Capillary electrophoresis using triple layer modified capillary facilitating salivary ion analyses: Application to search for potential stress markers induced by cold pressure test.

In this study, we introduce a novel approach for the analysis of salivary ions using capillary electrophoresis (CE) with a triple-layer coated capillary. The capillary is sequentially coated with cationic silylating reagents, poly(vinylsulfonate), and polybrene to form a custom designed surface that effectively inhibits adsorption of protein matrix on the capillary inner wall and allows for reproducible ion analysis. For the CE with capacitively coupled contactless conductivity detection, we used suitable background electrolytes (BGEs) for salivary ion analysis. Anions were separated using a mixture of 2-(N-morpholino)ethanesulfonic acid and l-arginine, and cations were separated using that with 18-crown-6. This setup enabled rapid separation, within 4 min, together with sensitive detection. We quantified nine common anions and five cations typically found in saliva samples using this CE method, both before and after a cold pressure test (CPT, a standard stress test). The CE system demonstrated consistent ion separation across 30 consecutive measurements without requiring capillary replacement. Notably, the salivary ion balance remained predominantly anion-rich, regardless of the CPT. Cold water exposure induced greater variation in the total anion concentration than in the total cation concentration. Further analysis using multiple regression analysis revealed strong relationships between nitrate and nitrite, formate and phosphate, and potassium and nitrate, before and after the CPT. Notably, potassium and nitrate ions exhibited variations in response to stress. These results provided a method for assessing salivary ion composition and insights into the potential of salivary ions as biomarkers for stress.

Effects of Cathode Direct-Current Electroacupuncture Stimulus of the Promixal Anterior Lower Limbs on Heart Rate and Lumbar Blood Flow.

Objective: There has been little research on direct-current acupuncture. This study was conducted to verify the effect of acupuncture current intensity and polarity on heart rate (HR) when applying direct-current electroacupuncture (DCEA) to the proximal anterior part of the lower limbs and to clarify the effect of cathodes (-) on lumbar blood flow. Materials and Methods: This study was conducted at Teikyo Heisei University, Chiba, Japan. The subjects were 144 healthy male volunteers (21.7 ± 0.6 years' old). These participants were divided into 1 control, 1 manual acupuncture (MA), and 3 DECEA groups (-25 µA, +75 µA, and -75 µA). Acupuncture was performed on the lower limbs (at ST 36), and HR and intertissue blood flow (ITBF) were observed as the main outcome measures. Results: For intensity, HR was significantly lower in the -75 µA group than in the control (P < 0.001), MA (P < 0.001), and -25 µA (P < 0.01) groups. For polarity, HR was significantly lower in the -75 µA group than in the +75 µA group (P = 0.0028). For blood flow, the volume of HbO2 in the L-4-L-5 region at 1 minute after stimulation was significantly higher in the -75 µA stimulus group than in the MA group (P < 0.05). There was no change in the T-7-T-8 region. Conclusions: A -75 µA stimulus to ST 36 reduced HR and increased ITBF.

Effects of a Standardized Oligomerized-Polyphenol from Litchi chinensis Fruit Extract and Mixed Plant Extract Supplementation on Peripheral Circulation and Cold Sensitivity.

Certain individuals tend to suffer from a cold sensation-particularly in the lower extremities-despite most people not suffering from the same sensation. In Japan, this phenomenon is called "hie-sho" and reduces quality of life for several people, particularly women. A previous study has shown that a standardized oligomerized-polyphenol from Litchi chinensis fruit extract (OPLFE) reportedly causes a significant increase in body surface temperature. The present study aimed to investigate whether supplementation with OPLFE affected peripheral circulation and cold sensitivity. This randomized, double-blind, placebo-controlled trial was performed including 25 participants (age, 45.0±10.4 y; 3 males and 22 females) who were assigned to consume OPLFE, mixed plant extract with OPLFE, or placebo capsules for 14 d. Participants were instructed to relax for 60 min in a temperature-controlled room prior to obtaining measurements. Changes in skin temperature and peripheral blood flow of the middle finger were assessed immediately before and 1, 5, 10, 20, and 30 min after immersion in cold water (10ºC). Participants' height, weight, skin temperature, and blood flow in peripheral tissue were measured; furthermore, their "hie-sho" was measured using the Visual Analog Scale (VAS). Skin temperature and blood flow in peripheral tissue increased in the OPLFE and mixed plant extract with OPLFE groups on day 14 compared with those on day 1. In addition, cold sensitivity in these two groups significantly improved between day 1 and day 14. These findings suggest that OPLFE improves "hie-sho" by increasing peripheral blood flow and skin temperature.

Electroacupuncture of the Ophthalmic Branch of the Trigeminal Nerve: Effects on Prefrontal Cortex Blood Flow.

Objective: The current authors observed enhanced cerebral blood flow (CBF) in the prefrontal cortex (PFC) in response to 100-Hz electroacupuncture (EA) stimulation of the ophthalmic branch of the trigeminal nerve. However, it is not yet clear if responsiveness to 100-Hz EA depends on stimulus intensity. This study examined the effects of stimulus strength on PFC CBF during 100-Hz EA of the ophthalmic branch of the trigeminal nerve. Materials and Methods: Twelve subjects underwent 3 acupuncture sessions: I, control, no stimulation; II, 0.1 mA EA; and III, 0.2 mA EA). Needles were inserted 1 cm lateral of the head median line; the anterior insertion point was on the front hairline and the posterior insertion point was ∼7 cm behind the hairline. Stimulation frequency was set to 100-Hz. PFC CBF was measured in terms of oxygenated, deoxygenated, and total hemoglobin (OxyHb, DeoxyHb, TotalHb, respectively), using 16-channel (Ch) near-infrared spectroscopy. Results: Stimulation of 0.2 mA was associated with significant elevation of OxyHb levels in the 0.1 mA condition in Chs 6, 10, and 12. Ch 2-6, 10, 12 signals were notably higher than in the control condition. Stimulation of 0.2 mA and 0.1 mA were associated with significant declines in DeoxyHb levels, compared to the control condition in Ch 4. Finally, 0.2 mA stimulation in Chs 12 and 13 was associated with significant elevation of TotalHb levels in the control condition. Conclusions: Using 0.2-mA stimulation, 100-Hz EA of the ophthalmic nerve enhances PFC CBF more strongly than 0.1-mA stimulation.

Acupuncture Relaxation, Vigilance Stage, and Autonomic Nervous System Function: A Comparative Study of Their Interrelationships.

Objective: During acupuncture stimulation, autonomic nervous system (ANS) function changes toward being parasympathetic-dominant, with a transient decrease in heart rate (HR). The aim of this research was to determine the relationships between cortical relaxation and vigilance as observed on background electroencephalograms (EEGs), HR, and ANS function during deep acupuncture. Materials and Methods: This comparative study was conducted at Teikyo Heisei University, in Toshima-ku, Tokyo, Japan, with 27 healthy male volunteers. After resting for 20 minutes, the men received manual acupuncture at LI 10 on the left forearm for 2 minutes at a depth of 15-20 mm at a 1-Hz frequency while undergoing concurrent EEG and electrocardiography (ECG) monitoring. Each subject described his level of comfort during acupuncture. HR; power ratios (normalized units [n.u.]) of EEG alpha, beta, delta, and theta waves; and HR variability (HRV) indices were calculated. Results: In the subgroup who experienced discomfort, delta and theta n.u. were decreased while alpha n.u. were increased, indicating increased vigilance and decreased relaxation. In the subgroup who experienced comfort, there were no significant changes. HRV indices suggested parasympathetic-dominant changes in both subgroups. Weak correlations were observed between a decrease of theta n.u. and sympathetic-dominant changes in HRV indices. Conclusions: Alterations in background EEG activities were not the primary factors changing ANS function to parasympathetic-dominant and decreasing HR, but these alterations related to a weak secondary factor changing ANS function. EEG activity by which cortical relaxation and vigilance were represented was the weak secondary factor changing ANS function during acupuncture; the primary factor might be supraspinal reflection.

Effects of Acupuncture Sensations on Transient Heart Rate Reduction and Autonomic Nervous System Function During Acupuncture Stimulation.

Objective: During acupuncture stimulation, heart rate (HR) transiently decreases and autonomic nervous system (ANS) function becomes parasympathetic-dominant. To clarify the effect of acupuncture sensations (pain, De Qi), the effects of deep acupuncture sensations on HR and ANS functions were determined. Materials and Methods: In this comparative study at Teikyo Heisei University, Tokyo, Japan, 40 healthy, male student volunteers rested for 20 minutes before undergoing manual acupuncture to the Shousanli (LI 10) acupoint on the left forearm for 2 minutes at a frequency of 1 Hz, with concurrent electrocardiography. Depth of stimulation was 15-20 mm. These subjects described their subjective acupuncture sensations. Calculations were performed, using HR variability analysis to find HR and low-frequency (LF) normalized units (nu), the ratio of LF components to total components (as an index of sympathetic nervous system function), high-frequency (HF)nu, the ratio of HF components to total components (as an index of parasympathetic nervous system function), and LF/HF (as an index of sympathetic and parasympathetic balance). Results: For the final analysis, data were available for 32 subjects. Compared to before acupuncture, HR decreased during acupuncture. HR decreased when no acupuncture sensations or when weak De Qi sensations were perceived, and remained unchanged when acupuncture sensations without De Qi or strong De Qi were perceived. LFnu decreased, HFnu increased, and LF/HF decreased, regardless of pain or De Qi. Conclusions: Acupuncture stimulation reduced HR even without De Qi sensations and caused ANS function to be parasympathetic-dominant, irrespective of the perception of acupuncture sensations.

Genetic transformation of the dermatophyte, Trichophyton mentagrophytes, based on the use of G418 resistance as a dominant selectable marker.

BACKGROUND: Dermatophytes are closely related keratinophilic fungal pathogens and are the causative agents of a superficial cutaneous infection called dermatophytosis (ringworm). A lack of gene manipulation techniques has prevented detailed analyses of the mechanisms of host invasion by dermatophytes. We have introduced the tetracycline-regulatable (TR) gene expression system into dermatophytes to facilitate functional analyses of genes essential for growth and virulence. As the TR gene expression system consists of two plasmid vector components, two dominant selectable markers are required for genetic transformation. In dermatophytes, only the hygromycin B phosphotransferase gene (hph) is available as a selectable marker. OBJECTIVE: We investigated the possibility of G418 resistance as a secondary selectable marker for genetic transformation in dermatophytes. METHODS: A series of plasmid vectors carrying the neomycin phosphotransferase gene (nptII) were introduced into the protoplasts of Trichophyton mentagrophytes, one of the most clinically important dermatophyte species, by polyethylene glycol (PEG)-mediated transformation. Transformants were selected on selective medium containing G418 at 300-500 microg/ml. RESULTS: Molecular biological analyses indicated that colonies appearing on the selective medium harbored nptII in their chromosomes. Colonies produced from protoplasts transformed with the enhanced green fluorescent protein (eGFP) gene-T. mentagrophytes cyclophilin cDNA (TmcypB) fusion vector also exhibited GFP fluorescence throughout their mycelia, but accumulation of the GFP-TmCYPB fusion protein in specific intracellular compartments was not observed. CONCLUSIONS: This study has provided a new selectable marker for genetic transformation in dermatophytes.

Morphological relationships between peptidergic nerve fibers and immunoglobulin A-producing lymphocytes in the mouse intestine.

Immunoglobulin A (IgA) lymphocytes are present close to the nerve fibers in the lamina propria of the small intestine, and the administration of lipopolysaccharides (LPSs) increases the number of these cells and IgA secretion to the lumen. In the present study, we demonstrated that the nerve fibers immunoreactive for vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) were close to the IgA lymphocytes in the mouse ileum lamina propria. Three hours after intraperitoneal administration of LPSs, IgA lymphocytes close to VIP nerve fibers, those close to basement membrane, and those close to both VIP nerve fibers and basement membrane were increased in number. Further, all IgA lymphocytes seen in the ileum lamina propria expressed the receptors for VIP, VIPR1, and VIPR2. Electron microscopy revealed that varicosities were in close apposition to the lymphocyte plasma membrane. The present study suggests that VIP/NPY/CGRP neurons in the submucosal plexus have a close anatomical relationship to IgA lymphocytes, playing a role in the secretion of IgA and intestinal fluid in response to stimulation by lipopolysaccharides, pathogens, or toxins.

Effects of AHCC® on Immune and Stress Responses in Healthy Individuals.

AHCC® is a functional food from the basidiomycete Lentinula edodes. We evaluated the effects of AHCC® on subjects under different kinds of stress and at rest. Physical stress was imposed using an active standing test, known as Schellong's test. Sympathetic nervous activity in the standing position was significantly greater in AHCC®-treated subjects than in a placebo group. In contrast, AHCC® significantly increased parasympathetic nervous activity at rest. Under mental stress, AHCC® increased sympathetic nervous activity, with no difference in the parasympathetic nervous system. In subjects with chronic mental stress, self-reported "initiation and maintenance of sleep" was significantly greater in the AHCC®-intake period than in the placebo intake period, and natural killer cell activity also increased after AHCC® intake, suggesting a possible mechanism of action of AHCC®. Our findings indicate that AHCC® is potentially effective in stress management and may be useful in the treatment of depression.

Evaluation of Autonomic Nervous System Function Using Heart Rate Variability Analysis During Transient Heart Rate Reduction Caused by Acupuncture.

Objective: Human studies have demonstrated that heart rate (HR) decreases during acupuncture stimulation, and pharmacologic studies have shown that this autonomic nervous system (ANS) response is parasympathetic-dominant. It has become clear that significant changes occur in the ANS after acupuncture, based on HR variability (HRV). However, it is inconclusive, according to HRV analysis, if acupuncture induces a significant change in autonomic function during stimulation. The aim of this study was to investigate ANS function using HRV analysis during HR reduction induced by manual acupuncture stimulation to the muscles. Materials and Methods: In this study, electrocardiograms of 25 adult men were analyzed. After resting for 20 minutes, participants underwent 15-20-mm deep acupuncture stimulation at the Shousanli (LI 10) point at 1 Hz for 2 minutes. Instantaneous HR was recorded. The index of parasympathetic nervous activity high-frequency (HF) normalized units (HFnu) and the ratio of sympathovagal balance (low frequency [LF]/HF) were calculated by HRV analysis. Results: HR during acupuncture was significantly lower, compared to HR both before and after acupuncture. HFnu during acupuncture were significantly higher, compared to HFnu both before and after acupuncture. The LF/HF ratio during acupuncture was significantly lower, compared to the ratio before acupuncture, and remained low after acupuncture, compared to before acupuncture. Conclusions: Acupuncture stimulation to the muscle can effectively reduce HR, increase HFnu, and decrease LF/HF that depends on autonomic regulation of both sympathovagal balances.

Effects of electroacupuncture to the trigeminal nerve area on the autonomic nervous system and cerebral blood flow in the prefrontal cortex.

BACKGROUND: The autonomic nervous system and trigeminal nerve are involved in adjusting flow through diverging cerebral arteries in the prefrontal cortex. The purpose of this study was to examine the effect of 100 Hz electroacupuncture (EA) to the trigeminal nerve area on cerebral blood flow and autonomic nervous system function. METHOD: This was a randomised crossover study of 16 healthy volunteers who were assigned to an EA or control group. Stimulation (in the EA group) was performed five times, each after 1 min of rest. Needles were inserted at the inner edge of the eyebrows and 1 cm from the front hairline midpoint. We used high-frequency (HF) and low-frequency (LF) components of heart rate (HR) variability to assess autonomic nervous system function. HF and LF/HF ratio were taken as indicators of parasympathetic and sympathetic nervous system activity, respectively. We measured cerebral blood flow using a two-channel near-infrared spectroscope. RESULTS: In the EA group, HR significantly decreased (p=0.004) and HF significantly increased (p=0.006) relative to baseline. By contrast, there were no significant changes in HR or HF within the control group (p>0.05). Accordingly, HR tended to be lower (p=0.087) and HF greater (p=0.071) in the EA group versus the control group. There were no significant differences in LF/HF ratio within/between groups. Compared with the control group, cerebral blood flow was significantly greater in the left (p=0.048) and right (p=0.016) prefrontal cortex in the EA group. CONCLUSIONS: Delivery of 100 Hz EA to the trigeminal nerve area reduces HR and increases parasympathetic nervous activity and cerebral blood flow.

Effect of Enzyme-Treated Asparagus Extract (ETAS) on Psychological Stress in Healthy Individuals.

The aim of this study was to examine the effectiveness of Enzyme-Treated Asparagus Extract (ETAS) on improving stress response. A randomized, double-blind, placebo-controlled cross-over trial was undertaken in healthy volunteers. ETAS (150 mg/d) or a placebo was consumed for 28 d, with a washout period. Psychological parameters were examined using a self-report scale questionnaire and psychological stress was applied using the Uchida-Kraepelin (U-K) test. During the stress load, autonomic nervous function was analyzed. After the stress load, a profile of mood states (POMS) psychological rating was performed, and serum cortisol, plasma catecholamine, salivary secretory immunoglobulin A (sIgA), and salivary cortisol were analyzed. ETAS intake improved the self-reported rating for the items "Feel tired," "Hard to get up," and "Feel heavy" in the psychological questionnaire; ameliorated the self-reported rating for the items "Depression-Dejection" and "Fatigue" in the POMS questionnaire; and increased salivary sIgA levels after the U-K test. In contrast, serum and salivary cortisol levels, and plasma catecholamine did not change. During the U-K test, ETAS significantly upregulated the sympathetic nerve activity. Furthermore, ETAS intake significantly increased the number of answers and the number of correct answers in the U-K test, suggesting that it might improve office work performance with swiftness and accuracy under stressful conditions. In conclusion, ETAS supplementation reduced feelings of dysphoria and fatigue, ameliorated quality of sleep, and enhanced stress-load performance as well as promoted stress response by increasing salivary sIgA levels. These data suggest ETAS intake may exert beneficial effects, resulting from well-controlled stress management, in healthy individuals.

Effects of a Gentle, Self-Administered Stimulation of Perineal Skin for Nocturia in Elderly Women: A Randomized, Placebo-Controlled, Double-Blind Crossover Trial.

BACKGROUND: Somatic afferent nerve stimuli are used for treating an overactive bladder (OAB), a major cause of nocturia in the elderly. Clinical evidence for this treatment is insufficient because of the lack of appropriate control stimuli. Recent studies on anesthetized animals show that gentle stimuli applied to perineal skin with a roller could inhibit micturition contractions depending on the roller's surface material. We examined the efficacy of gentle skin stimuli for treating nocturia. METHODS: The study was a cross-over, placebo-controlled, double-blind randomized clinical study using two rollers with different effects on micturition contractions. Participants were elderly women (79-89 years) with nocturia. Active (soft elastomer roller) or placebo (hard polystyrene roller) stimuli were applied to perineal skin by participants for 1 min at bedtime. A 3-day baseline assessment period was followed by 3-day stimulation and 4-day resting periods, after which the participants were subjected to other stimuli for another 3 days. The primary outcome was change in the frequency of nighttime urination, for which charts were maintained during each 3-day period. RESULTS: Twenty-four participants were randomized, of which 22 completed all study protocols. One participant discontinued treatment because of an adverse event (abdominal discomfort). In participants with OAB (n = 9), change from baseline in the mean frequency of urination per night during the active stimuli period (mean ± standard deviation, -0.74 ± 0.7 times) was significantly greater than that during placebo stimuli periods (-0.15 ± 0.8 times [p < 0.05]). In contrast, this difference was not observed in participants without OAB (n = 13). CONCLUSIONS: These results suggest that gentle perineal stimulation with an elastomer roller is effective for treating OAB-associated nocturia in elderly women. Here the limitation was a study period too short to assess changes in the quality of sleep and life. TRIAL REGISTRATION: UMIN Clinical Trial Registry (CTR) UMIN000015809.

Morphological analysis of the relation between immunoglobulin A production in the small intestine and the enteric nervous system.

To investigate the relation between the enteric nervous system and immunocytes, fecal immunoglobulin (Ig) A and the distribution of IgA-positive cells close to nerve fibers in the lamina propria around the crypts of the small intestine were examined after intraperitoneal injection of lipopolysaccharides (LPS). In mice, IgA in the feces 1 h (LPS1) and 6 h (LPS6) after LPS injection increased compared to that in the control group. In the ileum lamina propria of the LPS1 group, the number of IgA-positive cells close to the epithelial basement membrane was increased. In the LPS6 group, on the other hand, there was a significant increase in the number of IgA-positive cells close to both the basement membrane and the nerve fibers. Our data suggest that LPS induced an increase in the number of IgA-positive cells migrating to the nerve fibers, and that migration to the nerve fibers is as important as migration to the basement membrane for IgA production.

Effect of Forefoot Strike on Lower Extremity Muscle Activity and Knee Joint Angle During Cutting in Female Team Handball Players.

BACKGROUND: The purpose of this study is to examine the effects of different strike forms, during cutting, on knee joint angle and lower limb muscle activity. METHODS: Surface electromyography was used to measure muscle activity in individuals performing cutting manoeuvres involving either rearfoot strikes (RFS) or forefoot strikes (FFS). Three-dimensional motion analysis was used to calculate changes in knee angles, during cutting, and to determine the relationship between muscle activity and knee joint angle. Force plates were synchronized with electromyography measurements to compare muscle activity immediately before and after foot strike. RESULTS: The valgus angle tends to be smaller during FFS cutting than during RFS cutting. Just prior to ground contact, biceps femoris, semitendinosus, and lateral head of the gastrocnemius muscle activities were significantly greater during FFS cutting than during RFS cutting; tibialis anterior muscle activity was greater during RFS cutting. Immediately after ground contact, biceps femoris and lateral head of the gastrocnemius muscle activities were significantly greater during FFS cutting than during RFS cutting; tibialis anterior muscle activity was significantly lower during FFS cutting. CONCLUSIONS: The results of the present study suggest that the hamstrings demonstrate greater activity, immediately after foot strike, during FFS cutting than during RFS cutting. Thus, FFS cutting may involve a lower risk of anterior cruciate ligament injury than does RFS cutting.

Monoaminergic and peptidergic axonal projections to the vagal motor cell column of a teleost, the filefish Stephanolepis cirrhifer.

In an immunohistochemical study, the vagal motor nucleus of a teleost, the filefish Stephanolepis cirrhifer, could be divided into a rostral part and a caudal part, and the former into a dorsolateral group and a ventromedial group. The dorsolateral group consisted of neurons immunoreactive for calcitonin gene-related peptide, whereas the ventrolateral-caudal group was negative for calcitonin gene-related peptide. The latter group was retrogradely labeled after dextran amine injection to the visceral ramus of the vagus nerve, suggesting that it is a general visceral efferent column, made up of parasympathetic preganglionic neurons, whereas the dorsolateral rostral group is a special visceral efferent column. In the general visceral efferent column, a dense concentration of nerve fibers immunoreactive for serotonin, tyrosine hydroxylase, cholecystokinin-8, and substance P, and a small number of fibers immunoreactive for neuropeptide Y was observed. Perikarya in contact with varicose terminals immunoreactive for these substances were frequently seen. In contrast, in the special visceral efferent column, only a moderate concentration of neuropeptide Y-immunoreactive nerve fibers and a sparse distribution of fibers immunoreactive for tyrosine hydroxylase were observed. Perikarya in contact with varicose terminals immunoreactive for these substances were rare. These results suggest that the vagal parasympathetic preganglionic neurons might receive multiple inputs of monoaminergic and peptidergic fibers involved in the regulation of the visceral organs. On the other hand, monoaminergic and peptidergic afferent fibers might be of much less significance in the activity of the special visceral efferent component of the vagus nerve.

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.

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.

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.