Opposing effects of bronchopulmonary C-fiber subtypes on cough in guinea pigs.

We have addressed the hypothesis that the opposing effects of bronchopulmonary C-fiber activation on cough are attributable to the activation of C-fiber subtypes. Coughing was evoked in anesthetized guinea pigs by citric acid (0.001-2 M) applied topically in 100-µl aliquots to the tracheal mucosa. In control preparations, citric acid evoked 10 ± 1 coughs cumulatively. Selective activation of the pulmonary C fibers arising from the nodose ganglia with either aerosols or continuous intravenous infusion of adenosine or the 5-HT3 receptor-selective agonist 2-methyl-5-HT nearly abolished coughing evoked subsequently by topical citric acid challenge. Delivering adenosine or 2-methyl-5-HT directly to the tracheal mucosa (where few if any nodose C fibers terminate) was without effect on citric acid-evoked cough. These actions of pulmonary administration of adenosine and 2-methyl-5-HT were accompanied by an increase in respiratory rate, but it is unlikely that the change in respiratory pattern caused the decrease in coughing, as the rapidly adapting receptor stimulant histamine also produced a marked tachypnea but was without effect on cough. In awake guinea pigs, adenosine failed to evoke coughing but reduced coughing induced by the nonselective C-fiber stimulant capsaicin. We conclude that bronchopulmonary C-fiber subtypes in guinea pigs have opposing effects on cough, with airway C fibers arising from the jugular ganglia initiating and/or sensitizing the cough reflex and the intrapulmonary C fibers arising from the nodose ganglia actively inhibiting cough upon activation.

Distribution pattern of dorsal root ganglion neurons synthesizing nitric oxide synthase in different animal species.

The main aim of the present review is to provide at first a short survey of the basic anatomical description of sensory ganglion neurons in relation to cell size, conduction velocity, thickness of myelin sheath, and functional classification of their processes. In addition, we have focused on discussing current knowledge about the distribution pattern of neuronal nitric oxide synthase containing sensory neurons especially in the dorsal root ganglia in different animal species; hence, there is a large controversy in relation to interpretation of the results dealing with this interesting field of research.

Mice and rats differ with respect to activity-dependent slowing of conduction velocity in the saphenous peripheral nerve.

We assess in mice, the electrophysiological criteria developed in humans and rats in vivo for unmyelinated (C) fibre differentiation into sub-classes, derived from the activity-induced latency increase ("slowing") in response to electrical stimulation during 6 min at 0.25 Hz followed by 3 min at 2 Hz. Fibres are considered nociceptors if they show more than 10% slowing at 2 Hz; nociceptors are further divided into mechanosensitive ("polymodal") and mechanoinsensitive ("silent") ones according to a latency shift of less and more than 1% during the first minute at 0.25 Hz, respectively. Sympathetic postganglionics are recognised by 2-10% slowing at 2 Hz; units slowing less than 2% at 2 Hz remain uncategorised. For assessment of these criteria, we also developed a novel in vivo technique for recording of peripheral single-fibres in the mouse. We compared the theoretical slowing-rate discriminator criteria with experimental data obtained from mice in vivo/in vitro and rats in vitro. Out of 69 cutaneous mouse C-fibres in vitro and 19 in vivo, only 38 (67%) and 9 (47%) met the above 1% criterion, respectively; sympathetics were not identified. In contrast, out of 20 rats nerve fibres in vitro, 19 (95%) met this criterion. We conclude that (A) our novel electrophysiological technique is a practical method for examining mouse cutaneous single-fibres in vivo and (B) the published criterion for identifying silent nociceptors in rats and humans is not applicable in mice.

Development and validation of the QUT corneal nerve grading scale.

PURPOSE: The aim of this study was to develop and validate a grading scale to facilitate the estimation of the amount of nerve tissue in images of the corneal subbasal nerve plexus captured using in vivo laser scanning corneal confocal microscopy (LSCCM). METHODS: Images of the corneal subbasal nerve plexus obtained using a Heidelberg LSCCM were sourced from a large image bank at the Queensland University of Technology (QUT). These images were used to construct a grading scale for depicting the amount of nerve tissue, ranging from 0 (sparse) to 4 (extensive). Twenty-five observers graded 20 images of a known corneal nerve fiber length (defined as the total length of nerves per unit area) on 2 occasions, at least 2 weeks apart. An equivalent calculated grade was determined for each test image from known values of corneal nerve fiber length. RESULTS: The intraclass correlation coefficient for repeat gradings was 0.88 (P < 0.001). Intraobserver and interobserver repeatabilities were unrelated to the calculated grade (P = 0.467 and P = 0.530, respectively). Grading can be performed with average 95% confidence limits of ±1.2 grading units. Overall grading estimates did not differ between observers (P = 0.998). There was a strong agreement between the estimated and calculated grades (intraclass correlation coefficient = 0.92, P < 0.001). CONCLUSIONS: We have developed the QUT Corneal Nerve Grading Scale, which is demonstrated to be repeatable, reliable, precise, and accurate. This tool provides clinicians and researchers with a simple and convenient pictorial reference for assessing, comparing, and monitoring the corneal subbasal nerve plexus with reference to LSCCM images.

Differential effects of low dose lidocaine on C-fiber classes in humans.

UNLABELLED: The nonselective sodium channel blocker lidocaine is widely used as a local anesthetic but also systemically for treatment of postoperative and neuropathic pain. Voltage-gated sodium channels are crucial for action potential generation and conduction, and their availability controls the amount of activity-dependent conduction velocity slowing. This important axonal property, as assessed by microneurography, is used to differentiate human mechanoinsensitive (silent) nociceptors from the classical polymodal nociceptors. In the current study, microneurography was used to assess axonal properties of the 2 main nociceptor classes in humans, before and after intradermal injection of lidocaine .1% or control saline solution in the receptive field. In mechanosensitive nociceptors, lidocaine reduced baseline conduction velocity and turned activity-dependent slowing into speeding of conduction. In contrast, mechanoinsensitive fibers were not affected in their baseline conduction velocity or their activity-dependent slowing, but probability of conduction block with repetitive stimulation increased. Recovery cycles showed reduced hyperpolarization in all C-fiber classes after lidocaine injections. These results support our hypothesis that sodium channel subtypes are differentially expressed in the 2 nociceptor classes of mechanosensitive C-fibers (CMs) and mechanoinsensitive C-fibers (CMis). PERSPECTIVE: This study reveals that microneurography can be used to assess pharmacological effects on single C-fibers directly in humans.

Specific paucity of unmyelinated C-fibers in cutaneous peripheral nerves of the African naked-mole rat: comparative analysis using six species of Bathyergidae.

In mammalian peripheral nerves, unmyelinated C-fibers usually outnumber myelinated A-fibers. By using transmission electron microscopy, we recently showed that the saphenous nerve of the naked mole-rat (Heterocephalus glaber) has a C-fiber deficit manifested as a substantially lower C:A-fiber ratio compared with other mammals. Here we determined the uniqueness of this C-fiber deficit by performing a quantitative anatomical analysis of several peripheral nerves in five further members of the Bathyergidae mole-rat family: silvery (Heliophobius argenteocinereus), giant (Fukomys mechowii), Damaraland (Fukomys damarensis), Mashona (Fukomys darlingi), and Natal (Cryptomys hottentotus natalensis) mole-rats. In the largely cutaneous saphenous and sural nerves, the naked mole-rat had the lowest C:A-fiber ratio (∼1.5:1 compared with ∼3:1), whereas, in nerves innervating both skin and muscle (common peroneal and tibial) or just muscle (lateral/medial gastrocnemius), this pattern was mostly absent. We asked whether lack of hair follicles alone accounts for the C-fiber paucity by using as a model a mouse that loses virtually all its hair as a consequence of conditional deletion of the ß-catenin gene in the skin. These ß-catenin loss-of function mice (ß-cat LOF mice) displayed only a mild decrease in C:A-fiber ratio compared with wild-type mice (4.42 compared with 3.81). We suggest that the selective cutaneous C-fiber deficit in the cutaneous nerves of naked mole-rats is unlikely to be due primarily to lack of skin hair follicles. Possible mechanisms contributing to this unique peripheral nerve anatomy are discussed.

Why do axons differ in caliber?

CNS axons differ in diameter (d) by nearly 100-fold (∼0.1-10 µm); therefore, they differ in cross-sectional area (d(2)) and volume by nearly 10,000-fold. If, as found for optic nerve, mitochondrial volume fraction is constant with axon diameter, energy capacity would rise with axon volume, also as d(2). We asked, given constraints on space and energy, what functional requirements set an axon's diameter? Surveying 16 fiber groups spanning nearly the full range of diameters in five species (guinea pig, rat, monkey, locust, octopus), we found the following: (1) thin axons are most numerous; (2) mean firing frequencies, estimated for nine of the identified axon classes, are low for thin fibers and high for thick ones, ranging from ∼1 to >100 Hz; (3) a tract's distribution of fiber diameters, whether narrow or broad, and whether symmetric or skewed, reflects heterogeneity of information rates conveyed by its individual fibers; and (4) mitochondrial volume/axon length rises ≥d(2). To explain the pressure toward thin diameters, we note an established law of diminishing returns: an axon, to double its information rate, must more than double its firing rate. Since diameter is apparently linear with firing rate, doubling information rate would more than quadruple an axon's volume and energy use. Thicker axons may be needed to encode features that cannot be efficiently decoded if their information is spread over several low-rate channels. Thus, information rate may be the main variable that sets axon caliber, with axons constrained to deliver information at the lowest acceptable rate.

Urodynamic effects of the bladder C-fiber afferent activity modulation in chronic model of overactive bladder in rats.

Previous studies have suggested that, different types of unmyelinated bladder afferent C-fibres, such as capsaicin-sensitive and capsaicin-resistant mediate the voiding reflex in overactive bladder (OAB). Considering its polymodal features, we explored the urodynamic effect of primary afferent neurons modulation on detrusor activity in normal and OAB rats. Experiments were performed on 48 female rats. OAB was induced by intraperitoneal administration of cyclophosphamide. All the surgical procedures and urodynamic studies were performed under urethane anaesthesia. Cystometry was done after a 1 h recovery period from the surgical procedure. All animals were randomly divided into six groups: control, chronic OAB, chronic OAB after capsaicin or lidocaine instillation, control capsaicin or lidocaine instillation. The measurements represent the average of five bladder micturition cycles. We analyzed: basal, threshold, micturition voiding pressure; intercontraction interval; compliance; functional bladder capacity; motility index; detrusor overactivity index. We used chronic cyclophosphamide OAB model for further investigations. In healthy rats, intravesical instillation of capsaicin caused complete inhibition of detrusor contractility preventing from proper voiding function of the bladder. Contrary, lidocaine has no influence on micturition cycles in intact animals. Also, intravesical instillation of capsaicin and lidocaine reduced the severity of detrusor overactivity of OAB rats leading to improvement of cystometric parameters.

Microneurographic assessment of C-fibre function in aged healthy subjects.

Physiological changes in the nervous system occur with ageing. Both a decline of function and a decrease in the number of C-fibres in the skin have been reported for healthy aged subjects. With the use of microneurographic recordings from single C-fibres in humans we have compared the sensory and axonal properties of these neurones in young and aged healthy subjects. A total of 146 C-fibres were recorded from the common peroneal nerve in young subjects (mean age 24.7 years) and 230 C-fibres were recorded in aged subjects (mean age 56.2 years). In aged subjects, changes were found in the composition of the C-fibre population and in sensory and axonal properties. The relative incidence of afferent to efferent C-fibres was relatively constant independent of the age of subjects. The ratio of mechano-responsive to mechano-insensitive nociceptors was approximately 8 : 2 in the young controls while in aged subjects it was 7 : 3. In aged subjects 13% of the fibres showed atypical discharge characteristics, while this was not observed in young subjects. Spontaneous activity, sensitization and loss of sensory function were found regularly. Changes in functions of the conductile membrane were also observed in fibres from aged subjects. The degree of activity-dependent conduction velocity slowing in response to high frequency stimulation (2 Hz) was more pronounced, while the normalization of conduction velocity subsequent to high frequency stimulation was protracted. We found that both sensitization and desensitization or degeneration of afferent C-fibres occur with age, but are still rare compared to patients with neuropathy. The changes in the axonal properties of C-fibres in aged subjects are compatible with hypoexcitability of the fibres. These findings are important for the understanding and differential diagnoses regarding pathological processes and normal ageing.

Separate peripheral pathways for pruritus in man.

Recent findings suggest that itch produced by intradermal insertion of cowhage spicules in human is histamine independent. Neuronal mechanisms underlying nonhistaminergic itch are poorly understood. To investigate which nerve fibers mediate cowhage induced itch in man, action potentials were recorded from cutaneous C-fibers of the peroneal nerve in healthy volunteers using microneurography. Mechano-responsive and -insensitive C-nociceptors were tested for their responsiveness to cowhage spicules, histamine, and capsaicin. Cowhage spicules induced itching and activated all tested mechano-responsive C-units (24/24), but no mechano-insensitive C-fibers (0/17). Histamine also induced itch, but in contrast to cowhage, it caused lasting activation only in mechano-insensitive units (8/12). In mechano-responsive C-units, histamine caused no or only short and weak responses unrelated to the time course of itching. Capsaicin injections activated four of six mechano-responsive fibers and three of four mechano-insensitive C-fibers. Cowhage and histamine activate distinctly different nonoverlapping populations of C-fibers while inducing similar sensations of itch. We hypothesize that cowhage activates a pathway for itch that originates peripherally from superficial mechano-responsive (polymodal) C-fibers and perhaps other afferent units. It is distinct from the pathway for histamine-mediated pruritus and does not involve the histamine-sensitive mechano-insensitive fibers.

Fiber composition of the rat sciatic nerve and its modification during regeneration through a sieve electrode.

Recovery after peripheral nerve transection is seldom complete, the outcome depending both on lesion and repair conditions, and on the type and neurochemical properties of axons. The interposition between the stumps of a perforated, or regenerative electrode (RE) is a promising avenue in the use of chronic nerve bioimplants, but represents an additional challenge to regeneration. We applied stereological methods to ultrathin and immunostained semithin sections to examine quantitatively the axon types that make up the sciatic nerve in control adult rats, and their changes 2 months after an RE implant. The number of myelinated axons (MAx) increased proximal to RE, but fell to 10% a few millimeters distal. This decrease affected more severely motor fibers, characterized by immunoreactivity to cholinacetyltransferase (ChAT+), than sensory (ChAT-) fibers. Regenerating MAx and myelin sheaths also changed notably in thickness. Unmyelinated axons (UAx) showed a moderate reduction in number distal to the implant. This reduction affected more tyrosine hydroxylase-immunoreactive axons (mostly vaso- and pilomotor fibers), than axons expressing ChAT and/or vasoactive intestinal peptide (mostly sudomotor fibers). Taken together with previous findings [Negredo, P., Castro, J., Lago, N., Navarro, X., Avendaño, C., 2004. Differential growth of axons from sensory and motor neurons through a regenerative electrode: a stereological, retrograde tracer, and functional study in the rat. Neuroscience 128, 605-615.], this study shows that regeneration through the RE is much less successful for MAx than UAx, that motor axons regenerate more poorly than sensory axons, and that some subclasses of sympathetic fibers regenerate better than others. The study also proves the value of the combined methodological approach presented here to assess the fiber composition of a nerve under normal, pathological or experimental conditions.

Endothelin 1 activates and sensitizes human C-nociceptors.

Microneurography was used to record action potentials from afferent C-fibers in cutaneous fascicles of the peroneal nerve in healthy volunteers. Afferent fibers were classified according to their mechanical responsiveness to von Frey stimulation (75g) into mechano-responsive and mechano-insensitive nociceptors. Various concentrations of Endothelin1 (ET1) and Histamine were injected into the receptive fields of C-fibers. Activation and heat sensitization were monitored. Axon reflex flare and psychophysical ratings were assessed after injection of ET1 and codeine into the forearms after pre-treatment with an H1 blocker or sodium chloride. 65% of mechanosensitive nociceptors were activated by ET1. One-third showed long lasting responses (>15min). In contrast, none of thirteen mechano-insensitive fibers were activated. Sensitization to heat was observed in 62% of mechanosensitive and in 46% of mechano-insensitive fibers. Injection of ET1 produced a widespread axon reflex flare, which was suppressed by pre-treatment with an H1 receptor blocker. In addition, pain sensations were induced more often than itching by ET1 in contrast to codeine. No wheal was observed after injection of ET1. Both itching and pain were decreased after H1 blocker treatment. In summary: (1) In humans ET1 activates mechanosensitive, but not mechano-insensitive, nociceptors. (2) Histamine released from mast cells is not responsible for all effects of ET1 on C-nociceptors. (3) ET1 could have a differential role in pain compared to other chemical algogens which activate additionally or even predominantly mechano-insensitive fibers.

Properties of neurons in the trigeminal nucleus caudalis responding to noxious dural and facial stimulation.

Extracellular single unit recordings were made in the rat trigeminal nucleus caudalis (Vc) from cells with Adelta and C-fibre latency responding to electrical stimulation of the thinned cranium overlying the middle meningeal artery (MMA). The neurons had an ipsilateral facial receptive field (FRF) that mainly extended over areas innervated by the first and second division of the trigeminal nerve but in some cases also included areas innervated by the third division of the trigeminal nerve. No wind-up of either long latency C-fibre or short latency Adelta responses was seen during trains of electrical stimulation. Sensitisation of mechanical stimulation of the FRF could also not be observed at any time during dural stimulation. In contrast, extracellular single unit recordings in the Vc activated by electrical stimulation of the facial skin resulted in a significant wind-up response of long latency response in six of ten cells studied. The facial-elicited wind-up response was significantly enhanced, 18 min after the electrical stimulation protocol was started, indicating that the process of wind-up had generated central excitability. The findings in this study demonstrate a clear difference between the effects of electrical stimulation of cutaneous and non-cutaneous inputs. In the trigeminal system, this has implications for the study of pathways such as those involved in headache, where it is believed that an enhanced dural input to the Vc may generate central sensitisation and partly explain the hyperalgesia and allodynia reported by patients.

Subtypes of vagal afferent C-fibres in guinea-pig lungs.

An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C-fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C-fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C-fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C-fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C-fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C-fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C-fibre population responded with action potential discharge to the P2X selective receptor agonist alpha,beta-methylene-ATP. Whole cell patch clamp recording of capsaicin-sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C-fibre neurones express functional P2X receptors, whereas lung specific jugular C-fibres do not. The data support the hypothesis that both neural crest-derived neurones (jugular ganglia) and placode-derived neurones (nodose ganglia) project C-fibres in the vagus, and that these two C-fibre populations represent distinct phenotypes.

Active "itch fibers" in chronic pruritus.

An itch-specific neuronal pathway was recently discovered in healthy humans and animals. Here the authors report that activity in this specific pathway coincides with itch under pathophysiologic conditions in a patient with chronic pruritus. Microneurographic recordings from the symptomatic area revealed spontaneous activity in six single C-fiber afferents that had the characteristic features of "itch fibers." Itch may be caused by activity in a specific subpopulation of C-fiber afferents.