Venom composition of Trimeresurus albolabris, T. insularis, T. puniceus and T. purpureomaculatus from Indonesia

Background: Several studies have been published on the characterization of Trimeresurus venoms. However, there is still limited information concerning the venom composition of Trimeresurus species distributed throughout Indonesia, which contributes to significant snakebite envenomation cases. The present study describes a comparative on the composition of T. albolabris, T. insularis, T. puniceus, and T. purpureomaculatus venoms originated from Indonesia. Methods: Protein content in the venom of four Trimeresurus species was determined using Bradford assay, and the venom proteome was elucidated using one-dimension SDS PAGE nano-ESI- LCMS/MS shotgun proteomics. Results: The venom of T. albolabris contained the highest protein content of 11.1 mg/mL, followed by T. puniceus, T. insularis and T. purpureomaculatus venom with 10.7 mg/mL, 8.9 mg/mL and 5.54 mg/mL protein, respectively. In total, our venomic analysis identified 65 proteins belonging to 16 protein families in T. purpureomaculatus; 64 proteins belonging to 18 protein families in T. albolabris; 58 different proteins belonging to 14 protein families in T. puniceus; and 48 different proteins belonging to 14 protein familiesin T. insularis. Four major proteins identified in all venoms belonged to snake venom metalloproteinase, C-type lectin, snake venom serine protease, and phospholipase A2. There were 11 common proteins in all venoms, and T. puniceus venom has the highest number of unique proteins compared to the other three venoms. Cluster analysis of the proteins and venoms showed that T. puniceus venom has the most distinct venom composition. Conclusions: Overall, the results highlighted venom compositional variation of four Trimeresurus spp. from Indonesia. The venoms appear to be highly similar, comprising at least four protein families that correlate with venom's toxin properties and function. This study adds more information on venom variability among Trimeresurus species within the close geographic origin and may contribute to the development of optimum heterologous antivenom.(AU)

Benefits of Early In-Hospital Antivenom Administration to Patients with Protobothrops mucrosquamatus Envenomation.

Protobothrops mucrosquamatus is one of the common venomous snakes in Southeast Asia. This retrospective cohort study conducted in six medical institutions in Taiwan aimed to obtain information on the optimal management strategies for P. mucrosquamatus snakebite envenomation. Data were extracted from the Chang Gung Research Database from January 2006 to December 2016. The association between early antivenom administration and patient demographics, pain requiring treatment with analgesic injections, and hospital length of stay was analyzed. A total of 195 patients were enrolled; 130 were administered antivenom within 1 hour after emergency department arrival (early group), whereas 65 were treated later than 1 hour after arrival (late group). No in-hospital mortality was identified. The difference in surgical intervention rates between the early and late groups was statistically insignificant (P = 0.417). Compared with the early group, the late group showed a higher rate of antivenom skin test performance (46.9% versus 63.1%, respectively, P = 0.033), longer hospital stay (42 ± 62 hours versus 99 ± 70 hours, respectively, P = 0.016), and higher rate of incidences of pain requiring treatment with analgesic injections (29.2% versus 46.2%, respectively, P = 0.019). After adjusting for confounding factors, early antivenom administration was associated with decreased pain requiring treatment with analgesic injections (adjusted odds ratio: 0.51, 95% CI: 0.260-0.985). Antivenom administration within 1 hour of arrival was associated with a decreased likelihood of experiencing pain and hospital length of stay in patients with P. mucrosquamatus snakebites. Antivenom skin testing was associated with delays in antivenom administration.

Trimeresurus albolabris snakebite treatment implications arising from ontogenetic venom comparisons of anticoagulant function, and antivenom efficacy.

Does the venom of Trimeresurus albolabris (white-lipped pit viper) differ between neonate and adults? This species is responsible for most snakebites within south and southeast Asia, yet it is unknown whether ontogenetic variation in venom composition occurs in this species, or how this might affect antivenom efficacy. Using a coagulation analyser robot, we examined the anticoagulant activity of T. albolabris venom from eight individuals across multiple age classes. We then compared the efficacy of Thai Red Cross Green Pit Viper Antivenom across these age classes. Venoms from all age classes were equally potent in their pseudo-procoagulant, fibrinogenolytic activity, in that fibrinogen was cleaved to form weak, unstable fibrin clots that rapidly broke down, thus resulting in a net anticoagulant state. Similarly, this coagulotoxic activity was well neutralised by antivenom across all venoms. Given that coagulotoxicity is the primary serious pathology in T. albolabris envenomations, we conclude that Thai Red Cross Green Tree Pit Viper Antivenom is a valid treatment for envenomations by this species, regardless of age or sex of the offending snake. These results are relevant for clinical treatment of envenomations by T. albolabris.

Comprehensive Snake Venomics of the Okinawa Habu Pit Viper, Protobothrops flavoviridis, by Complementary Mass Spectrometry-Guided Approaches.

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Structural characteristics and evolution of the Protobothrops elegans pancreatic phospholipase A2 gene in contrast with those of Protobothrops genus venom phospholipase A2 genes.

The nucleotide sequence of the gene encoding Protobothrops elegans (Crotalinae) pancreatic phospholipase A(2) (PLA(2)), abbreviated PePancPLA(2), was determined by means of inverted PCR techniques. Since its deduced amino acid sequence contains a pancreatic loop and shows high similarity to that of Laticauda semifasciata (Elapinae) group IB pancreatic PLA(2), PePancPLA(2) is classified into group IB PLA(2). The nucleotide sequences of the PePancPLA(2) gene, the L. semifasciata group IB pancreatic PLA(2) gene, and the L. semifasciata group IA venom PLA(2) gene are similar to one another but greatly dissimilar to those of Protobothrops genus (Crotalinae) group II venom PLA(2) genes, suggesting that the Elapinae group IB PLA(2) gene and the group IA PLA(2) gene appeared after Elapinae was established, and that the Crotalinae group II venom PLA(2) genes came into existence before Elapinae and Crotalinae diverged. A phylogenetic analysis of their amino acid sequences confirms this.

Isolation, characterization and antigenic cross-reactivities of the major hemorrhagin from Cryptelytrops purpureomaculatus venom.

The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 microg in mice) but was not lethal to mice at a dose of 1 microg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.

TA-2, a thrombin-like enzyme from the Chinese white-lipped green pitviper (Trimeresurus albolabris): isolation, biochemical and biological characterization.

Through three chromatographic steps, a new thrombin-like enzyme (TLE), named TA-2, from the venom of the Chinese white-lipped green pitviper (Trimeresurus albolabris) has been isolated and purified to homogeneity. TA-2 was a single-chain glycoprotein with about 6% sugar, pI 3.9 and a molecular weight of 38.8 kD. Its N-terminal sequence (VVGGDECNIN) showed high sequence conformity with many other TLEs. In vitro, it coagulated bovine fibrinogen (108.6 NIH units/mg) and cleaved the Aα and Bß chains of bovine fibrinogen-releasing fibrinopeptide A and B, but did not degrade bovine fibrin; displayed high stability at different temperature, pH, and presence of several divalent cations and inhibitors; also exhibited strong activity towards casein (192.3 units/mg) and high esterase activity upon Nα-p-tosyl-L-arginine methyl ester (11 units/mg); and behaved as a promoter to platelet aggregation induced by ADP or collagen. In vivo, TA-2 caused dose-dependent prolongation of bleeding time in mice, but had no hemorrhagic and edema-inducing activities even at high concentrations.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents.

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes Aα followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Jerdonuxin, a novel snaclec (snake C-type lectin) with platelet aggregation activity from Trimeresurus jerdonii venom.

Serious clinical symptoms of Trimeresurus jerdonii bite are mainly caused by abnormalities of blood system. We have previously identified and characterized several bioactive components affecting human blood system, such as serine proteases, metalloproteinases and disintegrins. But few snaclec was characterized in the T. jerdonii venom. In this study, a novel snaclec, named jerdonuxin, was isolated, molecular cloned and characterized as a human platelet agonist. On SDS-polyacrylamide gel electrophoresis, jerdonuxin showed a single band with an apparent molecular weight of 120 kDa under non-reducing conditions and two distinct bands with apparent molecular weights of 18 kDa (α-subunit) and 14 kDa (ß-subunit) under reducing conditions. The cDNA sequence of each subunit of jerdonuxin was identified. The precursors of both subunits contain a 23-amino acid residue signal peptide and the mature proteins are composed of 135 and 125 amino acids for α- and ß-subunits, respectively. The N-terminal amino acid sequences of each subunit determined by Edman degradation were consistent with deduced amino acid sequences of cDNA. Jerdonuxin dose-dependently induced human platelet aggregation. The phosphorylation profile pattern induced by jerdonuxin showed similar with mucetin (a platelet agonist via glycoprotein Ib), but different from stejnulxin (an agonist via glycoprotein VI). The jerdonuxin-induced platelet aggregation was inhibited by the anti-GPIbα or anti-GPIIb polyclonal antibodies, but not by anti-GPVI polyclonal antibodies. In summary, a novel snaclec of platelet agonist was purified and characterized from the T. jerdonii venom and our data also suggested that GPIb was involved in jerdonuxin-induced platelet aggregation.

Fast and furious: effects of body size on strike performance in an arboreal viper Trimeresurus (Cryptelytrops) albolabris.

Body size has a pervasive effect on animal functioning and life history with size dependent changes in performance and physiology throughout ontogeny being common in many ectothermic vertebrates. However, as selection on juvenile life history stages is strong, juveniles often offset the disadvantages of small body size by disproportionate levels of performance. Here, we investigate size-related changes in defensive strike performance in an arboreal pit viper, Trimerusurus (Cryptelytrops) albolabris. Our data show a significant negative allometry in the scaling of head dimensions and head mass to body mass. However, strike velocity and strike distance are independent of body mass, with juveniles in our sample striking as fast and as far as adults. In contrast to model predictions suggesting that acceleration capacity should decrease with increasing body mass, acceleration capacity increases with snake body mass. Our results suggest that this is the result of a negative allometric scaling of head mass combined with an isometric scaling of the dorsal epaxial musculature. Finally, our data show a significant sexual dimorphism in body size and strike velocity with females being heavier and striking faster independent of the dimorphism in body size.

Bioenergetic modeling reveals that Chinese green tree vipers select postprandial temperatures in laboratory thermal gradients that maximize net energy intake.

With bioenergetic modeling, we tested the hypothesis that reptiles maximize net energy gain by postprandial thermal selection. Previous studies have shown that Chinese green tree vipers (Trimeresurus s. stejnegeri) have postprandial thermophily (mean preferred temperature T(p) for males =27.8 degrees C) in a linear thigmothermal gradient when seclusion sites and water existed. With some published empirical models of digestion associated factors for this snake, we calculated the average rate (E(net)) and efficiency (K(net)) of net energy gain from possible combinations of meal size, activity level, and feeding frequency at each temperature. The simulations consistently revealed that E(net) maximizes at the T(p) of these snakes. Although the K(net) peaks at a lower temperature than E(net), the value of K(net) remains high (>=0.85 in ratio to maximum) at the peak temperature of E(net). This suggested that the demands of both E(net) and K(net) can be attained by postprandial thermal selection in this snake. In conclusion, the data support our prediction that postprandial thermal selection may maximize net energy gain.

Purification and characterization of a new platelet aggregation inhibitor with dissociative effect on ADP-induced platelet aggregation, from the venom of Protobothrops elegans (Sakishima-habu).

A platelet aggregation inhibitor, named snake venom platelet aggregation dissociator (SV-PAD)-1, with a dissociative reaction of ADP-induced platelet aggregation, was purified from the venom of Protobothrops elegans (Sakishima-habu) by gel-filtration employing Sephadex G-100, and ion-exchange chromatographies using DEAE-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Mono S. By this procedure, about 1.5mg of purified protein was obtained from 1.0g of P. elegans venom. The purified protein showed a single protein band and the molecular weight was about 110kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed four-bands of 7.7, 7.8, 7.95, and 8.15. This protein strongly inhibited ADP-induced platelet aggregation in rabbit platelet-rich plasma (PRP), and its IC(50) was about 58nM. It inhibited ristocetin-induced platelet aggregation in rabbit PRP (IC(50): 100nM), but hardly blocked collagen-induced platelet aggregation. This protein promptly dissociated platelet aggregation in rabbit PRP stimulated by high-concentration ADP.

A unique group of inactive serine protease homologues from snake venom.

A number of inactive serine protease homologues (SPHs), which have poorly understood functions, have been identified in invertebrates and vertebrates. Recently, several SPH transcripts have been reported from snake venom glands, which provide potential new tools for the study of the functions of SPHs. Herein we report for the first time a snake venom serine protease homologue (svSPH) protein, designated as TjsvSPH, isolated from the venom of Trimeresurus jerdonii. Despite its high sequence similarity to snake venom serine proteases (SVSPs), TjsvSPH is devoid of arginine esterase and proteolytic activity. This is probably due to the replacement of Arg-43 by His-43 in the catalytic triad. TjsvSPH did not influence the coagulation time of human plasma, induce human platelet aggregation, inhibit adenosine diphosphate/thrombin-induced human platelet aggregation or increase capillary permeability. Phylogenetic analysis showed that svSPHs were separated from SVSPs and formed an independent group. Structural analysis revealed that the structures of svSPHs are quite different from those of SPHs previously reported. These results indicate that snake venoms contain a unique group of svSPH proteins.

Blood flow in snake infrared organs: response-induced changes in individual vessels.

OBJECTIVE: In the past the microkinetics of blood flow in the infrared pit organs of pit vipers has been studied with Doppler flowmetry using various infrared stimuli such as a human hand or soldering iron at various distances, lasers of various wavelengths, etc. Quick-acting variations in blood flow were recorded, and interpreted as a cooling mechanism for avoiding afterimage in the infrared receptors. However, the Doppler measurements provided only the summation of blood flow in a number of vessels covered by the sensing probe, but did not give data on flow in individual vessels. METHODS: In the present work the authors introduced into the bloodstream of Gloydius and Trimeresurus pit vipers fluorescent microspheres labeled with fluorescein isothiocyanate (FITC) contained in a solution of FITC-dextran in physiological saline. They observed the passage of the microspheres through individual pit organ vessels with a fluorescent microscope to which was attached a high-speed video camera and image intensifier. Output of the camera was recorded before, during, and after stimulus with a 810-nm diode laser. Recording was done at 250 frames/s on high-speed video apparatus and downloaded to a hard disk. Disk files were loaded into proprietary software and particles were tracked and average velocities calculated. The data were then tested for significance by ANOVA with post hoc tests. RESULTS: A significant (p<.05) increase in blood velocity was found at the focal point of the stimulus laser, but not anywhere removed from this point. Proximal severing of the pit sensory nerves caused degeneration of the pit receptor terminals and abolished stimulus-induced blood flow changes, but did not affect normal blood flow. CONCLUSIONS: The authors conclude that the receptors themselves are directly and locally controlling the smooth muscle elements of the blood vessels, in response to heating of the receptors by infrared radiation. They speculate that the heavy vascularization constitutes a cooling system for the radiation-encoding receptors, and further that the agent of control may be a volatile neuromediator such as nitric oxide.

Immunohistochemical study of endothelial nitric oxide synthase in the trigeminal ganglia of a crotaline snake Trimeresurus flavoviridis.

The immunoreactivity of constitutive endothelial nitric oxide synthase (eNOS) was studied in the trigeminal ganglia (TG) of a crotaline snake, Trimeresurus flavoviridis. eNOS immunoreactivity was found in TG neurons of different sizes. The percentage of eNOS-positive TG neurons was significantly higher in the mandibular division than in the infrared-related divisions, the maxillary division and ophthalmic ganglion (p<0.001). These findings suggest that eNOS in the TG of crotaline snakes is involved in constitutive neurotransmission in the TG, and is minimally involved in processing in the infrared-sensory system.

Ecological diversification in a group of Indomalayan pitvipers (Trimeresurus): convergence in taxonomically important traits has implications for species identification.

We analyse molecular and phenotypic evolution in a group of taxonomically problematic Indomalayan pitvipers, the Trimeresurus sumatranus group. Mitochondrial DNA sequencing provides a well-resolved phylogeny, with each species representing a distinct lineage. Multivariate morphological analysis reveals a high level of phenotypic differentiation, which is congruent between the sexes but does not reflect phylogenetic history. An adaptive explanation for the observed pattern of differentiation is supported by independent contrasts analysis, which shows significant correlations between current ecology and the characters that most account for the variation between taxa, including those that are presently used to identify the species. Reduced precipitation and altitude, and increased temperature, are correlated with higher numbers of scales on the head, body and tail. It is hypothesized that scale number plays an important role in heat and water exchange by influencing the area of exposed of interstitial skin, and that colour pattern variation reflects selection pressures involving camouflage and thermoregulation. Ecological convergence in traits used for classification is found to have important implications for species identification where taxa are distributed over varying environments.

Snake infrared receptors respond to dimethylsulfoxide in the blood stream.

1. We used extracellular recording of the infrared (IR)-sensitive trigeminal ganglion (TG) neurons (primary neurons) of a crotaline snake, Trimeresurus flavoviridis, which has very sensitive thermoreceptors, to examine changes in the IR response induced by dimethylsulfoxide (DMSO), in vivo. 2. The responses in the TG were recorded after each concentration of DMSO (1, 10, and 25%) was administered in the bloodstream. 3. At a constant temperature, DMSO dose-dependently potentiated the IR-triggered discharges of IR-sensitive TG neurons in this snake. 4. It is suggested that the increased IR response to DMSO is due to its chemical effect, or to an indirect effect via its vasoactive role in the thermoreceptors of IR-sensitive snakes.

Offshore insular variation in the diet of the Taiwanese bamboo viper Trimeresurus stejnegeri (Schmidt).

Dietary data were ascertained for 229 T. stejnegeri (snout vent length >300mm) from 36 localities throughout the main island of Taiwan and the outlying Orchid (Lanyu) and Green (Ludau) Islands. Twenty nine percent of the snakes were devoid of any prey, and of the snakes containing prey, 43% of the cases were unidentifiable. This relatively large proportion of unidentifiable prey items (observed in the hindgut) may reflect either rapid digestion of amphibian prey and/or rapid venting of feces as an evolutionary adaptation to arboreal life. Trimeresurus stejnegeri appears euryphagous, taking primarily amphibians, but additionally reptilian, mammalian and insect prey. There was no discrepancy in prey composition based on comparisons of where the prey item was recorded in the digestive tract. No sexual variation in diet composition was evident, although males were more likely to contain prey than females, indicating the utilisation of different foraging strategies on similar sympatric prey items. Variation in diet composition was observed between mainland Taiwan and offshore islands, which is most likely the result of differences in prey availability.

A new thrombin-like enzyme, flavoviridiobin from the venom of Trimeresurus flavoviridis (habu).

Fibrinopeptide A and B releasing enzyme, flavoviridiobin, was isolated from the venom of Trimeresurus flavoviridis using Q-Sepharose, CM-Cellulose, and Sephadex G-75 column chromatographies. Homogeneity was established by the formation of a single band in polyacrylamide gel electrophoresis, isoelectric focusing, and Ouchterlony immunodiffusion. The enzyme has a molecular weight of 48,000, isoelectric point of 8.1, consists of 237 total amino acid residues, and demonstrates clotting activity. However, no tosyl-L-arginine methyl ester (TAME) hydrolytic and kinin-releasing activities were observed. This clotting enzyme was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), benzamidine, and beta-mercaptoethanol, suggesting that serine, acidic amino acids, and disulfide bonds are involved in the expression of the enzyme's clotting activity. This thrombin-like enzyme hydrolyzes B beta-chain of human fibrinogen at first, followed by hydrolysis A alpha-chain. The enzyme was stable over the pH range of 7-10 and was shown to be heat resistant.

C mechanical nociceptive neurons in the crotaline trigeminal ganglia.

Using 32 Crotaline snakes, Trimeresurus flavoviridis, intrasomal recordings were made from 44 neurons of the trigeminal ganglia in vivo. They were 10 C neurons from 9 snakes and 34 A-delta mechanical nociceptive neurons from 23 snakes. 5 of the 10 C neurons were identified as mechanical nociceptive neurons. The neurons were labeled with iontophoretically injected HRP. Each of the 5 C nociceptive neurons had one receptive field, on which 1 spike was elicited by pricking the skin or mucosa with a pin. They were sensitized after repeated stimulation. The fields were insensitive to thermal stimulation. No background discharge was observed. Average conduction velocity was 0.95 m/s (+/- 0.4 S.D., n = 5). Mean resting potential was -62.5 mV (+/- 6.0 S.D., n = 4), and mean action potential amplitude was 88.0 mV (+/- 10.9 S.D., n = 4). Two somata were successfully visualized with HRP (22 microns x 20 microns, 20 microns x 18 microns). Total lengths of labeled axons were 1260 and 1480 microns peripherally to the edge of the section, and 1810 and 770 microns centrally. Neither of the neurons had branching of the peripheral or central axons in the ganglion.