Uncoupling protein 2 and aldolase B impact insulin release by modulating mitochondrial function and Ca2+ release from the ER.

Uncoupling protein 2 (UCP2), a mitochondrial protein, is known to be upregulated in pancreatic islets of patients with type 2 diabetes (T2DM); however, the pathological significance of this increase in UCP2 expression is unclear. In this study, we highlight the molecular link between the increase in UCP2 expression in ß-cells and ß-cell failure by using genetically engineered mice and human islets. ß-cell-specific UCP2-overexpressing transgenic mice (ßUCP2Tg) exhibited glucose intolerance and a reduction in insulin secretion. Decreased mitochondrial function and increased aldolase B (AldB) expression through oxidative-stress-mediated pathway were observed in ßUCP2Tg islets. AldB, a glycolytic enzyme, was associated with reduced insulin secretion via mitochondrial dysfunction and impaired calcium release from the endoplasmic reticulum (ER). Taken together, our findings provide a new mechanism of ß-cell dysfunction by UCP2 and AldB. Targeting the UCP2/AldB axis is a promising approach for the recovery of ß-cell function.

Aristolochic Acid Induces Renal Fibrosis and Senescence in Mice.

The kidney is one of the most susceptible organs to age-related impairments. Generally, renal aging is accompanied by renal fibrosis, which is the final common pathway of chronic kidney diseases. Aristolochic acid (AA), a nephrotoxic agent, causes AA nephropathy (AAN), which is characterized by progressive renal fibrosis and functional decline. Although renal fibrosis is associated with renal aging, whether AA induces renal aging remains unclear. The aim of the present study is to investigate the potential use of AAN as a model of renal aging. Here, we examined senescence-related factors in AAN models by chronically administering AA to C57BL/6 mice. Compared with controls, the AA group demonstrated aging kidney phenotypes, such as renal atrophy, renal functional decline, and tubulointerstitial fibrosis. Additionally, AA promoted cellular senescence specifically in the kidneys, and increased renal p16 mRNA expression and senescence-associated ß-galactosidase activity. Furthermore, AA-treated mice exhibited proximal tubular mitochondrial abnormalities, as well as reactive oxygen species accumulation. Klotho, an antiaging gene, was also significantly decreased in the kidneys of AA-treated mice. Collectively, the results of the present study indicate that AA alters senescence-related factors, and that renal fibrosis is closely related to renal aging.

OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.

Proteomic analysis of exosome-enriched fractions derived from cerebrospinal fluid of amyotrophic lateral sclerosis patients.

Exosomes contain many proteins associated with neurodegenerative diseases. To identify new candidate biomarkers and proteins associated with amyotrophic lateral sclerosis (ALS), we performed liquid chromatography-tandem mass spectrometry proteomic analysis of exosome-enriched fractions isolated from cerebrospinal fluid (CSF) of sporadic ALS patients using gel filtration chromatography. Proteomic data revealed that three proteins were increased and 11 proteins were decreased in ALS patients. The protein with the greatest increase in exosome-enriched fractions of CSF derived from ALS was novel INHAT repressor (NIR), which is closely associated with nucleolar function. By immunohistochemical analysis, we found that NIR was reduced in the nucleus of motor neurons in ALS patients. Our results demonstrate the potential utility of our methodology for proteomic analysis of CSF exosomes and suggest that nucleolar stress might play a role in sporadic ALS pathogenesis through the dysfunction of NIR.

Angiotensin II Type 1 Receptor-Associated Protein Regulates Kidney Aging and Lifespan Independent of Angiotensin.

BACKGROUND: The kidney is easily affected by aging-associated changes, including glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Particularly, renal tubulointerstitial fibrosis is a final common pathway in most forms of progressive renal disease. Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP), which was originally identified as a molecule that binds to AT1R, is highly expressed in the kidney. Previously, we have shown that ATRAP suppresses hyperactivation of AT1R signaling, but does not affect physiological AT1R signaling. METHODS AND RESULTS: We hypothesized that ATRAP has a novel functional role in the physiological age-degenerative process, independent of modulation of AT1R signaling. ATRAP-knockout mice were used to study the functional involvement of ATRAP in the aging. ATRAP-knockout mice exhibit a normal age-associated appearance without any evident alterations in physiological parameters, including blood pressure and cardiovascular and metabolic phenotypes. However, in ATRAP-knockout mice compared with wild-type mice, the following takes place: (1) age-associated renal function decline and tubulointerstitial fibrosis are more enhanced; (2) renal tubular mitochondrial abnormalities and subsequent increases in the production of reactive oxygen species are more advanced; and (3) life span is 18.4% shorter (median life span, 100.4 versus 123.1 weeks). As a key mechanism, age-related pathological changes in the kidney of ATRAP-knockout mice correlated with decreased expression of the prosurvival gene, Sirtuin1. On the other hand, chronic angiotensin II infusion did not affect renal sirtuin1 expression in wild-type mice. CONCLUSIONS: These results indicate that ATRAP plays an important role in inhibiting kidney aging, possibly through sirtuin1-mediated mechanism independent of blocking AT1R signaling, and further protecting normal life span.

Retrograde tracing technique for neonatal animals.

Tract tracing is a fundamental technique in neuroanatomy for examining fiber connections in the nervous system. After the introduction of horseradish peroxidase 40 years ago, many tracing substances have been used for neuroanatomical studies on various nervous systems. Here, we described retrograde tracing techniques using multiple fluorescent tracers, which make it possible to detect axonal collaterals. This technique is useful to study the development of axonal trajectories, as well as regenerative and compensatory mechanisms of animals that undergo neural damage at early stages.

Islet-1 expression in thoracic spinal motor neurons in prenatal mouse.

The LIM homeodomain protein Islet-1, an embryonic marker for motoneurons in the spinal cord, has been reported to be heterogeneously expressed among motoneuron groups in mouse. In the present study, we examined Islet-1 expression in the thoracic and rostral lumbar spinal cord in prenatal mice. In the thoracic spinal cord at embryonic day 12.5 (E12.5) and E13.5, strong Islet-1 immunoreactivity was observed in the lateral group of the ventral horn, whereas weaker immunoreactivity was observed in the ventral group. Strong Islet-1 immunoreactivity was also observed in the intermediolateral area and more medial part of the intermediate zone. In the rostral lumbar spinal cord at E12.5 and E13.5, strong Islet-1 immunoreactivity was observed in the lateral group of the ventral horn, and in the intermediolateral nucleus, whereas weaker immunoreactivity was observed in the ventral, and dorsolateral groups. At E14.5, the number of Islet-1 immunoreactive neurons was reduced in the spinal cord, but the distribution pattern was similar to that at E12.5 and E13.5. At E15.5, Islet-1 immunoreactivity was almost completely confined to the intermediolateral area. Some weakly immunoreactive neurons were observed in the ventral horn. The findings of the present study indicated that Islet-1 expression at embryonic stages differs among the motoneuron groups in the thoracic and rostral lumbar spinal cord.

A retrograde tracing study of compensatory corticospinal projections in rats with neonatal hemidecortication.

To examine the compensatory mechanisms in rats that underwent left decortication at postnatal day 7 (P7), we injected the retrograde tracers fluorescein isothiocyanate-cholera toxin B subunit (FITC-CTB) and Fast Blue (FB) into the right and left upper cervical spinal cord, respectively, at postoperative weeks 2, 3, 4, and 5 and counted the number of retrogradely labeled corticospinal neurons in the right cerebral cortex compared with that in normally developed rats. Significantly more ipsilaterally projecting neurons were labeled with FITC-CTB in the decorticated rats compared with normal rats at all time points examined. The number of labeled neurons was similar to that at P7 in normal rats. There were also some FITC-CTB and FB double-labeled neurons in both decorticated and normal rats. The number of double-labeled neurons in the decorticated rats increased each week and was significantly greater than that in normal rats at postoperative weeks 4 and 5. The present results suggest that the elimination of ipsilaterally projecting axons observed in normal rats was prevented in the decorticated rats, so that the cerebral cortex neurons on the unlesioned side projected corticospinal tracts to the ipsilateral spinal cord. Furthermore, the collaterals of the corticospinal tracts originating from the cerebral cortex on the unlesioned side also project to the ipsilateral spinal cord. These compensatory mechanisms might underlie the acquisition of motor function in these animals.

The chelonian spinal nerve ganglia are a conglomerate of the spinal nerve ganglia proper and the sympathetic ganglia.

A tyrosine hydroxylase-immunoreactive cell mass is found in the caudal portion of the dorsal nerve ganglion of the red-eared slider, Trachemys scripta elegans. The ganglion appears as a flat oval structure in the horizontal plane, where the major axis runs latero-medially, and the minor axis rostro-caudally in the ventral view. A communicating branch to the sympathetic chain diverges from the top of each tubercle which lies on the caudo-lateral side of the ganglion. A tyrosine hydroxylase- immunoreactive cell mass is located in this tubercle. This cell mass exists in both sexes. Tyrosine hydroxylase-immunoreactive cells, that contain Nissl bodies in cytoplasm and are enveloped by the satellite cells, are multipolar and their neural processes are distributed in a distal direction into the spinal nerve. The range of distribution of the synapsin I-immunoreactive structures is limited to the tyrosine hydroxylase-immunoreactive cell mass. The chelonian dorsal spinal nerve ganglia are a conglomerate of the spinal nerve ganglion proper and the sympathetic ganglion.

Differential Islet-1 expression among lumbosacral spinal motor neurons in prenatal mouse.

Onuf's nucleus in the lumbosacral spinal cord, comprising somatic motoneurons that innervate the pelvic floor muscles via the pudendal nerve, shares some characteristics with the autonomic preganglionic neurons and functions in coordination with the autonomic nervous system. In mouse, neurons projecting to the urethral sphincter and ischiocavernosus muscles form the dorsolateral (DL) nucleus at the caudal lumbar levels, whereas neurons projecting to the limb and hip joint muscles comprise the retrodorsolateral and ventral nucleus, as well as the DL nucleus at the rostral lumbar levels. The results of the present study in mouse revealed that the expression pattern of a LIM homeodomain protein Islet-1, an embryonic marker for motoneurons in the spinal cord, was different among motoneuronal groups at the prenatal stage (embryonic days 13.5-15.5); the highest expression was observed in the DL at the caudal lumbar cord, whereas there was little expression in the lateral part of the rostral DL. Islet-1 expression was also observed in the parasympathetic preganglionic neurons at the sacral spinal cord. These findings provide evidence that the DL neurons at the caudal lumbar cord, corresponding to Onuf's nucleus, are chemically distinct among the motoneuronal groups at the prenatal stages. This differential Islet-1 expression among the motoneuronal groups suggests that Islet-1 not only leads to a motoneuronal lineage, but also to the differentiation of motoneuronal subsets in the lumbosacral spinal cord.

Mediolateral and rostrocaudal topographic organization of the sympathetic preganglionic cell pool in the spinal cord of Xenopus laevis.

The sympathetic preganglionic cell pool in Xenopus laevis can be divided into four parts, i.e., the intercalated nucleus (IC) and the intermediolateral nucleus (IML) located respectively at the medial and the lateral borders of the lateral field, the lateral funiculus, and the ventral field within the thoracolumbar spinal segments. We compared the location of the preganglionic cells labeled following tracer application to the paravertebral sympathetic chain with those labeled following application to the celiac ganglion (CG), the adrenal gland (AG), and the splanchnic nerves (SNs) and found that their relative contribution differs depending on the sites. In tracer application to the paravertebral chain ganglia and the sympathetic trunk, 31.4-41.9% and 43.9-58.4% of labeled cells were detected respectively in the IC and in the IML, whereas application to the CG, AG, and on all the SNs, revealed that more than 84% of labeled cells were found in the IML and in the lateral funiculus with less than 8.6% in the IC. The contribution of the ventral field cells was less than 7.5% in all experiments. This type of topographic cytoarchitecture is a character shared with the mammalian preganglionic cell pool, but what distinguishes it from that of mammals is its systematic form throughout the entire longitudinal extent of the pool. In Xenopus, differences of mean soma areas and dendritic projections of labeled cells also suggest that the cell pools are distinguished not only by their location and axonal projections, but also by the morphology of their cells.

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.

Prenatal development of transient receptor potential vanilloid 1-expressing primary sensory projections to sacral autonomic preganglionic neurons.

The visceral reflexes of the pelvic organs are mediated by connections between primary afferents innervating the pelvic organs and parasympathetic preganglionic neurons in the intermediolateral column of the sacral spinal cord. The present immunohistochemical study revealed many varicosities expressing transient receptor potential vanilloid 1 (TRPV1) that were closely apposed to the preganglionic neuronal perikarya at embryonic day 16 in mice. Many, but not all, varicosities expressing TRPV1 in the intermediolateral column were also immunopositive for calcitonin gene-related peptide. In contrast, no nerve fibers expressing TRPV1 projected to the sympathetic preganglionic cell column in the lumbar spinal cord in prenatal stages. The results of the present study raised the possibility that the primary afferents transmit signals elicited by the activation of TRPV1 receptors to the sacral parasympathetic preganglionic neurons. Thus, the functional circuit for pelvic spinal reflexes, such as micturition induced by urine influx, might develop in the prenatal stages in mice.

Differential development of TRPV1-expressing sensory nerves in peripheral organs.

In mouse ontogeny, neurons immunoreactive for transient receptor potential vanilloid receptor 1 (TRPV1) were observed primarily in the dorsal root ganglia (DRG) at embryonic day 13 (E13). In the embryonic period, the number of TRPV1(+) neurons decreased, but then gradually increased postnatally. Some of TRPV1(+) neurons were also immunoreactive for calcitonin gene-related peptide (CGRP). At postnatal day 7 (P7), 66% of CGRP(+) neurons were TRPV1(+), and 55% of TRPV1(+) neurons were also CGRP(+) in the L4 DRG. In the peripheral organs, TRPV1-immunorective nerve fibers were transiently observed in the skin at E14. They were also observed in the urinary tract at E14, and in the rectum at E15. Many TRPV1(+) nerve fibers in these organs were also CGRP(+). At P1, TRPV1(+) nerve fibers were observed in the respiratory organs, and to a lesser extent in the stomach, colon, skin, and skeletal muscles. The number of TRPV1(+) nerve fibers on each organ gradually increased postnatally. At P7, TRPV1(+) nerve fibers were also observed in the small intestine and kidneys. The percentage of total TRPV1(+) nerve fibers that co-localized with CGRP was greater in most organs at P7 than at P1. The present results indicate that TRPV1 expression on peripheral processes differs among organs. The differential time course of TRPV1 expression in the cell bodies might be related to the organs to which they project. Co-localization of TRPV1 with CGRP on nerve fibers also varies among organs. This suggests that the TRPV1-mediated neuropeptide release that occurs in certain pathophysiologic conditions also varies among organs.

Medullary efferent and afferent neurons of the facial nerve of the pit viper Gloydius brevicaudus.

For the purposes of comparative anatomy, we used tracer techniques and immunohistochemistry to study the facial nerve in the pit viper Gloydius brevicaudus and obtained much new data applicable to the function of this nerve in snakes and, in particular, pit vipers. We were able to identify the superior salivatory nucleus in these snakes. Preganglionic fibers from this nucleus pass along the palatine nerve and an anterior communicating branch to reach the pterygopalatine ganglion attached to the deep branch of the trigeminal maxillary nerve. The palatine nerve also contains general somatic afferents and a very few special visceral afferents from some taste buds on the palate. In the mandibular direction, preganglionic fibers from the superior salivatory nucleus join special visceral efferents from the motor nucleus in the hyomandibular nerve, from which they pass into the chorda tympani to course together for a short distance. The special visceral efferents branch off outside the cranium, and the preganglionic fibers continue on to join the trigeminal mandibular nerve to project to small ganglia within the mandible. The chorda tympani also contains general somatic afferents from the mandibular region but no special visceral afferents. This is the first time that the superior salivatory nucleus and its adjuncts have been identified in a snake. The chorda tympani of these snakes is also distinguished from the mammalian condition by lacking any special visceral afferents and by branching outside the cranium.

Prenatal development of peptidergic primary afferent projections to mouse lumbosacral autonomic preganglionic cell columns.

To examine the prenatal development of spinal visceral reflexes, primary sensory nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) were examined in the spinal cord, particularly in the autonomic preganglionic nuclei of mouse embryos. On embryonic day 16 (E16), CGRP-immunoreactive fibers were first observed in the sacral intermediolateral nucleus (IML) of the parasympathetic division as well as in the lumbar central autonomic nucleus (CA) of the sympathetic division, where they appeared in proximity to preganglionic neuronal perikarya immunoreactive for choline acetyltransferase or nitric oxide synthase. Most of the CGRP-immunoreactive varicosities were negative for substance P. Substance P-immunoreactive varicosities were scattered in these nuclei, but no appositions were seen on the preganglionic neuronal perikarya. On E18, CGRP-immunoreactive fibers were more abundant in the sacral IML and the lumbar CA. Co-expression of substance P and CGRP was frequently observed in the varicosities very close to the preganglionic neuronal perikarya on E18. CGRP-immunoreactive fibers were also observed in the lumbar IML on E18, although significantly fewer were found in this nucleus compared with the sacral IML. In contrast to the upper lumbar level, no fibers immunoreactive for CGRP were observed in the IML at the thoracic level. These results suggest that peptidergic primary sensory fibers grow to project to the selective targets of autonomic preganglionic neurons during the embryonic period. The potential direct connections between the peptidergic primary sensory fibers and preganglionic neurons innervating the pelvic viscera might provide a circuit for spinal visceral reflexes active in embryos.

The microvasculature of python pit organs: morphology and blood flow microkinetics.

Boid snakes have infrared sensing pits that resemble crotaline pits in electrophysiological function and ultrastructure, but differ in gross morphology, number, and location: boids have three or more simple pits in the labial scales vs a single facial pair with more complex morphology in the crotalines. We studied the morphology of the capillary bed and the microkinetics of blood flow in a boid snake, the ball python, Python regius, and compared them with the already known condition in crotalines. We used a Doppler blood flow recorder in conjunction with an electrocardiograph to measure blood flow and heartbeat, and resin casts, transmission electron microscopy, and laser confocal microscopy to study capillary morphology. Blood flow in response to infrared stimulus was virtually identical in the two taxa, but the morphology of the capillary bed differed drastically. In the ball python pits, the capillary bed consisted of a forest of vertically oriented loops with a characteristic dome at the top in contact with the receptor layer of the fundus. Immunohistochemical staining showed pericytes constricting the capillaries and domes with smooth muscle alpha-actin-labeled processes. Since latency of response was as short as 1 ms, the capillaries were apparently responding under local control to provide both nutrition and cooling to the heat-sensitive receptors. We concluded that mitochondria-filled receptors provided with a swiftly responding cooling system were nature's most efficient way of attaining infrared imaging.

Central projections of thoracic splanchnic and somatic nerves and the location of sympathetic preganglionic neurons in Xenopus laevis.

The central and peripheral organization of thoracic visceral and somatic nervous elements was studied by applying dextran amines to the proximal cut ends of the thoracic splanchnic and somatic nerves in Xenopus laevis. Many labeled dorsal root ganglion cells of visceral afferents, and all somatic afferents, were located in a single ganglion of one spinal segment, and the two types of cells were distributed topographically within the ganglion. The labeled sympathetic preganglionic neurons were located predominantly in the same area of the thoracic spinal gray as in other frogs and in mammals. The labeled visceral afferents projected to Lissauer's tract and the dorsal funiculus. The visceral fibers of the tract ascended to the level of the subcerebellar area, supplying collateral branches to the lateral one-third of the dorsal horn and to the area of brainstem nuclei, including lateral cervical and descending trigeminal nucleus, and descended to the filum terminale. The visceral fibers of the dorsal funiculus were distributed to the dorsal column nucleus and the solitary tract. A similar longitudinal projection was also seen in the somatic afferents. The dual central pathway of thoracic primary afferents in the anuran spinal cord is a property held in common with mammals, but the widespread rostrocaudal projection through Lissauer's tract may be a characteristic of the anuran central nervous system. In frogs, the direct transmission of primary afferent information to an extremely wide area of the central nervous system may be important for prompt assessment of environmental factors and control of body functions.

Choline acetyltransferase immunoreactive sympathetic ganglion cells in a teleost, Stephanolepis cirrhifer.

The present study showed neurons immunoreactive for choline acetyltransferase (ChAT) in the cranial sympathetic ganglia lying close to the trigeminal-facial nerve complex of the filefish. In these ganglia, less than 1% of ganglion cells were positive for choline acetyltransferase. Choline acetyltransferase-positive neurons were significantly larger than the randomly sampled neurons in this ganglion. The majority of choline acetyltransferase-positive neurons were negative for tyrosine hydroxylase, but many of them were positive for galanin (GAL). Some neurons were positive for both choline acetyltransferase and tyrosine hydroxylase, but these neurons were rarely immunoreactive for dopamine beta hydroxylase, suggesting that they are not adrenergic. In the cranial sympathetic ganglia and the celiac ganglia, many nerve fibers immunoreactive for galanin were seen, and varicose terminals were in contact selectively with neurons negative for both choline acetyltransferase and tyrosine hydroxylase, but not with those positive for choline acetyltransferase or tyrosine hydroxylase. Nerve fibers immunoreactive for choline acetyltransferase were found to be present in contact with the deep layer of chromatophores, which was observed only in the labial region. These results suggest that cholinergic postganglionic neurons are present in the filefish cranial sympathetic ganglia, and that they also contain galanin. As few cholinergic sympathetic neurons express tyrosine hydroxylase and none express dopamine beta hydroxylase, they are unlikely to synthesize noradrenaline or adrenaline.

Distribution of myelinated and unmyelinated nerve fibers and their possible role in blood flow control in crotaline snake infrared receptor organs.

We used transmission electron microscopic montages to examine the composition of nerve bundles serving the infrared pit organs of two species of crotaline snakes, Agkistrodon blomhoffii and A. brevicaudus. In the three main bundles, the myelinated fibers totaled 2,200-3,700, and unmyelinated fibers 2,400. We also discovered for the first time two accessory bundles composed almost entirely of unmyelinated fibers running alongside the main bundles, containing an average total of 3,300 unmyelinated fibers vs. an average of 10 myelinated fibers. Thus, the average total of unmyelinated fibers was nearly twice that of myelinated fibers. To study the nature of the unmyelinated fibers, we did double staining immunohistochemistry with antibodies for substance P (SP) and vasoactive intestinal polypeptide (VIP) in combination with and without capsaicin pretreatment. SP and VIP immunoreactive varicose fibers ran straight toward the center of the pit membrane in parallel with arterioles and venules, and also formed a dense network around the periphery of the membrane. There were three types of fibers: fibers containing only SP, fibers containing only VIP, and fibers containing both peptides. SP-only fibers were distributed singly throughout the pit membrane and in small bundles around the periphery. SP+VIP fibers were distributed sparsely in the pit membrane and around its periphery. VIP-only fibers were distributed throughout the pit membrane and were of smaller diameter than SP and SP+VIP fibers. After treatment with capsaicin, most of the three types of varicose fibers disappeared from the central part of the pit membrane, but those around the periphery remained unaffected. The capsaicin-sensitive fibers may be unmyelinated sensory types, and the unaffected ones may be autonomic nerve fibers.