The effect of nerve activity on the distribution of synaptic vesicles

Certain gymnotid fish (apteronotids) continuously emit a high-frequency electric-organ discharge and thus continuously drive their electroreceptor afferents at high rates. Electroreceptor afferents terminate in one lamina of the electrosensory lateral line lobe (ELL) and can be readily sampled. Normally these terminals have many small vesicles clustered adjacent to the presynaptic membrane. When afferent activity is blocked for 24 hr by an injection of tetrodotoxin (TTX) into the electroreceptor nerve, the density of vesicles adjacent to the synaptic membrane declines; the volume of the remaining vesicles increases. If the nerve of a TTX-treated fish is stimulated proximal to the injection site, these changes can be reversed. These results imply that the migration of vesicles toward the presynaptic membrane is influenced by the level of activity in the nerve.     

Evidence for transmitter operated electrical synapses (TOES) in ampullary electroreceptor organs

Afferent fibres of ampullary electroreceptor organs in electrosensitive fish fire spontaneously, that is, they fire without external stimulus. In the past it has been postulated that the spontaneous activity originates from a sustained level of neurotransmitter release delivered by the electroreceptor cells. The spontaneous activity can be modulated by electrical stimuli. Blocking of the chemical synapse, however, reduces the susceptibility to electrical stimuli to 2% or less, but the spontaneous activity to 60% only. By evaluating existing experimental evidence it is concluded that spontaneous firing of afferents is based on two processes. (1) A membrane bound oscillator, which does not depend on transmitter release, is almost free of frequency fluctuations, and is described by Hodgkin/Huxley-equations (HH-equations). (2) Release of neurotransmitter, which increases the firing level, adds frequency noise, and raises the susceptibility of the afferent to electrical stimuli. There is evidence that neurotransmitter release acts as a gating process, which makes the generator area of the afferents directly accessible to electrical stimuli from the outside. Apparently, the activated synapse behaves as a transmitter operated electrical synapse (TOES).     

End buds: non-ampullary electroreceptors in adult lampreys

Summary Shared anatomical and physiological characters indicate that the low-frequency sensitive electrosensory system of lampreys is homologous with those of non-teleost fishes and amphibians. However, the ampullary electroreceptor organs which characterize all of these gnathostomes are not found in lampreys. Experimental anatomical and physiological studies reported here demonstrate that the epidermal end buds are the electroreceptors of adult lampreys.End buds, consisting of both sensory and supporting cells, are goblet-shaped with the top (25–60 m diameter) at the epidermal surface and the stem directed toward the dermis (Fig. 1A). Short lines or clusters of 2–8 end buds (Fig. 1B) are distributed over both trunk and head. Injections of horseradish peroxidase (HRP) into vitally-stained end buds labeled anterior lateral line afferents terminating in the ipsilateral dorsal nucleus (Fig. 2A) — the primary electrosensory nucleus of the lamprey medulla. Conversely, after HRP injection into the dorsal nucleus HRP-filled fibers and terminals were present on ipsilateral end buds (Fig. 2B).End buds are usually not visible without staining. However, in adult sea lampreys the presence of end buds was histologically confirmed in skin patches containing the receptive fields of electroreceptor fibers recorded in the anterior lateral line nerve. Additionally, in the rare instance of two silver lampreys in which end buds were visible without staining, electrosensory activity indistinguishable from that of the primary electroreceptor afferents was recorded from the end bud surface (Figs. 3, 4).End buds were initially characterized as chemoreceptors (Johnston 1902) but were later correctly advanced as lateralis receptors based on the presence of presynaptic dense bodies in the receptor cells (Whitear and Lane 1981). Unlike all other low-frequency electroreceptors, end buds lack canals. The receptor cells contact the epidermal surface and possess apical microvilli rather than the kinocilium of most gnathostomes with homologous electrosensory systems of the primitive (non-teleost) type.Larval lampreys and newly transformed adults lack end buds although at least the latter are electroreceptive. End buds, therefore, may be the form taken by electroreceptors only in the final portion of a lamprey's life.     

RAPID DEGENERATION OF AMPULLARY ELECTRORECEPTOR ORGANS AFTER DENERVATION

Electroreceptors (ampullary organs) of the transparent catfish (Kryptopterus bicirrhus) lie in the epidermis, and contain spherical receptor cells that receive purely afferent innervation from the lateral line nerve. Section of this nerve causes rapid degenerative changes to occur in the receptors. Fine structural alterations occur in the receptor cell synapses and nerve fiber 6–12 h postoperatively. Disruption of the receptor cells begins by 18 h and most are lost by 48 h. By 72 h supporting cells and secretory cells also show marked degeneration, and by 96 h they may be totally lost. The rapid degeneration of the electroreceptor organs of Kryptopterus should make them a useful preparation for analysis of neurotrophic functions.     

Simultaneous measurements of calcium mobilization and afferent nerve activity in electroreceptor organs of anesthetized Kryptopterus bicirrhis

The transduction pathway of ampullary electroreceptor organs involves ionic currents. It has been shown that calcium, as well as sodium and potassium play important parts in this process. In this study we examine the stimulus-evoked changes in the Fura-2 ratio in electroreceptor cells. Furthermore, we recorded stimulus-evoked Fura-2 ratio changes while Na+ and K+ channels were blocked by amiloride and TEA. Simultaneously, extracellular recordings of the afferent spike activity were made. The results show the presence of stimulus evoked fluctuations in the Fura-2 ratio. These fluctuations can be abolished by the application of Cd2+, TEA, and amiloride. The stimulus-evoked activity of the afferent nerve was decreased due to application of these drugs. We conclude that the transduction current is carried by Na+, K+, and probably Ca2+. This fits the existing model on transduction in electroreceptors.     

The development of the hindbrain afferent projections in the axolotl: evidence for timing as a specific mechanism of afferent fiber sorting

The aim of this study is to reveal the timing and growth pattern of central octavolateral projection development in the Mexican axolotl, Ambystoma mexicanum. In this amphibian species the development of the inner ear occurs first, followed by mechanosensory lateral line organs, and finally by ampullary electroreceptors. Several hypotheses have been proposed about how the development of peripheral organs, including differential projections of the ear, might relate to the development of central projections. Our data suggest that the sequence of maturation of the ear, mechanosensory lateral line, and ampullary electroreceptive organs is closely accompanied by the timed development of the trigeminal, inner ear, mechanosensory lateral line organs, and the ampullary electroreceptor afferent projections in the axolotl. Our data suggest that segregation of central termination within the alar plate is a function of time and space: later forming organs are likely innervated by later forming ganglia that project centrally later and to more dorsal areas of the alar plate that have not yet received any other afferents. Later forming ganglia of the same type may grow along existing pathways of earlier formed neurons.     

Microvilli in electroreceptor organs in Ictalurus nebulosus play a part in signal filtering

Ampullary electroreceptor organs of catfish show a band-pass-filter characteristic on sinusoidal electric stimulation. The structures and processes which are responsible for the frequency characteristics are not fully understood. To investigate the role of the apical membrane and its microvilli in signal filtering, the ampullary organs were apically exposed to the actin filament disrupting agent cytochalasin B. Electrophysiological data showed that cytochalasin B treatment reduced the absolute sensitivity to about 20% over the whole frequency range. The decrease in sensitivity at 20 Hz, however, was less than at other frequencies. The phase lags at 14 and 20 Hz became less negative, indicating a relatively better transduction at high frequencies. Calculations with an electric equivalent circuit of an electroreceptor cell indicated that a reduction in apical surface area in combination with a reduction of the number or the conductivity of apical ion channels can explain such effects. We conclude that, although only the basal membrane is thought to be involved in stimulus transduction, the apical membrane contributes considerably to the frequency characteristics of ampullary electroreceptor organs.     

Resistance of the European catfish (Silurus glanis) to channel catfish virus

European catfish (Silurus glanis) fingerlings (2 to 4 g each) were tested for susceptibility to channel catfish virus (CCV). They had supported CCV replication at 2 days after intraperitoneal injection with 0.1 ml of saline containing 10⁵ TCID₅₀. Homogenized visceral organs (liver, kidney and spleen) contained 10⁴ TCID₅₀/0.1 ml at 2 days post inoculation (PI) but at 4 days the titer decreased to 10¹ TCID₅₀. Bathing European catfish in CCV yielded only one positive sample with à titer of 10^0.83 TCID₅₀ per 0.1 ml of tissue. No clinical signs of CCV developed and no virus related deaths occurred.     

Reversible,long-term inhibition of ovulation with a gonadotropin-releasing hormone antagonist in the marmoset monkey (Callithrix jacchus)

The effects of weekly injections of a gonadotropin-releasing hormone (GnRH) antagonist (GnRHa) ([N-acetyl-DβNal1-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8D-Ala10] NH2 GnRH) on pituitary and ovarian function were examined in the marmoset monkey, Callithrix jacchus. In experiment 1, five cyclic females were given weekly injections of vehicle (50% propylene glycol in saline) for 6 weeks followed by GnRHa for 20 weeks, animals receiving either 200 μg GnRHa/injection (n = 2) or 67 μg GnRHa/injection (n = 3) for 10 weeks, after which the treatment was reversed. Bioactive luteinizing hormone (LH) and progesterone (Po) were measured in blood samples (0.2–0.4 ml) collected twice weekly until at least 8 weeks after the last GnRHa injection. GnRHa treatment, timed to begin in the midluteal phase, caused a rapid decline in LH and Po and luteal regression after a single injection (both doses). Po levels were consistently low (<10 ng/ml), and ovulation was inhibited throughout 200 μg treatment in all animals. Short periods of elevated Po (>10 ng/ml) were, however, occasionally seen during 67 μg treatment, indicating incomplete ovarian suppression. Mean LH levels were significantly lower during GnRHa treatment compared with the period of vehicle injection (all animals 200 μg; three animals 67 μg), and there were significant differences in LH levels between GnRHa treatments (200 μg vs. 67 μg) in four animals. Four animals resumed normal ovarian cycles after the end of GnRHa treatment (15/16 days, three animals; 59 days, one animal); the fifth animal died of unknown causes 32 days after the last GnRHa injection. In a second experiment, pituitary responsiveness to exogenous GnRH was tested 1 day after a single injection of vehicle or antagonist (200 or 67 μg). Measurement of bioactive LH indicated that pituitary response to 200 ng native GnRH was significantly suppressed in animals receiving the antagonist, the degree of suppression being dose related. A third experiment examined the effect of four weekly injections of 200 μg GnRHa on follicular size and granulosa cell responsiveness to human follicle-stimulating hormone (hFSH) in vitro. Follicular development beyond 1 mm was inhibited by GnRHa treatment (preovulatory follicles normally 2-4 mm) although granulosa cell responsiveness to FSH during 48 hr of culture was not impaired. These results suggest that the GnRHa-induced suppression of follicular development and ovulation was mediated primarily by an inhibition of pituitary gonadotropin secretion and not by a direct action at the level of the ovary.     

Fate of intraperitoneally injected fluorescent microspheres in developing Ictalurus punctatus

Fluorescent microspheres (FMS) were injected intraperitoneally into channel catfish fry at 2 days post hatch (dph), 1, 2, 3, 4 and 8 weeks post hatch (wph). The FMS were observed in the vasculature almost immediately after injection in all age groups except 2 dph. Fluorescent microspheres were observed within mononuclear phagocytes in the vasculature after 0.16 dph in all age groups. Fluorescent microspheres were first phagocytized in the coelomic cavity immediately after injection, while the majority of coelomic FMS were phagocytized between 0.16 and 1 dph for all ages. Enzyme cytochemical staining indicated that both polymorphonuclear (neutrophilic granulocytes) and mononuclear phagocytes had phagocytized FMS in the coelomic cavity and organs, with a predominance of FMS found in mononuclear phagocytic cells in all age groups across all sample periods. The predominant organs associated with the observed cellular responses were the posterior kidney, spleen, and anterior kidney. Splenic organization and melanomacrophage development and activity were more pronounced as the fish aged from 2 wph on. Particulate clearance rates were faster in the 2 dph and 1 wph fish than the older ages of fish. These results suggest that to facilitate particulate retention, channel catfish should be vaccinated at 4 wph or older.     

Afferent proprioreceptive fibers in the rat and their distribution in the dorsal roots

The distribution in the dorsal roots of proprioceptive afferent fibres from tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of the rat and the physiological characteristics of the related nervous endings have been investigated. Axons of proprioceptive endings from TA and EDL were found mainly in L4, only a few in L5. Afferent proprioceptive fibres from posterior superficial crural muscles (gastrocnemius, soleus, plantaris) pass mainly through root L5; axons of extrafusal motor units are distributed in a similar way. Sensory endings in TA and EDL were examined, after identification, by means of their static threshold to passive stretch. Almost all steady-state responses to passive stretch, within the physiological extension range, came from muscle spindles. 1-2 to 20 g loads were necessary to obtain steady-state discharges from these receptors. Spindle endings were classified as primary or secondary by measuring the conduction velocity of the afferent fibres, and according to the features of their passive behaviour. Threshold difference cannot be regarded as a fundamental characteristic, because of the considerable overlapping of the values obtained from the two types of endings. Conduction velocities of 50 to 80 m/sec for primary and of 20 to 40 m/sec for secondary afferent fibres were observed. Afferent fibres conducting at intermediate velocity often behave like primary ones. As a rule, tendon organs showed a higher static threshold to passive stretch; the loads employed only rarely elicited a steady-state response. As for these receptors, which usually showed marked adaptation characteristics, passive force is a less effective stimulus than active contraction. The conduction velocity range of afferent fibres from tendon organs is the same as that of primary afferents. The results are discussed.     

Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying

Intestinal perfusion with carbohydrates inhibits gastric emptying via vagal and spinal capsaicin-sensitive afferent pathways. The aim of the present study was to determine the role of 1) 5-hydroxytryptamine (5-HT)(3) receptors (5-HT(3)R) in mediating glucose-induced inhibition of gastric emptying and 2) 5-HT(3)R expression in vagal and spinal afferents in innervating the duodenum. In awake rats fitted with gastric and duodenal cannulas, perfusion of the duodenum with glucose (50 and 100 mg) inhibited gastric emptying. Intestinal perfusion of mannitol inhibited gastric emptying only at the highest concentration (990 mosm/kgH(2)O). Pretreatment with the 5-HT(3)R antagonist tropisetron abolished both glucose- and mannitol-induced inhibition of gastric emptying. Retrograde labeling of visceral afferents by injection of dextran-conjugated Texas Red into the duodenal wall was used to identify extrinsic primary afferents. Immunoreactivity for 5-HT(3)R, visualized with an antibody directed to the COOH terminus of the rat 5-HT(3)R, was found in >80% of duodenal vagal and spinal afferents. These results show that duodenal extrinsic afferents express 5-HT(3)R and that the receptor mediates specific glucose-induced inhibition of gastric emptying. These findings support the hypothesis that enterochromaffin cells in the intestinal mucosa release 5-HT in response to glucose, which activates 5-HT(3)R on afferent nerve terminals to evoke reflex changes in gastric motility. The primary glucose sensors of the intestine may be mucosal enterochromaffin cells.     

Identification of Different Types of Spinal Afferent Nerve Endings That Encode Noxious and Innocuous Stimuli in the Large Intestine Using a Novel Anterograde Tracing Technique

In mammals, sensory stimuli in visceral organs, including those that underlie pain perception, are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRG). One of the major challenges in visceral organs has been how to identify the different types of nerve endings of spinal afferents that transduce sensory stimuli into action potentials. The reason why spinal afferent nerve endings have been so challenging to identify is because no techniques have been available, until now, that can selectively label only spinal afferents, in high resolution. We have utilized an anterograde tracing technique, recently developed in our laboratory, which facilitates selective labeling of only spinal afferent axons and their nerve endings in visceral organs. Mice were anesthetized, lumbosacral DRGs surgically exposed, then injected with dextran-amine. Seven days post-surgery, the large intestine was removed. The characteristics of thirteen types of spinal afferent nerve endings were identified in detail. The greatest proportion of nerve endings was in submucosa (32%), circular muscle (25%) and myenteric ganglia (22%). Two morphologically distinct classes innervated myenteric ganglia. These were most commonly a novel class of intraganglionic varicose endings (IGVEs) and occasionally rectal intraganglionic laminar endings (rIGLEs). Three distinct classes of varicose nerve endings were found to innervate the submucosa and circular muscle, while one class innervated internodal strands, blood vessels, crypts of lieberkuhn, the mucosa and the longitudinal muscle. Distinct populations of sensory endings were CGRP-positive. We present the first complete characterization of the different types of spinal afferent nerve endings in a mammalian visceral organ. The findings reveal an unexpectedly complex array of different types of primary afferent endings that innervate specific layers of the large intestine. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the large intestine.     

The antimicrobial peptide, epinecidin-1, mediates secretion of cytokines in the immune response to bacterial infection in mice

Epinecidin-1, an antimicrobial peptide which encodes 21 amino acids, was isolated from a marine grouper (Epinephelus coioides). In this study, we investigated its immunomodulatory functions in mice co-injected with Pseudomonas aeruginosa. In vivo results showed that the synthetic epinecidin-1 peptide induced significant secretion of immunoglobulin G1 (IgG1) in mice co-injected with P. aeruginosa. Moreover, after injection of 40, 100, 200, or 500 μg epinecidin-1/mouse, we detected IgM, IgG, IgG1, and IgG2a in mice treated for 1, 2, 3, 7, 14, 21, and 28 days. Results showed that there were no significant differences in IgM, IgG, or IgG2a between mice injected with epinecidin-1 alone. IgG1 increased to a peak at 24 h, 7 days, and 28 days after an epinecidin-1 (40 μg/mouse) injection. Injection of 500 μg epinecidin-1/mouse increased IgG1 to peaks at 2 and 3 days; injection of 100 μg epinecidin-1/mouse increased IgG1 to a peak at 21 days. This supports epinecidin-1 being able to activate the Th2 cell response (enhance IgG1 production) against P. aeruginosa infection. Treatment with different concentrations of epinecidin-1 in mice elevated plasma interleukin (IL)-10 to initial peaks at 24 and 48 h, and it showed a second peak at 16 days. In RAW264.7 cells, treatment with epinecidin-1 alone did not produce significant changes in tumor necrosis factor (TNF)-α protein secretion at 1, 6, or 24h after treatment with 3.75, 7.5, or 15 μg/ml epinecidin-1 compared to the lipopolysaccharide group.     

Peripheral electrosense physiology: a review of recent findings.

Advances, since 1974, in understanding the physiology of electroreceptors are reviewed. In brief: 1. In fish that produce a weak electric discharge with electric organs, the tuberous electroreceptors are generally most sensitive to stimulus frequencies near the species', individual's, and even local, waveform of the electric organ discharge; there is a good match between receptor sensitivity and the normal stimulus. 2. The ability of tuberous electroreceptors to detect field distortions produced by reasonably sized objects is limited; an object must be closer than a body-length to be detected, and the afferent response is a negative power function of object distance. 3. The second major electroreceptor class, the ampullary electroreceptors, is sensitive to low frequency, low intensity electric fields, and this acute sensitivity results in the ability of the receptors in marine species to detect magnetic fields on the order of the Earth's. 4. The calcium ion is essential for normal functioning of ampullary electroreceptors.     

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.     

Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis

Background

Recent data have suggested a relationship between acute arthritic pain and acid sensing ion channel 3 (ASIC3) on primary afferent fibers innervating joints. The purpose of this study was to clarify the role of ASIC3 in a rat model of osteoarthritis (OA) which is considered a degenerative rather than an inflammatory disease.

Methods

We induced OA via intra-articular mono-iodoacetate (MIA) injection, and evaluated pain-related behaviors including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test, histology of affected knee joint, and immunohistochemistry of knee joint afferents. We also assessed the effect of ASIC3 selective peptide blocker (APETx2) on pain behavior, disease progression, and ASIC3 expression in knee joint afferents.

Results

OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase.

Conclusions

Local ASIC3 immunoreactive nerve is strongly associated with weight-bearing pain and secondary hyperalgesia in MIA-induced OA model. APETx2 inhibited ASIC3 upregulation in knee joint afferents regardless of the time-point of administration. Furthermore, early administration of APETx2 prevented cartilage damage. APETx2 is a novel, promising drug for OA by relieving pain and inhibiting disease progression.     

Evidence for a direct effect of androgens upon electroreceptor tuning

Tuberous electroreceptors of individual wave type weakly electric fish are tuned to the fundamental frequency of that fish's electric organ discharge (EOD). EOD frequency and receptor best frequency (BF) are both lowered following systemic injection of 5-alpha-dihydrotestosterone (DHT). A previous study (Meyer et al. 1984) showed that the effect of DHT on the EOD generating circuitry was independent of an ongoing EOD and suggested that its effect on electroreceptor tuning was indirect, possibly mediated by the electric field. We have continued these studies to determine the factors which influence electroreceptor tuning. Baseline recordings of EOD frequency, receptor oscillations, and single afferent tuning curves were taken. After fish were electrically silenced by spinal cord transection they were injected daily with either DHT or saline or were implanted with either DHT-filled or empty silastic capsules. As previously reported, the EOD frequency (determined from pacemaker nucleus recordings) was lowered in DHT-treated, transected fish and increased in control fish. Similarly, receptor tuning was lowered in the DHT-treated, silenced fish. Oscillation frequencies decreased in both treated and control groups, but significantly more in the hormone group. Single afferent best frequencies were lowered in both DHT groups and raised in their respective control groups. In another series of experiments exogenous electric fields capable of driving receptors in a 1-to-1 phase-locked manner were placed around silenced fish. We were unable to elicit any shift in pacemaker frequency or electroreceptor tuning regardless of stimulus field geometry. Four transected fish were injected with DHT and placed in exogenous electric fields of higher frequency than their original EOD. Even in the presence of a higher frequency electric field, DHT lowered EOD frequency and afferent BF. We conclude that androgens produce effects both on the EOD generating circuitry, probably at the level of the pacemaker nucleus, and on electroreceptors, probably, ultimately, on receptor cell membrane conductances. These effects occur in parallel allowing the two parameters to remain well matched. In contrast to former predictions, exogenous electric fields alone appear unable to shift receptor tuning.     

TRPV1 marks synaptic segregation of multiple convergent afferents at the rat medial solitary tract nucleus

TRPV1 receptors are expressed on most but not all central terminals of cranial visceral afferents in the caudal solitary tract nucleus (NTS). TRPV1 is associated with unmyelinated C-fiber afferents. Both TRPV1+ and TRPV1- afferents enter NTS but their precise organization remains poorly understood. In horizontal brainstem slices, we activated solitary tract (ST) afferents and recorded ST-evoked glutamatergic excitatory synaptic currents (ST-EPSCs) under whole cell voltage clamp conditions from neurons of the medial subnucleus. Electrical shocks to the ST produced fixed latency EPSCs (jitter<200 μs) that identified direct ST afferent innervation. Graded increases in shock intensity often recruited more than one ST afferent and ST-EPSCs had consistent threshold intensity, latency to onset, and unique EPSC waveforms that characterized each unitary ST afferent contact. The TRPV1 agonist capsaicin (100 nM) blocked the evoked TRPV1+ ST-EPSCs and defined them as either TRPV1+ or TRPV1- inputs. No partial responses to capsaicin were observed so that in NTS neurons that received one or multiple (2-5) direct ST afferent inputs--all were either blocked by capsaicin or were unaltered. Since TRPV1 mediates asynchronous release following TRPV1+ ST-evoked EPSCs, we likewise found that recruiting more than one ST afferent further augmented the asynchronous response and was eliminated by capsaicin. Thus, TRPV1+ and TRPV1- afferents are completely segregated to separate NTS neurons. As a result, the TRPV1 receptor augments glutamate release only within unmyelinated afferent pathways in caudal medial NTS and our work indicates a complete separation of C-type from A-type afferent information at these first central neurons.     

The Mast Cell Degranulator Compound 48/80 Directly Activates Neurons

Background

Compound 48/80 is widely used in animal and tissue models as a “selective” mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents.

Methodology/Principal Findings

We used in vivo recordings from extrinsic intestinal afferents together with Ca⁺⁺ imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca⁺⁺ and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca⁺⁺ transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H₁ and H₂ antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca⁺⁺ transients in mast cell-free enteric neuron cultures.

Conclusions/Significance

The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn.