal antibodies (MAbs), and their lectin recog...
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monoclonal antibodies against the tn specific Isolectin B4 from vicia villosa seeds characterization of the epitope of the blocking antibody vv34
Hybridoma and Hybridomics, 2004Co-Authors: Andrea Medeiros, Nora Berois, Henia Balter, Ana Robles, Ana Gimenez, Juan J. Calvete, Enrique Perezpaya, Eduardo OsinagaAbstract:: Vicia villosa Isolectin B4 (VVLB4) recognizes the Tn antigen (GalNAc-O-Ser/Thr) exposed in certain human carcinomas. We have produced anti-VVLB4 monoclonal antibodies (MAbs), and their lectin recognition selectivity was assessed by ELISA and Western blot against the purified Gal/GalNAc-specific lectins from Vicia villosa, Salvia sclarea, Helix pomatia, Arachis hypogaea, Glycine max, and Dolichos biflorus. The antibodies were also tested for their ability to block the binding of VVLB4 to the Tn antigen expressed on immobilized asialo ovine submaxillary mucin. Two MAbs, VV34 and VV2, specifically recognized VVLB4 and impaired the binding of the lectin to the Tn antigen by 98% and 21%, respectively. On the other hand, MAbs VV1 and VV22 cross-reacted with other purified lectins. The four antibodies recognized native and periodate-oxidized nonreduced as well as reduced VVLB4 after SDS-PAGE and Western blot, suggesting that they were recognizing continuous polypeptide epitopes. The VV34 antibody recognized two tryptic peptides (7-29 and 96-106) from VVLB4, which are contiguous in the three-dimensional structure of the lectin. The minimum structural determinant of the epitope was mapped to the polypeptide stretch (18)LILQED(23) using a set of overlapping synthetic peptides. This region of the molecule encompasses the C-terminal part of the loop joining strands beta1 and beta2 and the N-terminal part of beta2, and is located about 20-25 A away from the center of the Tn-combining site.
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The crystal structure of a plant lectin in complex with the Tn antigen.
FEBS letters, 2003Co-Authors: Alvaro Babino, Eduardo Osinaga, Diana Tello, Adriana Rojas, Pedro M AlzariAbstract:The structure of the tetrameric Vicia villosa Isolectin B4 (VVLB4) in complex with a cancer antigen, the Tn glycopeptide (GalNAc-O-Ser), was determined at 2.7 A resolution. The N-acetylgalactoside moiety of the ligand binds to the primary combining site of VVLB4 in a similar way as observed for other Gal/GalNAc-specific plant lectins. The amino acid moiety of the Tn antigen is largely exposed to the solvent and makes few contacts with the protein. The structure of the complex provides a framework to understand the differences in the strength of VVLB4 binding to different sugars and emphasizes the role of a single protein residue, Tyr127, as a structural determinant of Tn-binding specificity.
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Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy.
FEBS Letters, 2000Co-Authors: Eduardo Osinaga, Diana Tello, Alvaro Babino, Otto Pritsch, Karine Assemat, Danièle Cantacuzene, Hiroshi Nakada, Pedro M AlzariAbstract:Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that Isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.
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amino acid sequence and three dimensional structure of the tn specific Isolectin B4 from vicia villosa
FEBS Letters, 1997Co-Authors: Eduardo Osinaga, Diana Tello, Carlos Batthyany, M A Bianchet, Gisele A Tavares, Rosario Duran, Carlos Cervenansky, Luc Camoin, A Roseto, Pedro M AlzariAbstract:Abstract The partial amino acid sequence of the tetrameric Isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded β-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.
G Grant - One of the best experts on this subject based on the ideXlab platform.
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anterograde transport of horseradish peroxidase conjugated Isolectin B4 from griffonia simplicifolia i in spinal primary sensory neurons of the rat
Brain Research, 1998Co-Authors: Fredrik H Wang, Brita Robertson, G GrantAbstract:Anterograde transport of the Isolectin B4 from Griffonia simplicifolia I (B4) conjugated to horseradish peroxidase (HRP) was investigated in rat somatic and visceral primary sensory neurons at different spinal levels. Injection of B4-HRP into the L5 dorsal root ganglion (DRG) resulted in labelling in the sural nerve, but not in the gastrocnemius nerves. Free nerve endings and lanceolate-like nerve endings were labelled in the lateral hindpaw skin. Labelled fibres were also observed in the greater splanchnic nerve following B4-HRP injection into the T10–11 DRGs. Electron microscopic examination of the labelled nerves showed that B4-HRP labelled exclusively unmyelinated axons. In the spinal cord, labelling was observed in the superficial dorsal horn, and additionally, although much more sparse, in the medial and lateral collateral projections following injections into the T10–11 DRGs. The results suggest that B4-HRP should be a suitable anterograde tracer of unmyelinated cutaneous and splanchnic but not muscle primary afferent fibres.
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retrograde and transganglionic transport of horseradish peroxidase conjugated cholera toxin b subunit wheatgerm agglutinin and Isolectin B4 from griffonia simplicifolia i in primary afferent neurons innervating the rat urinary bladder
Neuroscience, 1998Co-Authors: H F Wang, P Shortland, M J Park, G GrantAbstract:In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and Isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6–S1 spinal cord segments and in the T13–L2 and L6–S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit–horseradish peroxidase was injected. Cholera toxin B subunit–horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV–VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin–horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit–horseradish peroxidase. The Isolectin B4 from Griffonia simplicifolia I–horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit–horseradish peroxidase than with wheat germ agglutinin– or Isolectin B4–horseradish peroxidase. In the L6–S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and Isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with myelinated fibres). Double labelling with other neuronal markers showed that 71%, 43% and 36% of the cholera toxin B subunit-immunoreactive cells were calcitonin gene-related peptide-, Isolectin B4-binding- and substance P-positive, respectively. A few cholera toxin B subunit cells showed galanin-immunoreactivity, but none were somatostatin-, vasoactive intestinal polypeptide-, or neuropeptide Y-immunoreactive or contained fluoride-resistant acid phosphatase. The results show that cholera toxin B subunit–horseradish peroxidase is a more effective retrograde and transganglionic tracer for pelvic primary afferents from the urinary bladder than wheat germ agglutinin–horseradish peroxidase and Isolectin B4–horseradish peroxidase, but in contrast to somatic nerves, it is transported mainly by unmyelinated fibres in the visceral afferents.
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early decline and late restoration of spinal cord binding and transganglionic transport of Isolectin B4 from griffonia simplicifolia i after peripheral nerve transection or crush
Restorative Neurology and Neuroscience, 1996Co-Authors: Carl Molander, H F Wang, C Riveromelian, G GrantAbstract:Copyright (c) 1996 Elsevier Science B.V. All rights reserved. Isolectin B4 from Griffonia simplicifolia I (B4r has a high binding affinity to a large population of unmyelinated primary sensory neurons (Wang et al., Neuroscience 62 (1994r 539−551r. Using immunohistochemical techniques, binding and transganglionic transport of B4 in the spinal cord was investigated, both at short and long survival times, after sciatic nerve transection and ligation or crush in the adult rat. Nerve transection and ligation resulted in nearly complete disappearance of B4 immunolabelling in the sciatic nerve territory of the superficial dorsal horn after B4 binding, as well as after transganglionic transport of B4 by 2 weeks postinjury. Partial recovery of both B4 binding and B4 transport was found by 8 months, and nearly complete recovery by 16 months, indicating that reappearance of B4 binding is not critically dependent on peripheral reinnervation. Crush injury made by jewellers forceps resulted in partial depletion of binding and transport by 2 weeks and a nearly complete recovery by 10 weeks. The results show that binding and transganglionic transport of B4 can be used to label dorsal horn connections of unmyelinated primary afferents during the process of regeneration after crush injury. Furthermore, as B4 binding and transport recover at long survival times in the absence of reestablished peripheral connections, the same techniques can be used to study central primary afferent connections at long survival times after nerve transection. Binding and transganglionic transport of B4 offer alternatives to the use of previous techniques such as transganglionic transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRPr to study central connections of fine primary afferents after injury.
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transganglionic transport and binding of the Isolectin B4 from griffonia simplicifolia i in rat primary sensory neurons
Neuroscience, 1994Co-Authors: H F Wang, Brita Robertson, C Riveromelian, G GrantAbstract:The Isolectin B4 from Griffonia simplicifolia I binds to a subpopulation of rat small-diameter dorsal root ganglion neurons, and to fibres and presumed terminals in laminae I–II of the spinal cord dorsal horn. In the present study we investigated B4 and B4 conjugated to horseradish peroxidase as potential transganglionic tracers of somatic primary afferent neurons after injection into a peripheral nerve. We also tried to identify the specific subpopulation of dorsal root ganglion neurons that bind and transport B4. Following injection of B4 or B4-horseradish peroxidase into the sciatic nerve, labelled presumed terminals that reached peak labelling at two days were found exclusively in regions of the spinal cord gray matter known to receive unmyelinated primary afferent fibres. Almost all dorsal root ganglion cells that transported B4-horseradish peroxidase also bound B4. Cell counts showed that 51% of the dorsal root ganglion neurons were B4-positive and cell area measurements that these were all in the small size range. An extensive overlap was found between B4 and fluoride-resistant acid phosphatase (85%), and between B4 and calcitonin gene-related peptide (59%). Seventeen per cent of the B4-positive cells were substance P-immunoreactive and 9% were immunoreactive to somatostatin. Minimal overlap was seen between B4-positive cells and cells positive for RT97 (3%), a selective marker of primary afferent neurons with myelinated axons. All somatostatin-immunoreactive cells and almost all (95%) of the fluoride-resistant acid phosphatase-positive cells were contained within the B4-positive population. This comprised also 58% of the cells immunoreactive to calcitonin gene-related peptide and 42% of those immunoreactive to substance P. The results obtained show that B4 binds to a subpopulation of unmyelinated primary afferent neurons, and that B4 and B4-horseradish peroxidase can be used as selective transganglionic tracers of this specific cell subpopulation.
Pedro M Alzari - One of the best experts on this subject based on the ideXlab platform.
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The crystal structure of a plant lectin in complex with the Tn antigen.
FEBS letters, 2003Co-Authors: Alvaro Babino, Eduardo Osinaga, Diana Tello, Adriana Rojas, Pedro M AlzariAbstract:The structure of the tetrameric Vicia villosa Isolectin B4 (VVLB4) in complex with a cancer antigen, the Tn glycopeptide (GalNAc-O-Ser), was determined at 2.7 A resolution. The N-acetylgalactoside moiety of the ligand binds to the primary combining site of VVLB4 in a similar way as observed for other Gal/GalNAc-specific plant lectins. The amino acid moiety of the Tn antigen is largely exposed to the solvent and makes few contacts with the protein. The structure of the complex provides a framework to understand the differences in the strength of VVLB4 binding to different sugars and emphasizes the role of a single protein residue, Tyr127, as a structural determinant of Tn-binding specificity.
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Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy.
FEBS Letters, 2000Co-Authors: Eduardo Osinaga, Diana Tello, Alvaro Babino, Otto Pritsch, Karine Assemat, Danièle Cantacuzene, Hiroshi Nakada, Pedro M AlzariAbstract:Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that Isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.
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amino acid sequence and three dimensional structure of the tn specific Isolectin B4 from vicia villosa
FEBS Letters, 1997Co-Authors: Eduardo Osinaga, Diana Tello, Carlos Batthyany, M A Bianchet, Gisele A Tavares, Rosario Duran, Carlos Cervenansky, Luc Camoin, A Roseto, Pedro M AlzariAbstract:Abstract The partial amino acid sequence of the tetrameric Isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded β-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.
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Amino acid sequence and three‐dimensional structure of the Tn‐specific Isolectin B4 from Vicia villosa
FEBS Letters, 1997Co-Authors: Eduardo Osinaga, Diana Tello, Carlos Batthyany, M A Bianchet, Gisele A Tavares, Rosario Duran, Carlos Cervenansky, Luc Camoin, A Roseto, Pedro M AlzariAbstract:Abstract The partial amino acid sequence of the tetrameric Isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded β-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.
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Crystallization and Preliminary Crystallographic Analysis of a Tetrameric Isolectin from Vicia villosa, Specific for the Tn Antigen
Journal of Molecular Biology, 1993Co-Authors: Jean-luc Eiselé, Eduardo Osinaga, Diana Tello, A Roseto, Pedro M AlzariAbstract:Isolectin B4 isolated from Vicia villosa seeds is specific for the Tn antigen, a carcinoma-associated molecular marker. Crystals of the Isolectin grown in the presence of carbohydrate are tetragonal, space group P 41 (or P43), with a = 91·3 A, c = 151·7 A and one tetramer in the asymmetric unit. The crystals diffract X-rays to 2·8 A resolution and are suitable for high-resolution structural analysis.
Shao Rui Chen - One of the best experts on this subject based on the ideXlab platform.
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a type voltage gated k currents influence firing properties of Isolectin B4 positive but not Isolectin B4 negative primary sensory neurons
Journal of Neurophysiology, 2005Co-Authors: Amaresh Vydyanathan, Zi Zhen Wu, Shao Rui ChenAbstract:Voltage-gated K+ channels (Kv) in primary sensory neurons are important for regulation of neuronal excitability. The dorsal root ganglion (DRG) neurons are heterogeneous, and the types of native Kv currents in different groups of nociceptive DRG neurons are not fully known. In this study, we determined the difference in the A-type Kv current and its influence on the firing properties between Isolectin B4 (IB4)-positive and -negative DRG neurons. Whole cell voltage- and current-clamp recordings were performed on acutely dissociated small DRG neurons of rats. The total Kv current density was significantly higher in IB4-positive than that in IB4-negative neurons. Also, 4-aminopyridine (4-AP) produced a significantly greater reduction in Kv currents in IB4-positive than in IB4-negative neurons. In contrast, IB4-negative neurons exhibited a larger proportion of tetraethylammonium-sensitive Kv currents. Furthermore, IB4-positive neurons showed a longer latency of firing and required a significantly larger amoun...
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differential sensitivity of n and p q type ca2 channel currents to a μ opioid in Isolectin B4 positive and negative dorsal root ganglion neurons
Journal of Pharmacology and Experimental Therapeutics, 2004Co-Authors: Zi Zhen Wu, Shao Rui ChenAbstract:Opioids have a selective effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms for this preferential effect are not fully known. Two broad classes of nociceptors can be distinguished based on their growth factor requirements and binding to Isolectin B4(IB4). In this study, we determined the difference in the modulation of voltage-gated Ca2+ currents by [d-Ala2, N -Me-Phe4,Gly-ol5]-enkephalin (DAMGO, a specific μ opioid agonist) between IB4-positive and -negative small dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated DRG neurons in adult rats. Both 1–10 μM DAMGO and 1 to 10 μM morphine had a greater effect on high voltage-activated Ca2+ currents in IB4-negative than IB4-positive cells. However, DAMGO had no significant effect on T-type Ca2+ currents in both groups. The N-type Ca2+ current was the major subtype of Ca2+ currents inhibited by DAMGO in both IB4-positive and -negative neurons. Although DAMGO had no effect on L-type and R-type Ca2+ currents in both groups, it produced a larger inhibition on N-type and P/Q-type Ca2+ currents in IB4-negative than IB4-positive neurons. Furthermore, double labeling revealed that there was a significantly higher μ opioid receptor immunoreactivity in IB4-negative than IB4-positive cells. Thus, these data suggest that N-and P/Q-type Ca2+ currents are more sensitive to inhibition by the μ opioids in IB4-negative than IB4-positive DRG neurons. The differential sensitivity of voltage-gated Ca2+ channels to the μ opioids in subsets of DRG neurons may constitute an important analgesic mechanism of μ opioids.
Hiroyuki Ichikawa - One of the best experts on this subject based on the ideXlab platform.
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Trigeminal primary projection to the rat brain stem sensory trigeminal nuclear complex and surrounding structures revealed by anterograde transport of cholera toxin B subunit-conjugated and Bandeiraea simplicifolia Isolectin B4-conjugated horseradish
Neuroscience Research, 1997Co-Authors: Tomosada Sugimoto, Yoshiaki Fujiyoshi, Yi Fen He, Chun Xiao, Hiroyuki IchikawaAbstract:Trigeminal primary afferent neurons were labeled by injecting the rat trigeminal ganglion with either wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), cholera toxin B subunit (B)-HRP or Bandeiraea simplicifolia Isolectin B4 (IB4)-HRP. B-HRP stained medium to large cells (>600 μm2), while IB4-HRP mostly small cells (
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trigeminal primary projection to the rat brain stem sensory trigeminal nuclear complex and surrounding structures revealed by anterograde transport of cholera toxin b subunit conjugated and bandeiraea simplicifolia Isolectin B4 conjugated horseradish
Neuroscience Research, 1997Co-Authors: Tomosada Sugimoto, Yoshiaki Fujiyoshi, Yi Fen He, Chun Xiao, Hiroyuki IchikawaAbstract:Trigeminal primary afferent neurons were labeled by injecting the rat trigeminal ganglion with either wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), cholera toxin B subunit (B)-HRP or Bandeiraea simplicifolia Isolectin B4 (IB4)-HRP. B-HRP stained medium to large cells (>600 μm2), while IB4-HRP mostly small cells (<400 μm2). WGA-HRP labeled trigeminal ganglion neurons of all sizes. Cell bodies in the mesencephalic trigeminal tract nucleus were labeled with WGA-HRP and B-HRP but not IB4-HRP. B-HRP revealed dense projection to the entire brain stem sensory trigeminal nuclear complex (BSTC) except for lamina II of the medullary dorsal horn (MDH). Some contralateral projection was also seen in the caudal part of MDH. Non-trigeminal nuclei receiving B-HRP-labeled terminals included the paratrigeminal nucleus (paraV), solitary tract nucleus, supratrigeminal nucleus, Probst's nucleus and median accessory nucleus. Following IB4-HRP application, terminal label was found in more restricted regions within the BSTC. Modest terminal label was seen in the dorsal part of principal sensory nucleus and at the medial edge of subnucleus interpolaris, while relatively dense terminal fields were seen in the dorsal half of subnucleus oralis. The MDH laminae I and II contained dense terminal label. Non-trigeminal nuclei were almost devoid of the IB4-HRP-labeled terminals excepting the paraV that contained dense terminal label. The terminal areas revealed with WGA-HRP coincided with B-HRP-labeled and IB4-HRP-labeled areas combined.
