<p>(A) Fluorescent calcium responses to three bouts of electrical stimulation in a single neurite under control conditions (left panel), following exchange of extracellular Ca<sup>2+</sup> for equimolar... more
<p>(A) Fluorescent calcium responses to three bouts of electrical stimulation in a single neurite under control conditions (left panel), following exchange of extracellular Ca<sup>2+</sup> for equimolar Mg<sup>2+</sup> (centre panel) and after wash with re-establishment of the extracellular Ca<sup>2+</sup> concentration (right panel). (B) Pooled data for 14 neurite recordings comparing repeated electrical stimulation (grey bars, control) with the intervention of replacing extracellular Ca<sup>2+</sup> with Mg<sup>2+</sup> (filled bars). Calcium responses during Mg<sup>2+</sup> replacement were significantly reduced (interaction time*treatment F(2,24) = 8.84, **P < 0.01, repeated measures ANOVA). (C) Fluorescent calcium responses to three bouts of electrical stimulation in a single neurite under control conditions (left panel), following addition of EDTA (3 mM) to the perfusate (centre panel) and after wash with re-establishment of the extracellular Ca<sup>2+</sup> concentration (right panel). (D) Pooled data for 14 neurites comparing repeated electrical stimulation (grey bars, control) with the addition of EDTA (3 mM) to the extracellular solution (filled bars, EDTA). Calcium responses in the presence of EDTA (3 mM) were significantly reduced (interaction time*treatment F(2,24) = 6.45, **P < 0.01, repeated measures ANOVA).</p
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<p>Growth factor combinations and concentrations.</p
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<p>Top: Percentage of neurites with only thin endings (black bars), with one or more thick endings (grey bars), and undefined (white bars) under different culture conditions A—E. Insets show the allocation of growth factors in the... more
<p>Top: Percentage of neurites with only thin endings (black bars), with one or more thick endings (grey bars), and undefined (white bars) under different culture conditions A—E. Insets show the allocation of growth factors in the compartments of the culture chamber. Qualitative visual determination was done from microphotographs taken after 4 to 5 days in culture. Abbreviations indicate the growth factors (GF) in the chambers, e.g., G-N-G denotes GDNF in the lateral compartments and NGF in the central, somata-containing compartment. Bottom: Examples for thin (left) and thick (right) neurite endings are shown in microphotographs. Media conditions in the compartments as indicated. Number of animals: 3 to 5 for A—E, respectively.</p
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<p>CGRP release from potassium-stimulated neurites (A) or somata (C) and release in the adjacent soma (B) or neurite (D) compartment(s). (A) CGRP release in neurite compartment in response to potassium (60 mM) for 5 min (grey... more
<p>CGRP release from potassium-stimulated neurites (A) or somata (C) and release in the adjacent soma (B) or neurite (D) compartment(s). (A) CGRP release in neurite compartment in response to potassium (60 mM) for 5 min (grey shading) and (B) concomitant CGRP release in the unstimulated adjacent soma compartment. (C) CGRP release in the soma compartment in response to potassium (60 mM) for 5 min (grey shading) and (D) concomitant CGRP release in the adjacent neurite compartment. Insets: Total CGRP release (as AUC (pg/ml)) determined from the respective time course experiments, grey columns indicate total CGRP release from the stimulated compartment(s), white columns from the adjacent one(s). Symbols and abbreviations: Circles denote NGF in the lateral and central compartments (N-N-N); squares denote GDNF in the lateral compartments and NGF in the central compartment (G-N-G); triangles denote no growth factor in the lateral compartments and GDNF in the central compartment (Ø-G-Ø). A: ● n = 11/3, ■ n = 21/4, ▲ n = 18/3, B: ● n = 4/3, ■ n = 8/4, ▲ n = 8/3, C: ● n = 4/2, ■ n = 4/3, ▲ n = 5/2, D: ● n = 9/2, ■ n = 8/3, ▲ n = 11/2,(n = number of compartments/animals used). Error bars indicate ± SEM, asterisks indicate p<0.05.</p
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<p>(A) Schematic illustrating DRG neurons (green) cultured in the central compartment of a Campenot chamber that extended neurites into the lateral compartment. Fluorescent images were acquired from the lateral compartment. Constant... more
<p>(A) Schematic illustrating DRG neurons (green) cultured in the central compartment of a Campenot chamber that extended neurites into the lateral compartment. Fluorescent images were acquired from the lateral compartment. Constant current field stimulation (1s, 40mA, 20Hz) was applied in the central compartment. (B) Neurites were initially identified under brightfield illumination (B, left) and calcium (Fluo-8®, AM) fluorescence images (B, right) were acquired sequentially at 1Hz for 60s, with an increase in acquisition rate to 5Hz during the 5-10s period. A region of interest (ROI) was selected from the brightfield image (B, left) and used to determine average fluorescence (AU) within the ROI (C, red markers) for each image across time. A single exponential fit was used to determine baseline fluorescence (F<sub>0</sub>; C, solid grey line). The change in fluorescence (ΔF = F-F<sub>0</sub>; D, blue markers) was determined as the difference between raw (C, red markers) and fitted baseline fluorescence values (C, solid grey line). This difference signal was subsequently divided by the baseline fluorescence (C, solid grey line) to yield a fluorescence ratio (ΔF/F<sub>0</sub>) (E, black markers). In addition to average fluorescence values (C-E) the spatio-temporal distribution of fluorescence (panel F, color-coded) was determined by calculating the fluorescence ratio (ΔF/F<sub>0</sub>) for each individual pixel along the ROI (F, y-axis in μm) as a function of time, i.e. each image during the acquisition period (F, Time axis).</p
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<p>(A) Calcium responses to 5 sequential bouts of electrical stimulation shown as raw data for individual neurites (upper, individual response joined by lines) and as normalized averages (lower panels) for 14 test neurites (filled... more
<p>(A) Calcium responses to 5 sequential bouts of electrical stimulation shown as raw data for individual neurites (upper, individual response joined by lines) and as normalized averages (lower panels) for 14 test neurites (filled blue markers in upper panel and filled blue bars lower panel) and 7 control neurites (open blue bars in lower panel) from NGF cultured somata (left panels) and 11 test neurites (filled red markers in upper panel and filled red bars lower panel) and 7 control neurites (open red bars in lower panel) from GDNF cultured somata (right panels). Tetrodotoxin (TTX, 500nM, green shading) was applied to the test neurites (shaded bars in lower panels) during the second stimulus and similarly, for the test group of neurites lidocaine (Lido, 1mM, orange shading) was applied during the fourth stimulus period. (B) Individual calcium responses to electrical stimulation (grey shading) in NGF (left panel) and GDNF (centre panel) neurites in the presence of TTX (500nM). Applying the criterion for a positive calcium response (see methods) conduction in one of 14 NGF neurites (7%) was blocked by TTX (500nM) whereas 7 out of 11 GDNF neurites (64%) were blocked by TTX (right panel). (C) Averaged normalized calcium response to control electrical stimulation (grey shading) for 94 neurites (49 NGF, 45 GDNF) illustrating the bi-exponential decay of the calcium signal. Determined for each individual response, the average fast and slow time constants of decay did not differ between NGF or GDNF neurites (inset).</p
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<p>Schematic layout of culture chamber as top and side view and photograph.</p
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<p>(A) Time course of CGRP release in response to capsaicin stimulation (300 nM) for 5 min (grey shading). Neurites were cultured with NGF (● n = 13/2 compartments/animals) or GDNF (■ n = 11/2). Somata were cultured with NGF in both... more
<p>(A) Time course of CGRP release in response to capsaicin stimulation (300 nM) for 5 min (grey shading). Neurites were cultured with NGF (● n = 13/2 compartments/animals) or GDNF (■ n = 11/2). Somata were cultured with NGF in both cases. Inset: CGRP release as AUC (pg/ml) determined from the respective time course experiments. (B) Comparison between Capsaicin (300 nM, grey columns) and potassium (60mM, black columns) stimulated neurites, grown under NGF or GDNF, respectively. Somata were cultured with NGF in both cases. Data were taken from Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0203215#pone.0203215.g005" target="_blank">5B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0203215#pone.0203215.g006" target="_blank">6A</a>. Error bars indicate ± SEM. Asterisks indicate p<0.05.</p
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<p>(A) Number of lanes with outgrown neurites per lateral compartment. (B) Total length of the neurites per lateral compartment measured from microphotographs. (C) Frequency distribution of total neurite length for all single... more
<p>(A) Number of lanes with outgrown neurites per lateral compartment. (B) Total length of the neurites per lateral compartment measured from microphotographs. (C) Frequency distribution of total neurite length for all single lateral compartments. Single data same as in (B). Error bars indicate mean ± SEM; asterisks indicate p<0.05. Number of animals: N-N-N: 7; G-N-G: 10; Ø-G-Ø: 5.</p
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<p>Basal CGRP secretion; n: Number of compartments/animals; values represented as pg/ml (mean ± SEM).</p
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<p>(A) Overlay of Ca<sup>2+</sup> responses in a single neurite in response to sequential stimulation with 1, 5, 10, 20, 50 and 100 pulses over 1 second (grey bar). (B) Pooled data for average peak calcium response... more
<p>(A) Overlay of Ca<sup>2+</sup> responses in a single neurite in response to sequential stimulation with 1, 5, 10, 20, 50 and 100 pulses over 1 second (grey bar). (B) Pooled data for average peak calcium response (ΔF/F<sub>0</sub>, black markers) as a function of stimulus frequency with an exponential regression fit (red line). Averaged data derives from recordings from 30 neurites excepting data for 0 Pulses (n = 8), 1 Pulse (n = 29) and 50 Pulses (n = 19). Spatiotemporal profile of fluorescent calcium signals along the terminal 200μm of a single neurite (rightmost inset) in response to stimulation with 1 current pulse (left panels), 5 pulses/s (centre panels) and 10 pulses/s (right panels). Fluorescent images were acquired at 25 Hz. The upper (blue) panels show fluorescence intensity coded as color along the length of the neurite (ordinate) and as a function of time (abscissa). Lower panels show the average fluorescence signal (ΔF/F<sub>0</sub>, open markers) determined from all pixels along the neurite ROI as a function of time. Individual electrical stimuli (1ms, 40mA) are shown as vertical grey lines. (C) (D) Fluorescence images of a neurite before (top), during electrical stimulation (centre) and during application of ionomycin (10μm) (bottom). (E) Calcium responses for the neurite in panel D as a function of time (upper) and spatiotemporally (lower) in response to electrical stimulation (20Hz, left) and ionomycin (10μM, right).</p
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Aim of Investigation: Mechanically insensitive (silent) C nociceptors play a major role in chronic pain and are characterized in humans by a pronounced activity-dependent slowing of the conduction velocity. We studied axonal properties of... more
Aim of Investigation: Mechanically insensitive (silent) C nociceptors play a major role in chronic pain and are characterized in humans by a pronounced activity-dependent slowing of the conduction velocity. We studied axonal properties of C-fibres in pigs using identical stimulation parameter as tested in humans before.Methods: The experiments were conducted according to the German and international regulations on animal care. Domestic pigs (28-40 kg) were anaesthetized, ventilated and paralyzed. Using the teased fibre technique, extracellular single unit recordings from the saphenous nerve were performed. Axonal excitability changes upon repetitive electrical stimulation were assessed with electrical protocols: 1) progressive increase in stimulation frequency at low levels (1/8 - 1/4 - 1/2 Hz); 2) continuous stimulation at 2 Hz for 3 min; 3) recovery cycles. Subsequently, the fibre's responsiveness to natural (mechanical and thermal) stimuli was assessed.Results: We recorded 14...
Silent and polymodal nociceptors differ, e. g., in response patterns to electrical stimulation. One underlying mechanism could be differential expression of HCN channels, which presumably contribute to stabilizing resting membrane... more
Silent and polymodal nociceptors differ, e. g., in response patterns to electrical stimulation. One underlying mechanism could be differential expression of HCN channels, which presumably contribute to stabilizing resting membrane potential and controlling cell excitability.We used isolated DRG neurons from a non-rodent species, Sus scrofa domesticus. Neurons from piglets were investigated immunocytochemically and electrophysiologically.Single labeling revealed 54% capsaicin sensitive and 29% IB4 positive neurons; 83% expressed HCN-1 and 88% HCN-2 channels. Double labeling showed: (i) Out of the capsaicin sensitive neurons, 42% were IB4 positive, 80% were HCN-1 and 77% HCN-2 immunoreactive. (ii) Out of the HCN-1 or HCN-2 negative neurons, 94% and 97 % were capsaicin positive, 90% and 85% were IB4 negative, respectively. Triple labeling revealed that the vast majority of neurons lacking HCN channel subtypes 1 and/or 2 are capsaicin positive and IB4 negative. The soma sizes of these n...
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Research Interests: Endocrinology, Neuroscience, Chronic Pain, Biology, Medicine, and 15 moreInternal Medicine, Animals, Male, Neuronal Plasticity, Nerve Growth Factor, Bovine Serum Albumin, Ligands, Neurosciences, Binding Site, Nerve Injury, Bradykinin, Phosphate Buffer Saline, binding sites, High density, and Ligation
Activity-dependent slowing of conduction velocity (ADS) differs between classes of human nociceptors. These differences likely reflect particular expression and use-dependent slow inactivation of axonal ion channels and other mechanisms... more
Activity-dependent slowing of conduction velocity (ADS) differs between classes of human nociceptors. These differences likely reflect particular expression and use-dependent slow inactivation of axonal ion channels and other mechanisms governing axonal excitability. In this study, we compared ADS of porcine and human cutaneous C-fibers. Extracellular recordings were performed from peripheral nerves, using teased fiber technique in pigs and microneurography in humans. We assessed electrically-induced conduction changes and responsiveness to natural stimuli. In both species, the group of mechano-insensitive C-fibers showed the largest conduction slowing ( approximately 30%) upon electrical stimulation (2Hz for 3min). In addition, we found mechano-insensitive cold nociceptors in pig that slowed only minimally (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10% at 2Hz), and a similar slowing pattern was found in some human C-fibers. Mechano-sensitive afferents showed an intermediate conduction slowing upon 2Hz stimulation (pig: 14%, human 23%), whereas sympathetic efferent fibers in pig and human slowed only minimally (5% and 9%, respectively). In fiber classes with more pronounced slowing, conduction latencies recovered slower; i.e. mechano-insensitive afferents recovered the slowest, followed by mechano-sensitive afferents whereas cold nociceptors and sympathetic efferents recovered the fastest. We conclude that mechano-insensitive C-fiber nociceptors can be differentiated by their characteristic pattern of ADS which are alike in pig and human. Notably, cold nociceptors with a distinct ADS pattern were first detected in pig. Our results therefore suggest that the pig is a suitable model to study nociceptor class-specific changes of ADS.
Research Interests: Biophysics, Chemistry, Pain, Medicine, Humans, and 15 moreFemale, Animals, Male, Mechanoreceptors, Adult, Conduction Velocity, Action Potentials, Nerve Conduction Velocity, Hyperalgesia, Nociceptors, extracellular recording, Medical and Health Sciences, Efferent, Electric stimulation, and Cold Temperature
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Research Interests: Neuroscience, Chemistry, Calcium, Atomic Force Microscopy, Medicine, and 14 moreOptical Imaging, Keratinocytes, Animals, Capsaicin, Behavioral Animal Models, Clinical Sciences, Mechanical Stress, Neurite, Swine, Stimulation, Cell communication, Potassium Chloride, Adenosine Triphosphate, and coculture techniques
Selected naturally occurring unsaturated dialdehyde sesquiterpenes and related bioactive terpenoids were assayed for vanilloid-like activity. Out of the 25 compounds tested, eight inhibited completely the specific binding of... more
Selected naturally occurring unsaturated dialdehyde sesquiterpenes and related bioactive terpenoids were assayed for vanilloid-like activity. Out of the 25 compounds tested, eight inhibited completely the specific binding of [3H]resiniferatoxin by rat spinal cord membranes: binding affinities ranged from 0.6 microM for cinnamodial to 19.0 microM for hebelomic acid F. These values were comparable to the binding affinity of capsaicin (2.7 microM). With the exception of four ligands, compounds that inhibited resiniferatoxin binding to rat spinal cord membranes were also pungent on the human tongue where they showed cross-tachyphylaxis with capsaicin. As expected from their reactive nature, these compounds possess additional sites of action, as reflected in the complex behavior of the stimulation of calcium influx by cinnamodial and cinnamosmolide at high concentrations. This observation might explain the unexpectedly weak membrane depolarization by cinnamodial compared to capsaicin. We conclude that a range of sesquiterpene dialdehydes and related terpenoids, both pungent and non-pungent, may function as vanilloids. These compounds may represent a new chemical lead for the development of vanilloid drugs, structurally unrelated to either capsaicin or resiniferatoxin.
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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to stabilizing resting membrane potential, thus controlling neuron excitability. Subclasses of nociceptive neurons differ in their excitability, therefore,... more
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to stabilizing resting membrane potential, thus controlling neuron excitability. Subclasses of nociceptive neurons differ in their excitability, therefore, these channels could be a distinguishing marker. We investigated isolated dorsal root ganglion neurons from a non-rodent species, the pig, Sus scrofa domesticus. Single labeling revealed capsaicin-induced cobalt-uptake in 54.3% and transient receptor potential V1 (TRPV1) immunoreactivity in 55.1% of all neurons. Ruthenium red and capsazepine suppressed capsaicin-induced cobalt-uptake. HCN-1 and HCN-2 channel isoform immunoreactivity was detected in 82.6% and 88.3%, respectively, and binding of IB4 in 29.4% of all neurons. Double labeling revealed that out of the capsaicin-positive neurons, 42.3% were IB4-positive, 80.0% immunoreactive for the HCN-1, and 77.3% for the HCN-2 channel isoform, respectively. Neurons lacking HCN-1 or HCN-2 channel isoforms were mostly capsaicin-positive and IB4-negative. The soma size of neurons lacking HCN-1 and/or HCN-2 channels was small to medium. Western blot analysis showed protein products of sizes similar to those of HCN-1 and HCN-2 channel isoforms. Functionally, in patch-clamp experiments, some neurons were unresponsive to membrane hyperpolarization, thus, probably lacking HCN channels. In conclusion, in porcine dorsal root ganglion neurons there is a subset of capsaicin-positive, IB4-negative neurons lacking HCN-1 and/or HCN-2 channel isoforms.
Research Interests: Chemistry, Medicine, Ion Channels, Patch-clamp and imaging techniques, Western blotting, and 14 moreFemale, Animals, Male, Plant Lectins, Capsaicin, Clinical Sciences, European, Fluorescent Antibody Technique, Potassium Channels, Membrane Potential, Sus Scrofa, Neurosciences, Nociceptors, and Pharmacology and pharmaceutical sciences
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A [3H]‐resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral,... more
A [3H]‐resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl‐scutigeral, ovinal, neogrifolin, and methyl‐neogrifolin), scutigeral (Ki=19 μM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. Scutigeral induced a dose‐dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 μM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 μM). [3H]‐RTX binding isotherms were shifted by scutigeral (10–80 μM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 μM. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements i...
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Bradykinin receptors are believed to contribute to hyperalgesia under conditions of neuropathic pain. Using calcium imaging we investigated responses to B1 and B2 agonists on isolated rat dorsal root ganglion neurons. No response to the... more
Bradykinin receptors are believed to contribute to hyperalgesia under conditions of neuropathic pain. Using calcium imaging we investigated responses to B1 and B2 agonists on isolated rat dorsal root ganglion neurons. No response to the B1 agonist was detected, whereas 12% of neurons responded to the B2 agonist. Northern blot analysis confirmed the lack of B1 receptor expression in dorsal root ganglia, as B1 mRNA was neither detected under normal conditions nor after nerve injury. In the calcium imaging experiments, agonists were applied with an elevated superfusion flow rate to avoid tachyphylaxis to the drug. Normal external solution applied at this flow rate constituted a mechanical stimulus causing a response in some neurons. Thus, in comparable set-ups mechanosensitivity has first to be tested to avoid masking effects.