Maximum axonal following frequency separates classes of cutaneous unmyelinated nociceptors in the pig

Abstract

Key points
C‐nociceptors are generally assumed to have a low maximum discharge frequency of 10–30 Hz. However, only mechano‐insensitive ‘silent’ C‐nociceptors cannot follow electrical stimulation at 5 Hz (75 pulses) whereas polymodal C‐nociceptors in the pig follow stimulation at up to 100 Hz without conduction failure.
Sensitization by nerve growth factor increases the maximum following frequency of ‘silent’ nociceptors in pig skin and might thereby contribute in particular to intense pain sensations in chronic inflammation.
A distinct class of C‐nociceptors with mechanical thresholds >150 mN resembles ‘silent’ nociceptors at low stimulation frequencies in pigs and humans, but is capable of 100 Hz discharge and thus is suited to encode painfulness of noxious mechanical stimuli.

Abstract
Using extracellular single‐fibre recordings from the saphenous nerve in pig in vivo, we investigated peak following frequencies (5–100 Hz) in different classes of C‐nociceptors and their modulation by nerve growth factor. Classes were defined by sensory (mechano‐sensitivity) and axonal characteristics (activity dependent slowing of conduction, ADS).
Mechano‐insensitive C‐nociceptors (CMi) showed the highest ADS (34% ± 8%), followed only 66% ± 27% of 75 pulses at 5 Hz and increasingly blocked conduction at higher frequencies. Three weeks following intradermal injections of nerve growth factor, peak following frequency increased specifically in the sensitized mechano‐insensitive nociceptors (20% ± 16% to 38% ± 23% response rate after 72 pulses at 100 Hz). In contrast, untreated polymodal nociceptors with moderate ADS (15.2% ± 10.2%) followed stimulation frequencies of 100 Hz without conduction failure (98.5% ± 6%). A distinct class of C‐nociceptors was exclusively sensitive to strong forces above 150 mN. This class had a high ADS (27.2% ± 7.6%), but displayed almost no propagation failure even at 100 Hz stimulation (84.7% ± 17%). Also, among human mechanosensitive nociceptors (n = 153) those with thresholds above 150 mN (n = 5) showed ADS typical of silent nociceptors. C‐fibres with particularly high mechanical thresholds and high following frequency form a distinct nociceptor class ideally suited to encode noxious mechanical stimulation under normal conditions when regular silent nociceptors are inactive. Sensitization by nerve growth factor increases maximum discharge frequency of silent nociceptors, thereby increasing the frequency range beyond their physiological limit, which possibly contributes to excruciating pain under inflammatory conditions.

Full text

J Physiol 599.5 (2021) pp 1595–1610 1595
The Journal of Physiology
Maximum axonal following frequency separates classes of
cutaneous unmyelinated nociceptors in the pig
Fiona Werland1,MichaelHirth
1,RomanRukwied
1,MatthiasRingkamp
2, Brian Turnquist3,
Ellen Jorum5,6,BarbaraNamer
4,MartinSchmelz
1and Otilia Obreja1
1Department of Experimental Pain Research, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
2Department of Neurosurgery, Johns Hopkins University, Baltimore, MD, USA
3Faculty of Mathematics and Computer Science, Bethel University, MN, USA
4IZKF Neuroscience Research Group, University Hospital RWTH Aachen and Department of Physiology and Pathophysiology, University of
Erlangen-Nuremberg, Erlangen, Germany
5Section of Clinical Neurophysiology, Department of Neurology, Oslo University Hospital, Oslo, Norway
6Institute of Clinical Medicine, University of Oslo, Oslo, Norway
Edited by: Harold Schultz & Carole Torsney
Key points
rC-nociceptors are generally assumed to have a low maximum discharge frequency of 10–30 Hz.
However, only mechano-insensitive ‘silent’ C-nociceptors cannot follow electrical stimulation at
5 Hz (75 pulses) whereas polymodal C-nociceptors in the pig follow stimulation at up to 100 Hz
without conduction failure.
rSensitization by nerve growth factor increases the maximum following frequency of ‘silent’
nociceptors in pig skin and might thereby contribute in particular to intense pain sensations in
chronic inammation.
rA distinct class of C-nociceptors with mechanical thresholds >150 mN resembles ‘silent’
nociceptors at low stimulation frequencies in pigs and humans, but is capable of 100 Hz discharge
and thus is suited to encode painfulness of noxious mechanical stimuli.
Abstract Using extracellular single-bre recordings from the saphenous nerve in pig in vivo,we
investigated peak following frequencies (5–100 Hz) in dierent classes of C-nociceptors and their
modulation by nerve growth factor. Classes were dened by sensory (mechano-sensitivity) and
axonal characteristics (activity dependent slowing of conduction, ADS).
Mechano-insensitive C-nociceptors (CMi) showed the highest ADS (34% ±8%), followed only
66% ±27% of 75 pulses at 5 Hz and increasingly blocked conduction at higher frequencies.
Three weeks following intradermal injections of nerve growth factor, peak following frequency
increased specically in the sensitized mechano-insensitive nociceptors (20% ±16% to 38% ±23%
response rate after 72 pulses at 100 Hz). In contrast, untreated polymodal nociceptors with moderate
ADS (15.2% ±10.2%) followed stimulation frequencies of 100 Hz without conduction failure
(98.5% ±6%). A distinct class of C-nociceptors was exclusively sensitive to strong forces above
Fiona Werland studied medicine at Heidelberg University from 2009 to 2014 and since then has begun her clinical career in
surgery and gynaecology at Diakonissenkrankenhaus Speyer. For the past 3 years she has been conducting research work in
the Department of Experimental Pain Research, Heidelberg University, that has included numerous single bre recordings,
theensuingcomplexanalysisofsinglebreresponsesandnallyanalysisoftheexperimentalprotocols.Thispublicationis
a crucial part of her medical thesis.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. DOI: 10.1113/JP280269
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which
permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no
modications or adaptations are made.

1596 F. Werland and others J Physiol 599.5
150 mN. This class had a high ADS (27.2% ±7.6%), but displayed almost no propagation failure even
at 100 Hz stimulation (84.7% ±17%). Also, among human mechanosensitive nociceptors (n=153)
those with thresholds above 150 mN (n=5) showed ADS typical of silent nociceptors. C-bres with
particularly high mechanical thresholds and high following frequency form a distinct nociceptor
class ideally suited to encode noxious mechanical stimulation under normal conditions when regular
silent nociceptors are inactive. Sensitization by nerve growth factor increases maximum discharge
frequency of silent nociceptors, thereby increasing the frequency range beyond their physiological
limit, which possibly contributes to excruciating pain under inammatory conditions.
(Received 3 June 2020; accepted after revision 17 December 2020; rst published online 28 December 2020)
Corresponding author M. Schmelz: Department of Experimental Pain Research, MCTN, Medical Faculty Mannheim,
Heidelberg University, Mannheim, 68167 Germany. Email: martin.schmelz@medma.uni-heidelberg.de
Introduction
Traditionally, nociceptor classes have been dened
based on the function of sensory endings leading to
the classication of polymodal nociceptors responsive
to heat, mechanical and chemical stimuli (Perl,
1996), mechano-insensitive ‘silent’ nociceptors (Meyer
et al. 1991; Schmidt et al. 1994, 1995; Wooten et al.
2014) or nociceptors responding to noxious cold
(LaMotte & Thalhammer, 1982; Campero et al. 1996;
Simone & Kajander, 1997; Serra et al. 2009). For
cold nociceptors, a specic link to axonal expression
of the voltage-sensitive sodium channel NaV1.8 has
been established (Zimmermann et al. 2007). Other
axonal features, such as activity-dependent slowing of
conduction, have been used to classify functional classes
of nociceptors, originally in the rat (Raymond et al. 1990;
Gee et al. 1996). The slowing patterns of unmyelinated
bres dier to such an extent that they predict the class
withhighprecisionandcanthereforebeusedtoidentify
corresponding nociceptor classes in humans (Serra et al.
1999; Schmelz et al. 2000a) and pigs (Obreja et al. 2010)
with identical stimulation protocols.
Currently, classication of primary aerents is focusing
on the single cell expression pattern (Usoskin et al.
2015; Haring et al. 2018; Zeisel et al. 2018), suggesting
10 to 14 subpopulations of sensory aerents in dorsal
root ganglia (Li et al. 2016, 2018). Very recently,
dierences in potassium channel expression in single
somatosensory neurons have been shown to separate
low threshold mechano-sensitive C-bres (‘C-touch’),
peptidergic and non-peptidergic nociceptors in mice
(Zheng et al. 2019). Interestingly, the authors found
dierential potassium channel expression correlating to
maximum discharge frequency upon current injection
into the somata of dorsal root ganglia cells, thereby
translating transcriptome patterns to intrinsic functional
properties including ring frequency (Zheng et al. 2019).
This nding provides a molecularly dened proof for
the fundamental hypothesis that, in addition to sensory
transduction, axonal properties determine the ability
of primary somatosensory neurons to encode specic
noxious or non-noxious stimuli. This hypothesis was put
forwardmorethan25yearsagobasedondierential
activity-dependent slowing of conduction in nociceptors
and non-nociceptors (Thalhammer et al. 1994; Gee et al.
1996).
Both in extracellular axonal recordings (Thalhammer
et al. 1994; Gee et al. 1996) of single bres and in patch
clamp studies (Djouhri et al. 2001; Zheng et al. 2019)
relatively low maximum discharge frequencies (about
10–30 Hz) of C-nociceptors have been reported, while
C-touch bres were capable of generating 30–100 Hz,
with dierential expression of Kv4.1 (non-peptidergic
nociceptors) vs. Kv4.3 (C-touch) possibly contributing
to this dierence (Zheng et al. 2019). However, these
frequencies might not directly translate to actual in
vivo discharge patterns based on general dierences
in geometry and temperature, but also on possible
dierences in membrane expression of voltage-gated
channels between axons and soma. Previous studies have
shown that nerve growth factor (NGF) treatment in the
pig increased axonal nociceptor excitability in human
and pig skin (Obreja et al. 2011, 2018) and increased
maximum following frequency of C-nociceptors in the
guinea-pig (Djouhri et al. 2001). We therefore set out
to test maximum discharge frequencies in dierent
functional classes of C-aerents and their modulation by
NGF in the pig in vivo andaddedreanalysedsinglebre
recordings from healthy volunteers.
Methods
Ethical approval
All experimental interventions in pigs were approved
by the regional ethics council in Karlsruhe,
Baden-Wuerttemberg, Germany (G-231/12, G-78/18).
The microneurography experiments performed in
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 599.5 Maximum following frequency separates nociceptor classes 1597
healthy human subjects were approved by the local
ethics committees in Erlangen, Germany (1995/544),
Uppsala, Sweden and Oslo, Norway and conformed to
the Declaration of Helsinki, except for registration in a
database.
Animal model and treatments
We used data from 53 neutered male pigs (Sus scrofa
domesticus,age:2—4months)providedtotheuniversity
facilities by a local farmer. Animals were fed twice daily
and had access to water ad libitum with the exception
of the day preceding the surgical interventions when
food was withdrawn. Four additional pigs were used for
pharmacological tests. In these animals, after 1 week
of housing, NGF was injected intradermally into the
innervation territory of the saphenous nerve on one hind
limb (see below). Animals did not show behavioural
changes related to the NGF-injected limb. This result
was expected based on previous studies with comparable
intracutaneous and intramuscular injection of NGF in
humans not resulting in local inammation or ongoing
pain. Extracellular recordings were performed 3 weeks
later by laboratory members who were blinded to the
treatment.
Anaesthesia and surgery in pigs
Before surgery, pigs were pre-medicated as previously
described (Obreja et al. 2010) by intramuscular injection
of 2 mg/kg azaperone (Stresnil, Janssen Pharmaceutica,
Beerse, Belgium), 0.015 mg/kg atropine (Eifelfango,
Bad Neuenahr, Germany) and 1 mg/kg midazolam
(Dormicum, Roche, Basel, Switzerland). An intravenous
indwelling venous catheter was placed in a suitable
ear vein to induce general anaesthesia with 2 mg/kg
propofol (Fresenius, Bad Homburg, Germany), which was
maintained with 8–14 mg/kg/h pentobarbital (Narcoren,
Merial, Halbergmoos, Germany). Vital parameters (heart
rate, core temperature, oxygen saturation, PETCO2) were
continuously monitored. Adequate depth of anaesthesia
was controlled by heart rate, absence of motor responses
to noxious stimulation, and eye position. Animals were
mechanically ventilated (respiratory rate 15/min; tidal
volume 10—15 ml/kg). Muscle relaxation was achieved by
.. administration of 0.5 mg/kg Rocuronium (Esmeron,
Organon International, BH OSS, Netherlands) followed
by 7–8 mg/kg/h succinylcholine (Lysthenon, Nycomed,
Unterschleissheim, Germany), which was tapered o after
4—6 h. A length of about 6 cm of the saphenous nerve
was exposed and then dissected (‘teased’) into very thin
laments as described previously (Meyer & Campbell,
1988; Lynn et al. 1995; Obreja et al. 2010).
Electrophysiological equipment and recordings
In vivo extracellular recordings from the ‘teased’
saphenous nerves were performed using DAPSYS
software (www.dapsys.net) (Turnquist et al. 2004). A
detailed description of the technique can be found in pre-
vious papers (Campbell & Meyer, 1983; Lynn et al. 1995;
Obreja et al. 2010). Briey, electrical rectangular pulses
(0.5 ms duration; 20 mA intensity; 0.25 Hz) were delivered
by a constant current stimulator (DS7A, Digitimer Ltd,
Herts, UK) via two non-insulated microneurography
electrodes (FHC Inc., Bowdoin, ME, USA), inserted intra-
dermally at sites where time-locked action potentials with
long latencies could be elicited. Both saphenous nerves
were recorded in one experimental session (10–36 h). At
the end of experiment, pigs were killed by .. injection
of 10 ml Tanax (T-61, Intervet Deutschland GmbH) and
death was conrmed by induction of lasting electrical
silence on ECG and disappearance of carotid pulse.
Mechanical testing and identication of
unmyelinated nociceptors
Under ongoing electrical stimulation at 0.25 Hz,
mechanical responsiveness was assessed with a set of
calibrated Semmes-Weinstein monolaments (Stoelting,
Chicago, IL, USA) according to the standardized
procedure described elsewhere (Obreja et al. 2011).
All bres were screened for responses to a mechanical
force of 150 mN, which separates the subclass of
mechano-sensitive nociceptors (‘high-threshold C-units’;
HT) from the mechano-insensitive nociceptors (CMi;
unresponsive to 150 mN stimulus). Low-threshold
mechanoreceptors were identied by their response to
innocuous skin stimulation with a paintbrush, but were
not included in this report. In the mechano-sensitive
nociceptors, we assessed the borders of their receptive
elds (RF) by repetitive 4 s applications of a 150 mN
monolament on a large skin area around the stimulation
needles. The edges were marked by a felt tip pen. The
mappingstartedinadistanceofabout5cmfromthe
stimulation needles, slowly progressing inwards until the
edge of the RF was met. In order to avoid sensitization,
stimulation with a 150 mN force was used exclusively
to mark the borders of the RF. Following mechanical RF
mapping, monolaments of incremental stiness (i.e.
force) starting from 10 mN were used to determine the
mechanical thresholds inside the mechanical RF. The
weakest monolament evoking discharges upon at least
2 out of 4 consecutive stimulations at one hot spot was
marked as mechanical threshold. The spike response was
additionally veried by the induced ‘marking’ (i.e. swift
increase of response latency due to activity-dependent
slowing induced by mechanically evoked discharge,
followed by an exponential recovery: Torebjörk & Hallin,
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

1598 F. Werland and others J Physiol 599.5
1974). Fibres that were unresponsive to 150 mN were
exposed to stronger mechanical stimulation up to 1 N or
bypinchingtheskinwithanon-serratedforceps.Incase
of no mechanically evoked response, an electrical search
protocolwasusedtomaptheRF(Meyeret al. 1991).
Axonal excitability testing
Conduction velocity. Subsequent to the RF mapping, the
stimulation needles were placed inside the RF and the
stimulation intensity was set at 2×the electrical threshold
of the recorded unit. The shortest distance between
the stimulation electrode and the recording electrode
was measured and divided by the rst response latency
assessedaftera2minpause(i.e.‘latencypost-wait’)in
order to calculate the conduction velocity (CV). In this
study, the CV of all C-bres was below 2 m/s.
Peak following frequency. Peak following frequency was
tested by trains of rectangular electrical pulses (0.5 ms
duration; 2×electrical threshold) applied in the receptive
eld of the characterized nociceptors at 5, 10, 20, 50
and 100 Hz via bipolar intracutaneous needle electro-
des. For each stimulation frequency, the response rate was
assessed as percentage of successful electrical stimulations
(Fig. 1). Trains of 25 consecutive pulses were used for
5–20 Hz stimulation whereas stimuli at 50 and 100 Hz
wereappliedin6burstsof4pulseswith200msbetween
the bursts to avoid overlap between stimulus artefact and
action potential recording. These stimulation blocks were
repeated 3 times with an interval of 10 s (Fig. 1) and a
2 min pause was kept between dierent frequencies.
Activity-dependent slowing (ADS). The
activity-dependent slowing (ADS) of conduction
velocity was tested by continuous stimulation at dierent
frequencies (from 0.125 Hz to 100 Hz) and intensity twice
the electrical threshold. All stimulation protocols were
preceded by a 2 min pause. The change in response latency
during each stimulation protocol was assessed. The rst
protocol consisted of 5 min continuous stimulation at
incremental frequencies: 0.125 Hz (20 pulses), then
0.25 Hz (20 pulses), then 0.5 Hz (30 pulses). The second
protocol consisted of continuous stimulation at 2 Hz for
3 min (i.e. 360 pulses), followed by 25 pulses at 0.25 Hz
in order to assess the recovery kinetics (Serra et al. 1999;
Weidne r et al. 1999). Sympathetic eerent bres showed a
characteristic relative speeding in this protocol (Campero
et al. 2004; Obreja et al. 2010). The new protocol entailed
the stimulations at higher frequencies (5–100 Hz; see
Fig. 1).
Recovery cycles. Conduction latencies evoked by the rst
two stimulations at dierent frequencies (2, 5, 10, 20,
50, and 100 Hz) were used to assess early post-spike
excitability changes of the axon (‘recovery cycles’: Bostock
et al. 2003; George et al. 2007; Ringkamp et al. 2010).
The‘inter-spikeinterval(ISI)’wascomputedasthe
latency dierence between the spike evoked by the
second versus the rst stimulation, and then plotted
against the ‘inter-pulse interval (IPI)’ (e.g. 200 ms for
the 5 Hz stimulation protocol). Positive values denote
slowing of the second action potential and negative values
indicate speeding (‘supernormal conduction’) (Bostock
et al. 2003).
NGF administration
After 1 week of housing, NGF was injected intradermally
into one hind limb in four pigs. Recombinant nerve
growth factor (1 mg NGF, Sigma Deisenhofen, Germany)
was dissolved in 1 ml sterile phosphate buer containing
0.1% bovine serum albumin, aliquoted (33 μl/aliquot)
andstoredat−20°C. On the treatment day an aliquot
was defrosted and 10 μl(containing10μg) NGF was
dissolved in 3 ml sterile NaCl 0.9%. Prior to NGF
Figure 1. Stimulation protocol to assess response rate in pig bres
Intracutaneous electrical pulses (0.5 ms duration) were applied in the receptive eld of the unit of interest and
were preceded by a 2 min pause. Stimulations at 5, 10 and 20 Hz were given as 25 consecutive pulses. For 50 and
100 Hz we used 6 bursts of 4 pulses separated by a 200 ms interval. Each stimulation block was applied 3 times
with 10 s pause between the blocks. When each electrical pulse evoked one action potential, the response rate
was 100% (centre). Blocked action potentials (grey) – occurring more often at higher stimulation frequency (right
panel) – reduced the response rate.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 599.5 Maximum following frequency separates nociceptor classes 1599
injections, animals were sedated with .. injections
of 2 mg/kg azaperone (Stresnil, Janssen Pharmaceutica,
Beerse, Belgium) and 1 mg/kg midazolam (Dormicum,
Roche, Basel, Switzerland). NGF (3 ml) was intradermally
injected (30G insulin syringes, BD Heidelberg, Germany)
in fractions of 20 μl into the innervation territory of
one saphenous nerve (i.e. medial aspect of the hind
limb), as described before (Hirth et al. 2013). Extracellular
recordings were performed 3 weeks after intradermal
injection of NGF by laboratory members who were
blinded to the treatment.
Microneurography in humans
Single nerve bre recordings obtained between 1998
and 2003 from deep peroneal nerve fascicles in healthy
human subjects (57 female, 43 male, 23 ±2years)
were reanalysed and 153 mechanosensitive nociceptors
in which mechanical thresholds had been assessed were
identied (Weidner et al. 1999; Schmelz et al. 2000b,
2003). Conduction velocity, activity-dependent slowing
of conduction upon repetitive electrical stimulation
(Weidner et al. 1999), heat activation thresholds and
electrical thresholds were analysed. The cumulative data
set is provided in Supporting information.
Statistical analysis
Oine analysis of the latency changes and
responses to natural stimulation were performed
using DAPSYS 8.0 and Microsoft Excel 2003. The
activity-dependent slowing (ADS) was quantied as
increase above the baseline latency induced by electrical
stimulation: e.g. after 360 pulses at 2 Hz, ADS2Hz =
(Latency360th pulse −Latency1st pulse)×100/Latency1st pulse )
(Obreja et al. 2010).
Statistical analysis and graphs were generated using
Microsoft Excel and Origin7 (Origin Lab Corp.,
Northampton, MA, USA) and Statistica 7.1 (StatSoft
Inc.,Tulsa,OK,USA).Dataarepresentedasmeans±SD.
Mechanical thresholds and the response rates at dierent
stimulation frequencies were analysed by non-parametric
tests (Kruskal-Wallis ANOVA, Mann-Whitney Utest
and Wilcoxon test). ADS dierences were assessed by
one-way ANOVA and changes in latencies were analysed
by repeated-measures ANOVA, with ‘pulse’ and ‘unit
type’ascategoricalfactors(ifnotspeciedotherwise).
Signicant dierences were located with the Scheé post
hoc test (if not specied otherwise). In microneurography
experiments all parameters were assessed by the Student’s
ttest. Dierences were considered signicant at Pvalues
below 0.05.
Results
Classication of unmyelinated nociceptors in pig skin
based on mechanical sensitivity and
activity-dependent slowing of conduction
According to their response to mechanical
stimulation, porcine C-nociceptors are classied
into mechano-sensitive (‘high-threshold’, HT) and
mechano-insensitive (‘silent’, CMi) nociceptors (Obreja
& Schmelz, 2010; Obreja et al. 2010). However, some
CMi-nociceptors could be activated by very strong
mechanical forces (≥150 mN) and were identied as ‘very
high threshold’ C-nociceptors (VHT) (Obreja et al. 2010).
In our sample, mechanical thresholds of HT nociceptors
were 40 mN (20–60; median, quartiles; n=59 in 50
pigs),whereasthoseoriginallyclassiedasCMiunitsbut
responding to high forces (VHT) had a median threshold
of 260 mN (260–600; quartiles; n=27) (Fig. 2).
The mechanical thresholds of these VHT nociceptors
were far beyond forces accepted as ‘cuto’ for mechanical
sensitivity in the literature (i.e. 100 mN; Meyer &
Campbell, 1988). Also, dierent to HT nociceptors, these
bres desensitize strongly upon repetitive stimulations at
threshold level, whereas repeated supra-maximal noxious
stimulation like pinching the skin sensitizes further
responses (data not shown). Despite their spurious
mechanical responsiveness (Obreja et al. 2010), we
initially included these very high threshold bres in the
groupofregularCMinociceptors(i.e.nomechanical
activation by stimulation with 1 N von Frey hair)
based on their similar resting conduction velocity,
activity-dependent slowing (ADS) of conduction upon
stimulation at low frequencies (from 0.125 Hz to 2 Hz) and
kinetics of recovery after 3 min continuous stimulation
at 2 Hz (Obreja et al. 2010). Here we pooled together
a large number of recordings from 2006 until 2013
Figure 2. Distribution of mechanical activation thresholds
The graph shows the distribution of mechanical activation thresholds
in conventional mechanosensitive nociceptors (HT, high threshold) in
the pig compared to those of silent nociceptors that showed some
mechanical responsiveness (VHT).
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

1600 F. Werland and others J Physiol 599.5
(published in Obreja et al. 2010, 2011, 2012) and
unpublished data from 2011–2013 to reanalyse for
potential dierences between regular silent nociceptors
and those with spurious mechanical responses.
We assessed class-specic dierences between
conduction velocities at resting conditions as well as
during prolonged stimulation at low frequencies (Fig. 3).
As expected, there was no dierence in the total ADS
at 0.125 Hz between VHT and CMI (0.9 ±0.6% vs.
1.07 ±0.6%; P=0.7; Scheé test). Upon completion
of this 5 min low-frequency stimulation, CMi and VHT
reachedasimilartotalADSandinbothcasesthiswas
signicantly higher than the level of HT (P<0.001;
Scheé test; Fig. 3A). Conduction velocities were similar
between CMi and VHT bres (0.9 ±0.24 m/s; n=38 in
34 pigs; vs. 0.80 ±0.27 m/s; n=19 in 17 pigs; P=0.63;
Figure 3. Activity-dependent slowing of conduction at
low stimulation frequencies
A, changes in response latency (mean ±SD) of different pig
C-bre classes (CMi, silent nociceptors; VHT, silent
nociceptors with a very high mechanical threshold; HT,
regular mechanosensitive nociceptors; symp, sympathetic
efferent bres) to electrical stimulation at incremental
frequencies (0.125 Hz, 0.25 Hz, 0.5 Hz). Note the similar
levels of ADS in CMi and VHT bres. Asterisks mark
signicant differences to the HT bres (CMi ∗∗∗P<0.0001,
VHT ∗∗P=0.007, symp ∗P=0.015; one-way ANOVA with
Scheffé post hoc test). B, resting conduction velocity in the
main C-nociceptor classes. Numbers in brackets show the
number of units recorded. Asterisks mark signicant
differences relative to HT bres (CMi ∗∗P=0.0015, VHT
∗∗∗P=0.00017, symp ∗∗∗ P<0.0001; one-way ANOVA
with Scheffé post hoc test). C, changes in response latency
in the C-bres stimulated for 3 min at 2 Hz. Note the
different levels of total ADS separating the three subclasses
of C-nociceptors. Asterisks mark signicant differences
relative to the HT bres (CMi ∗∗∗P<0.0001, VHT
∗∗∗P=0.0002, ∗∗∗ symp P<0.0001; ANOVA with Scheffé
post hoc test). D, immediately after the 2 Hz tetanus for
3 min, the response latencies recover at similar speeds in
both CMi and VHT units. In contrast, HT units recover
signicantly faster. Asterisks mark signicant differences
relative to the HT bres (CMi ∗∗∗P<0.0001, VHT
∗∗P=0.0047, symp ∗∗∗ P<0.0001; ANOVA with Scheffé
post hoc test). Raw data for latency changes of single bres
are plotted colour-coded for bre class.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 599.5 Maximum following frequency separates nociceptor classes 1601
Scheé test, Fig. 3B). In comparison, HT bres were
signicantly faster (1.16 ±0.37 m/s; n=57 in 48 pigs;
P=0.0014 vs.CMI;P=0.0002 vs.VHT;one-way
ANOVA with Scheé post hoc test; Fig. 3B).
Next, we assessed ADS development during a 2 Hz
stimulation. Both CMi and VHT bres accumulated more
ADS than HT bres (P<0.0001; one-way ANOVA with
Scheé test; Fig. 3C). However, our analysis revealed a
small but signicant dierence in total ADS between CMi
and VHT units (CMi: 32 ±12.0%; n=35 in 32 pigs; VHT:
27.2 ±9.4%; n=19 in 17 pigs; P=0.02; one-way ANOVA
with Scheé test).
In congruence with the 2010 report, CMi and VHT
bres displayed no dierence in the speed of the recovery
as assessed 20 s after the 2 Hz stimulation (CMi:
15.5 ±6.7%; n=35 in 32 pigs; VHT: 16.1 ±6.0%; n=19
in 17 pigs; P=0.97; one-way ANOVA with Scheé test;
Fig. 3D). In contrast, HT bres recovered signicantly
faster (25.8 ±6.8%; n=56 in 47 pigs; P<0.0001 vs.
CMi and 0.004 vs. VHT). Sympathetic bres were clearly
separated from the nociceptive bres by their extremely
fast recovery (Fig. 3D).
Peak following frequency discriminates between
classes of unmyelinated bres in pig
We assessed how reliably dierent bre classes follow
electrical stimulation of incremental frequencies between
5 and 100 Hz (Fig. 4). Most HT nociceptors faithfully
Figure 4. Maximum following frequencies separates C-bre classes
Percentage of electrical stimuli that successfully evoked action potentials in different classes of unmyelinated
bres in the pig (CMi, silent nociceptors; VHT, silent nociceptors with a very high mechanical threshold; HT,
regular mechanosensitive nociceptors; symp, sympathetic efferent bres) for incremental frequencies (5, 10, 20,
50, 100 Hz) depicted as median and quartiles. For each frequency, 3 repetitions (rep. 1, 2, 3) of bursts were
applied, separated by a 10 s pause. Note that a strong frequency-dependent conduction block occurred selectively
among the subclass of CMi nociceptors. Median and quartiles are shown with number of recorded bres for each
column. Analysis was done by Kruskal-Wallis ANOVA and Dunn’s multiple comparison post hoc test. Asterisks
mark signicant differences to CMi-bres (+HT, ∗VHT, #symp; one, two and three symbols represent P<0.05,
P<0.01 and P<0.001). The actual Plevels (5 Hz rep. 1, 2, 3/10 Hz rep. 1, 2, 3/50 Hz rep. 1, 2, 3/100 Hz rep.
1, 2, 3) were HT: 0.009, 0.00027, 0.00029/0.145, 0.0015, 0.0018/0.42, 0.15, 0.14/0.00012, <0.0001, <0.0001,
VHT: 0.045, 0.0016, <0.0001/0.58, 0.017, 0.023/0.62, 0.1, 0.08/0.046, 0.05, 0.034 and symp: 0.15, 0.015,
0.018/0.98, 0.033, 0.015/0.12, 0.018, 0.021/0.17, 0.0083, 0.00058. Decline of response rate in CMi nociceptors
upon repetitions was tested by Friedmann ANOVA (&P<0.05; difference to rst run). The actual Plevels (5 Hz
1:2, 1:3/10 Hz 1:2, 1:3/20 Hz 1:2, 1:3/ 100 Hz 1:2, 1:3) were 0.012, 0.013/0.058, 0.058/0.18, 0.02/0.019, 0.019.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

1602 F. Werland and others J Physiol 599.5
propagate pulses delivered at frequencies of up to 100 Hz
(median 100%) without conduction failures. In contrast,
‘true’ CMi nociceptors had signicantly lower maximum
following frequencies compared to all other bre classes,
withblockedactionpotentialsevenattherst25pulses
at 5 Hz (median response rate of 94% (Fig. 4). Repetition
of stimulation resulted in a further decrement of the
response rate to 66% and 52% for the second and third
repetition (P=0.003; Kruskal-Wallis ANOVA; Fig. 4).
At 100 Hz, the response rate decreased in a similar
manner from a median of 66% to 25%; P=0.006;
Kruskal-Wallis ANOVA, Fig. 4). Unexpectedly, VHT units
presented a completely dierent pattern. Very much
like HT nociceptors, VHT units faithfully followed all
electrical stimulations up to 100 Hz (median response
rate of 100%, 95% and 87.5% for the three repetitions at
100Hz;Fig.4).
A total of 11 sympathetic eerent bres (in 9 pigs)
were recorded for comparison. They followed electrical
stimulations up to 100 Hz to a similar degree as the HT
Figure 5. Activity-dependent slowing of conduction at high stimulation frequencies
A, activity-dependent slowing of response latency (mean ±SD) to three bursts of 25 pulses at 5 Hz with 10 s pause
discriminates between pig silent nociceptors ‘CMi’ on one hand and the remaining other bre classes (VHT, silent
nociceptors with a very high mechanical threshold; HT, regular mechanosensitive nociceptors; symp, sympathetic
efferent bres) on the other. CMi bres differed signicantly from each other bre class (###P<0.0001; repeated
measures ANOVA with Scheffé post hoc test). Number of bres included for the rst and last pulse in each burst
is given for each bre class. B, response latencies of the rst 4 pulses of the rst and the rst 2 pulses of the
second burst (after a 10 s pause) are shown for the different bre classes. CMi nociceptors had signicantly higher
increases of latency compared to VHT nociceptors at 5 (P<0.0001), 10 (P=0.005), 20 (P<0.0001) and 100 Hz
(P=0.0002) (##P<0.01; ### P<0.0001; repeated measures ANOVA with Scheffé post hoc tests). VHT and HT
nociceptors differed at 5 (P=0.037), 10 (P=0.048) and 100 Hz (P=0.003) stimulation (+P<0.05; ++P<0.01;
repeated measures ANOVA with Scheffé post hoc tests). Sympathetic bres showed a signicant drop in response
latency for the 2nd pulse at 5 (P=0.023), 10 (P=0.007) and 50 Hz (P=0.038) in the second stimulation sequence
(∗P<0.05, ∗∗P<0.01; repeated measures ANOVA with Scheffé post hoc tests). Raw data for latency changes of
single bres are plotted colour-coded for bre class.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 599.5 Maximum following frequency separates nociceptor classes 1603
units. Interestingly, those few bres that showed drop-outs
of action potentials in the rst 100 Hz burst became more
excitable upon repetition reecting activity-dependent
increase of excitability in contrast to the general tendency
in nociceptors that show decreasing excitability upon
repetitive activation.
Pattern of activity-dependent slowing of VHT nociceptors
switches towards HT at higher frequencies in pig. Since
the activity-dependent slowing (ADS) of conduction
at 2 Hz did not reliably dierentiate between CMi and
VHT nociceptors, we assessed ADS at higher frequencies.
Regular CMi nociceptors accumulated signicantly
more ADS than all other classes of nociceptors at 5 Hz
stimulation (P<0.0001, repeated-measures ANOVA;
Scheé post hoc;Fig.5A), whereas no signicant
dierence was found between VHT and HT nociceptors
(P=0.9, Scheé post hoc). Interestingly, all C-nociceptors
gradually increased their latency during electrical
stimulation, whereas sympathetic eerents displayed
an absolute speeding of the second pulse. The speeding
was exaggerated when the stimulation was repeated
after a 10 s pause when the units can be expected to be
hyperpolarized (Fig. 5A).
For higher stimulation frequencies, ADS accumulated
by the CMi nociceptors in the initial 4 stimuli was also
signicantly larger than in all bre classes (P<0.0001,
repeated-measures ANOVA; Scheé post hoc;Fig.5B).
This dierence was most obvious when focusing on the
ADS in the second stimulation block after the 10 s
pauseforallstimulationfrequencies.CMinociceptors
sharply diered from all other C-bre classes by showing
a delayed rst response after the 10 s pauses for each
stimulation frequency (Fig. 5B), reecting a long-lasting
ADS. VHT nociceptors displayed more ADS compared
to HT nociceptors at 5 (P=0.037), 10 (P=0.048) and
100 Hz (P=0.003) (repeated-measures ANOVA; Scheé
post hoc;Fig.5B), but did not dier signicantly at the
other stimulation frequencies.
Different recovery cycles in pig C-bre classes. Latency
changes during twin pulse stimulation at dierent inter-
vals reect post-spike excitability changes of the axons.
When the latency dierence from the rst to the second
pulseisplottedagainsttheinter-pulseinterval(IPI,
in milliseconds), supernormal conduction was evident
exclusively in the sympathetic eerents between 20 and
200 ms following the rst pulse. In contrast, the aerent
subclasses displayed dierent degrees of subnormality
(Fig. 6). While there was an obviously longer lasting
subnormality in CMi vs. HT nociceptors (P<0.0001,
ANOVA, Scheé post hoc for each inter pulse interval
except P=0.0029 for 20 ms), the pattern for VHT bres
was intermediate (Fig. 6).
NGF-evoked sensitization of pig CMi bres
After intradermal NGF injection (10 μg, 3 weeks
before recording) we have identied ‘sensitized CMi’
nociceptors that were characterized by pronounced ADS,
but had mechanical activation thresholds below 150 mN
(Hirth et al. 2013). Using identical criteria to identify
sensitized CMi nociceptors in our study, we compared
their peak following frequencies to the regular CMi and
the other nociceptor classes recorded in naive animals.
Considerably higher response rates were assessed for 5, 10
and20Hz.Forinstance,5Hzstimulationelicitedamedian
response rate of 100% in ‘sensitized CMi’ in the 1st and
2nd trials of 25 pulses and 96% in the 3rd trial. In control
CMi, we assessed for the same protocol median response
rates of 92%, 48% and 36% (1st run P=0.21, 2nd and 3rd
runs: P=0.03; Mann-Whitney Utest, Fig. 7A). Similar
behaviour was encountered for stimulations at 10 Hz and
20 Hz (Fig. 7A). No signicant changes were observed at
100 Hz.
Interestingly, control CMi units almost halved their
response rate from the 1st to the 2nd trials at all
stimulation frequencies, whereas ‘sensitized CMi’ were
less sensitive to repetition. Especially for the 5, 10 and
Figure 6. Time-dependent excitability changes after an action
potential was investigated by twin stimulation (‘recovery
cycles’) in pig bres
The response latency of the trailing action potential was assessed as
relative change to the rst action potential. Positive values denote
slower conduction or subnormality (above dotted line) whereas
negative values denote supernormal conduction (below dotted line).
Recovery cycles were assessed in silent nociceptors. CMi, silent
nociceptors; VHT, silent nociceptors with a very high mechanical
threshold; HT, regular mechanosensitive nociceptors; symp,
sympathetic efferent bres (mean ±SD; number of bres per
interval given). VHT had signicantly less subnormality compared to
CMi nociceptors at 10 and 200 ms (∗∗P=0.0045; ∗∗∗ P<0.0001;
ANOVA, Scheffé post hoc tests), but also more compared to HT
nociceptors at 200 ms (+P=0.013; ANOVA, Scheffé post hoc tests).
Raw data for latencies of single bres are plotted colour-coded for
bre class.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

1604 F. Werland and others J Physiol 599.5
20 Hz stimulation, the repetition diminished CMI’s
median response rates much more strongly. However, at
higher frequencies (50 and 100 Hz) ‘sensitized CMi’
also reduced their response rate upon repetition.
This behaviour of ‘sensitized CMi’ is clearly distinct
from the nearly 100% response rate observed in VHT
nociceptors with very high mechanical thresholds (see
Fig. 4). Therefore, our classication as ‘sensitized CMi’
nociceptors after NGF is more probable than ‘partially
desensitized VHT’ nociceptors.
In addition, we compared slowing patterns of the
bre classes at repetitive 5, 10 and 20 Hz stimulation
(Fig. 7B). Whereas VHT units slow as much as HT
units, the supposedly ‘sensitized CMi’ nociceptors follow
the trend of regular CMi nociceptors at all stimulation
frequencies, accumulating roughly 50% of the ADS of the
CMi nociceptors and have signicantly increased slowing
compared to VHT and HT nociceptors in particular at the
3rd repetition of the 5 Hz stimulus (Fig. 7B).
C-nociceptors with very high mechanical thresholds
and pronounced activity-dependent slowing in
humans
In our recordings we separated mechanosensitive
C-nociceptors, mechano-insensitive nociceptors and
sympathetic eerent bres based their characteristic
Figure 7. Maximum following frequency of silent nociceptors is increased 3 weeks after local injection
of nerve growth factor
A, following frequencies of regular silent nociceptors CMi in control pigs and CMi nociceptors sensitized after
injection of nerve growth factor (CMi sens.) are shown (median, quartiles). Note the increase in the response rate
of CMi sens. stimulated at 5 (P=0.033, P=0.034), 10 (P=0.057, P=0.04) and 20 Hz (P=0.042, P=0.043).
Differences were assessed by Mann Whitney Utest. Asterisks mark signicant differences (∗P<0.05). Number
of bres for 5/10/20/50/100 Hz: CMi, 7/7/4/4/6, in 4 pigs; CMi sens., 4/4/4/4/3/4, in 3 pigs. B, activity-dependent
slowing at 5, 10 and 20 Hz discriminates among CMi sens. and VHT nociceptors. Response latencies (mean ±SD)
increase during stimulation with 5 (left), 10 (middle) and 20 Hz stimulation (right). The slowing by the CMi sens.
nociceptors was less than in native CMi (P<0.0001 at 5 Hz, P=0.0056 at 10 Hz), but signicantly larger compared
to both VHT (P=0.79, P=0.050, P=0.030 at 5 Hz run 1, 2, 3) and HT nociceptors (P=0.06, P=0.033, P=0.027
at 5 Hz run 1, 2, 3, P=0.026 at 10 Hz). Asterisks mark signicant differences in comparison to the CMi sens.
(∗P<0.05; ∗∗P<0.01; ∗∗∗ P<0.001) (one-way ANOVA with Scheffé post hoc test, using ‘bre class’ as factor).
Number of bres at start of stimulus train (5 Hz run 1/5 Hz run 2/5 Hz run 3/10 Hz/20 Hz): CMi sens., 4/4/4/4/4,
in 3 pigs; CMi, 6/6/6/7/4, in 4 pigs; VHT, 5/5/5/4/4, in 3 pigs; HT, 5/5/5/8/7, in 4 pigs.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 599.5 Maximum following frequency separates nociceptor classes 1605
activity-dependent slowing to 70 electrical pulses of 1/8,1
4
and 1
2Hz (1.96 ±1.17%, n=141; 8.49 ±2.15%, n=108;
3.50 ±0.96%, n =20) and on their mechanical response.
As previously reported, the median mechanical threshold
was 30 mN (14–46 mN, quartiles, n =153). In 6 of these
mechanosensitive units, the activity-dependent slowing
exceeded 5%, i.e. the cut-o for silent nociceptors.
Interestingly, ve of them had mechanical thresholds
that fell far outside the regular range of polymodal
nociceptors (Fig. 8). Moreover, these units with high
mechanical thresholds also had a signicantly slower
conduction velocities (0.85 ±0.1, n=5vs. 0.98 ±0.1 m/s,
n=139; P=0.009, ttest; mean ±SD), a higher trans-
cutaneous electrical threshold (24.5 ±26 mA; n=4vs.
3.8 ±2.7 mA; n=99; P<0.0001, ttest) and a higher
activation threshold to radiant heat (44.3 ±4.7; n=5vs.
40.9 ±3.0°C; n=125; P=0.0001, ttest) than the regular
polymodal nociceptors. Their activity-dependent slowing
was 5.7 ±0.6% which was signicantly smaller than in
regular silent nociceptors of the same sample (8.5 ±2.1%;
n=108; P=0.019). In silent nociceptors, transcutaneous
electrical thresholds (61.3 ±20 mA, n=57; P=0.0009)
were also higher than in those nociceptors with very
high mechanical thresholds whereas dierences in heat
activation thresholds (silent nociceptors 48.2 ±7.8°C,
n=56) did not reach a signicant level (P=0.27, ttest).
Discussion
C-nociceptors have traditionally been assumed to have
a very limited capacity for high frequency discharge.
However, we demonstrate here that only silent nociceptors
are restricted to low following frequencies of about
5 Hz. In contrast, polymodal nociceptors reliably conduct
trains of action potentials at a frequency of 100 Hz.
Sensitization with intracutaneous nerve growth factor
(NGF) stepped up the maximum following frequency of
silent nociceptors, reecting increased axonal excitability.
Opening of such an axonal ‘low pass lter’ might allow
discharge of silent nociceptors to reach the spinal cord at
higher frequencies than could be generated under normal
conditions. In a sensitized state, they might therefore
contributespecicallytopainsensationsininammatory
pain.
Testing for peak following frequencies identied a sub-
population of C-nociceptors with a very high mechanical
threshold (>150 mN) that showed a pronounced
activity-dependent slowing similar to silent nociceptors
when stimulated at low frequencies (up to 2 Hz) in pig and
humans (see also Obreja et al. 2010), but these nociceptors
behaved like polymodal nociceptors when stimulated at
higher frequencies. This nociceptor class appears to be
optimally equipped to encode acute noxious mechanical
stimulation without prior sensitization.
Axonal excitability vs. sensory characteristics for
classication of nociceptor classes
Classication of nociceptors has traditionally been based
on sensory characteristics such as mechanical, chemical
and heat responsiveness (‘polymodal nociceptor’)
(Perl, 1996). Expression of corresponding transduction
proteins such as temperature-sensitive transient receptor
potential channels, e.g. TRPV1 or TRPM8, is therefore
deemed suitable to molecularly dene functional
classes (Belmonte & Viana, 2008; Julius, 2013). Recent
classication approaches investigating expression proles
of sensory bre classes also indicate that axonal ion
Figure 8. Histogram of mechanical thresholds of
human C-nociceptors recorded in healthy
volunteers between 1998 and 2003
In the inset the slowing of conduction ‘total slowing’
induced by 70 pulses applied at 0.125, 0.25 and
0.5 Hz is shown in relation to the mechanical
threshold. Fibres in which the total slowing exceeds
5% are marked with lled circles and their mechanical
thresholds in the histogram are shown in black (raw
data are provided in the spreadsheet Table S1).
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

1606 F. Werland and others J Physiol 599.5
channels contribute to their characteristic encoding
patterns (Zheng et al. 2019), supporting older ndings
that axonal characteristics such as activity-dependent
slowing patterns also segregate in sensory bre classes
(Raymond et al. 1990; Gee et al. 1996; Serra et al. 1999;
Schmelz et al. 2000a). This development conrms the
concept of Belmonte and Viana that a “characteristic
combinatorial expression of dierent ion channels in each
neuronal type … nally determines their transduction
and impulse ring properties” (Belmonte & Viana, 2008).
Therefore, the class-specic integration of transduction,
spike initiation and conduction within a single nociceptive
cell should be regarded as the ‘fundamental unit in pain’
(Reichling et al. 2013).
Both, sensory and axonal response patterns
concordantly separate polymodal and silent nociceptors
in a dichotomous way, whereas classication of very
high threshold mechanonociceptors is more complex.
Classically, units with extraordinary high thresholds were
classied as mechano-insensitive aerents (MIA). In
monkey,athresholdof6barwasset,whichisabout3×
the median threshold of polymodal nociceptors (Meyer
et al. 1991). In pig, a population of very high threshold
(>150 mN) mechanonociceptors was identied, but based
on their similar slowing pattern they were included into
the silent nociceptor class (Obreja et al. 2010). In humans,
only units not responding to 600 mN were classied as
silent nociceptors (Schmidt et al. 1995, 2000a). Based
on our results, such binary classication into mechano-
sensitive and silent nociceptors may prove problematic:
when units are dened as silent nociceptors by their
pronounced activity-dependent slowing, mechanical
responses to 600 mN have been suggested to indicate
mechanical sensitization (Orstavik et al. 2006). However,
these responses are ambiguous as they may also be
found in regular very high threshold units. Validation
of mechanical sensitization therefore requires sensitivity
to mechanical stimuli less than or equal to 150 mN.
Unfortunately, these tests were not performed at that
time. Moreover, a direct comparison of the dierences
in following frequencies shown in pig recordings is also
dicultassuchhighfrequentstimuliaretoopainfulfor
human subjects in microneurography experiments. A
shorter version might be developed for human studies.
Our data do not enable us to explain the peculiar
behaviour of very high threshold mechanosensitive
nociceptors showing pronounced slowing like silent
nociceptors at low frequencies (<2Hz)asopposed
to polymodal nociceptor-like slowing for higher
frequencies (>5 Hz). Activity-dependent excitability
changes are determined by a complex interaction of
hyperpolarization, sodium channel inactivation and
accumulation of intracellular sodium (De Col et al.
2008). According to modelling results, more depolarized
resting membrane potentials and an increase in density
of NaV1.8 channels leads to CMI-like behaviour
(Petersson et al. 2014). We might speculate that the
resting membrane potential of VHT units is similarly
depolarized to silent nociceptors, but the density
of NaV1.7 channels could be higher than in silent
nociceptors. Upon activity-dependent hyperpolarization
Nav1.7 may therefore predominate, leading to lower
sodium inux and less ADS (Tigerholm et al. 2014). At
higher frequencies, such as 5 Hz and more, we registered
a CMI-like ADS increase in VHT only for the rst two
pulses, followed by a limited ADS increase as in HT
nociceptors. At 5 Hz stimulation, the rst interstimulus
interval of 200 ms will not be sucient to deinactivate
Nav1.8 and deinactivation of NaV1.7 channels requires
hyperpolarization that is accumulating upon repetitive
stimulation.
The potential functional role of very high threshold
mechanosensitive nociceptors is a key question. There
is a puzzling inconsistency regarding pain induced
bypolymodalnociceptors:increasingheatstimuli
induce congruent increases in discharge in polymodal
nociceptors and in pain under normal and sensitized
conditions (Gybels et al. 1979; LaMotte et al. 1982;
Robinson et al. 1983; Torebjörk et al. 1984a;Tillmanet al.
1995). In contrast, the median activation threshold
of human polymodal nociceptors is only 30 mN,
which is far below the actual pain threshold. Spinal
inhibition of C-nociceptor input by concomitantly
activatedlowthresholdA-bres,asshowndirectlyin
humans (Torebjörk, 1985), could explain the dierence
between mechanical and heat pain. However, poly-
modal nociceptors also showed a clear saturation of their
response even at the level of the pain threshold in cat
(Garell et al. 1996) and monkey skin (Slugg et al. 2000). In
contrast, mechanonociceptors with very high thresholds
(‘mechanically insensitive aerents’, MIAs) encoded
intothepainfulrangeupto100g(Garellet al. 1996).
Thus, activation of high threshold mechanosensitive
nociceptors could contribute to pain inicted by strong
mechanical stimuli in normal skin. In this regard, their
ability to produce high discharge frequencies supports
the wide dynamic range required to encode intense
mechanical stimuli.
Peak axonal discharge frequency in C-bres –
modulation by NGF
The maximum discharge frequency of C-bres is generally
thought to be in a low range (about 10–30 Hz)
(Thalhammer et al. 1994; Gee et al. 1996; Belmonte &
Viana, 2008; Zheng et al. 2019). Peak frequencies of
180 Hz were assessed in human polymodal nociceptors
for trains of a few action potentials only under conditions
of supernormal conduction (Weidner et al. 2002). In
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 599.5 Maximum following frequency separates nociceptor classes 1607
ourstudy,peakfollowingfrequencieswereunexpectedly
high in polymodal and very high threshold mechano-
sensitive C-nociceptors. It is important to note that
these high frequencies were given as bursts of 24
pulses, whereas previous microneurography studies have
shown that stimulation periods of half a minute even
at 10 Hz induces adaptation of the pain response
and concomitant reduction of the microneurographically
assessed C-bre volley (Torebjörk et al. 1984b). Therefore,
our data are relevant for the interpretation of short-lasting
high-frequency stimulation protocols used for example
in the induction of spinal sensitization in human pain
models (Klein et al. 2004).
Onemightspeculatethataxonalinammation
modulates the lter characteristics, thereby potentially
sensitizing nociception purely by conductive mechanisms.
Actually, there are already examples of relevant
modulations of the nociceptive discharge pattern
by changes of axonal excitability. Moving the spike
initiation site closer to the sensory ending increased
the discharge frequency of corneal nociceptors after
capsaicin application (Goldstein et al. 2019). Moreover,
reduced conduction failure upon electrical stimulation
at 5–10 Hz was observed in polymodal nociceptors in
streptozocin-induced diabetic neuropathy in rat (Sun
et al. 2012). Interestingly, a reduced activity-dependent
slowing of C-aerents following complete Freund’s
adjuvants (CFA) arthritis was found even when measuring
conduction of the dorsal root (Dickie et al. 2017), which
indicates that the conduction properties of the entire axon
of the primary aerent can be modulated. More directly,
intradermal CFA injection in the guinea-pig increased
maximum following frequency of C-nociceptors
and reduced the width of the action potential and
after-hyperpolarization (Djouhri et al. 2001), and also
increased conduction velocity and reduced electrical
threshold (Djouhri & Lawson, 2001). Sequestering NGF
in this model normalized following frequency and action
potentialwidth,butnotafter-hyperpolarization(Djouhri
et al. 2001). Unfortunately, our extracellular recordings
do not allow us to assess these parameters and to unravel
the molecular mechanisms underlying the particular low
maximum following frequencies in silent nociceptors and
their modulation by NGF. However, higher expression
of NaV1.7 in silent nociceptors would be compatible
with reduced electrical thresholds, increased conduction
velocity and higher following frequency.
Ourdatashowanincreaseofmaximumfollowing
frequency in silent nociceptors following nerve growth
factor application. This result is in line with higher
maximum following frequencies in the CFA model
in guinea-pigs (Djouhri et al. 2001) and the reduced
activity-dependent slowing of conduction that we found
in silent nociceptors after NGF in pigs and humans
(Obreja et al. 2011, 2018). Functionally, we have already
shown mechanical sensitization in silent nociceptors in
pigs (Hirth et al. 2013) and humans (Obreja et al. 2018).
It must be noted that pain upon tonic pressure that has
been shown to correlate to activation of silent nociceptors
(Schmidt et al. 2000) is increased at the site of NGF
injection in humans, where squeezing is particularly
painful (Rukwied et al. 2010). It is therefore tempting to
postulate that sensitization of sensory endings of silent
nociceptors in combination with their increased axonal
excitability leads to the full-blown NGF hyperalgesia, both
in human experimental models (Rukwied et al. 2013)
and in actual chronic inammatory pain where anti-NGF
strategies have been proven to act as analgesics (Denk
et al. 2017). The protracted time course of hyperalgesia,
with a maximum at 3 weeks after intracutaneous injection
of NGF in humans (Rukwied et al. 2010), suggests
protein biosynthesis or structural changes as underlying
mechanisms. Among the NGF targets for upregulation
are nociceptor-specic ion channels, including the fast
repriming NaV1.8 (Bennett et al. 2019) and possibly
NaV1.7 (Gould et al. 2000; Schaefer et al. 2018), which
reduces activation thresholds; higher expression of both
channels is expected to increase peak following frequency.
However, is important to note that pain intensity is not
simply encoded by frequency, and that spike timing and
temporal pattern are also of crucial importance (Cho
et al. 2016; Barkai et al. 2020). Moreover, higher axonal
discharge frequencies as recorded in the periphery do
notnecessarilyreachthespinalcord,astheycanstillbe
ltered at the T-junction at the level of the dorsal root
ganglion (Du et al. 2017).
Our data provide evidence that there is a sub-
population of C-nociceptors in humans and pigs that is
distinct from classic polymodal and silent nociceptors,
characterized by a very high mechanical threshold and
a high maximum following frequency (about 100 Hz)
that would allow for encoding strong mechanical stimuli
in normal skin. In contrast, silent nociceptors have a
very low maximum following frequency (about 5 Hz)
under normal conditions, but can be sensitized by
NGF to conduct trains of action potentials at 10-fold
higher frequencies, thereby stepping up peak frequencies
reachingthespinalcord.Insummary,ourdatasupport
the concept that rather than just acting as passive cables,
nociceptive axons participate in the encoding process and
its modulation.
References
Barkai O, Butterman R, Katz B, Lev S & Binshtok AM
(2020). The input-output relation of primary nociceptive
neurons is determined by the morphology of the peripheral
nociceptive terminals. JNeurosci40, 9346–9363.
Belmonte C & Viana F (2008). Molecular and cellular limits to
somatosensory specicity. Mol Pain 4, 1744-8069-4-14.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

1608 F. Werland and others J Physiol 599.5
Bennett DL, Clark AJ, Huang J, Waxman SG & Dib-Hajj SD
(2019). The role of voltage-gated sodium channels in pain
signaling. Physiol Rev 99, 1079–1151.
Bostock H, Campero M, Serra J & Ochoa J (2003). Velocity
recovery cycles of C bres innervating human skin. JPhysiol
553, 649–663.
Campbell JN & Meyer RA (1983). Sensitization of
unmyelinated nociceptive aerents in monkey varies with
skin type. JNeurophysiol49, 98–110.
Campero M, Serra J, Bostock H & Ochoa JL (2004). Partial
reversal of conduction slowing during repetitive stimulation
of single sympathetic eerents in human skin. Acta Physiol
Scand 182, 305–311.
Campero M, Serra J & Ochoa JL (1996). C-polymodal
nociceptors activated by noxious low temperature in human
skin. JPhysiol497, 565–572.
ChoK,JangJH,KimSP,LeeSH,ChungSC,KimIY,Jang
DP & Jung SJ (2016). Analysis of nociceptive information
encoded in the temporal discharge patterns of cutaneous
C-bers. Front Comput Neurosci 10, 118.
De Col R, Messlinger K & Carr RW (2008). Conduction
velocity is regulated by sodium channel inactivation in
unmyelinated axons innervating the rat cranial meninges.
JPhysiol586, 1089–1103.
Denk F, Bennett DL & McMahon SB (2017). Nerve growth
factor and pain mechanisms. Annu Rev Neurosci 40, 307–325.
Dickie AC, McCormick B, Lukito V, Wilson KL &
Torsney C (2017). Inammatory pain reduces C ber
activity-dependent slowing in a sex-dependent manner,
amplifying nociceptive input to the spinal cord. JNeurosci
37, 6488–6502.
Djouhri L, Dawbarn D, Robertson A, Newton R & Lawson SN
(2001). Time course and nerve growth factor dependence of
inammation-induced alterations in electrophysiological
membrane properties in nociceptive primary aerent
neurons. JNeurosci21, 8722–8733.
Djouhri L & Lawson SN (2001). Increased conduction velocity
of nociceptive primary aerent neurons during unilateral
hindlimb inammation in the anaesthetised guinea-pig.
Neuroscience 102, 669–679.
Du X, Hao H, Yang Y, Huang S, Wang C, Gigout S, Ramli
R,LiX,JaworskaE,EdwardsI,DeucharsJ,Yanagawa
Y, Qi J, Guan B, Jae DB, Zhang H & Gamper N (2017).
Local GABAergic signaling within sensory ganglia controls
peripheral nociceptive transmission. JClinInvest127,
1741–1756.
Garell PC, McGillis SL & Greenspan JD (1996). Mechanical
response properties of nociceptors innervating feline hairy
skin. JNeurophysiol75, 1177–1189.
Gee MD, Lynn B & Cotsell B (1996). Activity-dependent
slowing of conduction velocity provides a method for
identifying dierent functional classes of C-bre in the rat
saphenous nerve. Neuroscience 73, 667–675.
George A, Serra J, Navarro X & Bostock H (2007). Velocity
recovery cycles of single C bres innervating rat skin. J
Physiol 578, 213–232.
Goldstein RH, Barkai O, Inigo-Portugues A, Katz B, Lev S
& Binshtok AM (2019). Location and plasticity of the
sodium spike initiation zone in nociceptive terminals in
vivo. Neuron 102, 801–812.e5 e805.
Gould HJ, Gould TN, England JD, Paul D, Liu ZP & Levinson
SR (2000). A possible role for nerve growth factor in the
augmentation of sodium channels in models of chronic
pain. Brain Res 854, 19–29.
Gybels J, Handwerker HO & Van HJ (1979). A comparison
between the discharges of human nociceptive nerve bres
and the subject’s ratings of his sensations. JPhysiol292,
193–206.
Haring M, Zeisel A, Hochgerner H, Rinwa P, Jakobsson JET,
Lonnerberg P, La Manno G, Sharma N, Borgius L, Kiehn O,
Lagerstrom MC, Linnarsson S & Ernfors P (2018). Neuronal
atlas of the dorsal horn denes its architecture and links
sensory input to transcriptional cell types. Nat Neurosci 21,
869–880.
Hirth M, Rukwied R, Gromann A, Turnquist B, Weinkauf B,
FranckeK,AlbrechtP,RiceF,HagglofB,RingkampM,
Engelhardt M, Schultz C, Schmelz M & Obreja O (2013).
Nerve growth factor induces sensitization of nociceptors
without evidence for increased intraepidermal nerve ber
density. Pain 154, 2500–2511.
Julius D (2013). TRP channels and pain. Annu Rev Cell Dev
Biol 29, 355–384.
Klein T, Magerl W, Hopf HC, Sandkuhler J & Treede RD
(2004). Perceptual correlates of nociceptive long-term
potentiation and long-term depression in humans. JNeuro-
sci 24, 964–971.
LaMotte RH & Thalhammer JG (1982). Response properties
of high-threshold cutaneous cold receptors in the primate.
Brain Res 244, 279–287.
LaMotte RH, Thalhammer JG, Torebjörk HE & Robinson CJ
(1982). Peripheral neural mechanisms of cutaneous hyper-
algesia following mild injury by heat. JNeurosci2, 765–781.
Li C, Wang S, Chen Y & Zhang X (2018). Somatosensory
neuron typing with high-coverage single-cell RNA
sequencing and functional analysis. Neurosci Bull 34,
200–207.
Li CL, Li KC, Wu D, Chen Y, Luo H, Zhao JR, Wang SS, Sun
MM,LuYJ,ZhongYQ,HuXY,HouR,ZhouBB,BaoL,
Xiao HS & Zhang X (2016). Somatosensory neuron types
identied by high-coverage single-cell RNA-sequencing and
functional heterogeneity. Cell Research 26, 967.
Lynn B, Faulstroh K & Pierau FK (1995). The classication
and properties of nociceptive aerent units from the skin of
the anaesthetized pig. Eur J Neurosci 7, 431–437.
Meyer RA & Campbell JN (1988). A novel electrophysiological
technique for locating cutaneous nociceptive and chemo-
specic receptors. Brain Res 441, 81–86.
Meyer RA, Davis KD, Cohen RH, Treede RD & Campbell
JN (1991). Mechanically insensitive aerents (Mias) in
cutaneous nerves of monkey. Brain Res 561, 252–261.
Obreja O, Hirth M, Turnquist B, Rukwied R, Ringkamp
M & Schmelz M (2012). The dierential eects of two
sodium channel modulators on the conductive properties
of C-bers in pig skin in vivo. Anesth Analg 115, 560–571.
Obreja O, Ringkamp M, Namer B, Forsch E, Klusch A,
Rukwied R, Petersen M & Schmelz M (2010). Patterns of
activity-dependent conduction velocity changes dierentiate
classes of unmyelinated mechano-insensitive aerents
includingcoldnociceptors,inpigandinhuman.Pain 148,
59–69.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 599.5 Maximum following frequency separates nociceptor classes 1609
Obreja O, Ringkamp M, Turnquist B, Hirth M, Forsch E,
Rukwied R, Petersen M & Schmelz M (2011). Nerve growth
factor selectively decreases activity-dependent conduction
slowing in mechano-insensitive C-nociceptors. Pain 152,
2138–2146.
Obreja O, Rukwied R, Nagler L, Schmidt M, Schmelz M &
Namer B (2018). Nerve growth factor locally sensitizes
nociceptors in human skin. Pain 159, 416–426.
Obreja O & Schmelz M (2010). Single-ber recordings of
unmyelinated aerents in pig. Neurosci Lett 470, 175–179.
Orstavik K, Namer B, Schmidt R, Schmelz M, Hilliges M,
Weidner C, Carr RW, Handwerker H, Jorum E & Torebjork
HE (2006). Abnormal function of C-bers in patients with
diabetic neuropathy. JNeurosci26, 11287–11294.
Perl ER (1996). Cutaneous polymodal receptors:
characteristics and plasticity. Prog Brain Res 113, 21–37.
Petersson ME, Obreja O, Lampert A, Carr RW, Schmelz M &
Fransen E (2014). Dierential axonal conduction patterns of
mechano-sensitive and mechano-insensitive nociceptors–a
combined experimental and modelling study. PLoS One 9,
e103556.
Raymond SA, Thalhammer JG, Popitz-Bergez F & Strichartz
GR (1990). Changes in axonal impulse conduction correlate
with sensory modality in primary aerent bers in the rat.
Brain Res 526, 318–321.
Reichling DB, Green PG & Levine JD (2013). The
fundamental unit of pain is the cell. Pain 154, S2–9.
Ringkamp M, Johanek LM, Borzan J, Hartke TV, Wu G,
Pogatzki-Zahn EM, Campbell JN, Shim B, Schepers RJ
& Meyer RA (2010). Conduction properties distinguish
unmyelinated sympathetic eerent bers and unmyelinated
primary aerent bers in the monkey. PLoS One 5, e9076.
Robinson CJ, Torebjörk HE & LaMotte RH (1983).
Psychophysical detection and pain ratings of incremental
thermal stimuli: a comparison with nociceptor responses in
humans. Brain Res 274, 87–106.
RukwiedR,MayerA,KluschinaO,ObrejaO,SchleyM
& Schmelz M (2010). NGF induces non-inammatory
localized and lasting mechanical and thermal hyper-
sensitivity in human skin. Pain 148, 407–413.
RukwiedR,WeinkaufB,MainM,ObrejaO&SchmelzM
(2013). Inammation meets sensitization – an explanation
for spontaneous nociceptor activity? Pain 154, 2707–2714.
Schaefer I, Prato V, Arcourt A, Taberner FJ & Lechner
SG (2018). Dierential modulation of voltage-gated
sodium channels by nerve growth factor in three major
subsets of TrkA-expressing nociceptors. Mol Pain 14,
174480691881464.
SchmelzM,SchmidR,HandwerkerHO&TorebjorkHE
(2000a). Encoding of burning pain from capsaicin-treated
humanskinintwocategoriesofunmyelinatednervebres.
Brain 123 560–571.
Schmelz M, Schmidt R, Handwerker HO & Torebjörk HE
(2000b). Encoding of burning pain from capsaicin-treated
humanskinintwocategoriesofunmyelinatednervebres.
Brain 123, 560–571.
SchmelzM,SchmidtR,WeidnerC,HilligesM,Torebjork
HE & Handwerker HO (2003). Chemical response pattern
of dierent classes of C-nociceptors to pruritogens and
algogens. JNeurophysiol89, 2441–2448.
SchmidtR,SchmelzM,ForsterC,RingkampM,TorebjörkHE
& Handwerker HO (1995). Novel classes of responsive and
unresponsive C nociceptors in human skin. JNeurosci15,
333–341.
Schmidt R, Schmelz M, Torebjork HE & Handwerker HO
(2000). Mechano-insensitive nociceptors encode pain
evoked by tonic pressure to human skin. Neuroscience 98,
793–800.
Schmidt RF, Schaible HG, Messlinger K, Hanesch U & Pawlak
M (1994). Silent and Active Nociceptors: Structure, Functions
and Clinical Implications, eds Gebhart GF, Hammond DL &
Jensen TS, pp. 213–250.IASP Press, Seattle, USA.
Serra J, Campero M, Ochoa J & Bostock H (1999).
Activity-dependent slowing of conduction dierentiates
functional subtypes of C bres innervating human skin. J
Physiol 515, 799–811.
SerraJ,SolaR,QuilesC,Casanova-MollaJ,PascualV,
Bostock H & Valls-Sole J (2009). C-nociceptors sensitized
to cold in a patient with small-ber neuropathy and cold
allodynia. Pain 147, 46–53.
Simone DA & Kajander KC (1997). Responses of cutaneous
A ber nociceptors to noxious cold. JNeurophysiol77,
2049–2060.
Slugg RM, Meyer RA & Campbell JN (2000). Response of
cutaneous A- and C-ber nociceptors in the monkey to
controlled-force stimuli. JNeurophysiol83, 2179–2191.
Sun W, Miao B, Wang XC, Duan JH, Wang WT, Kuang F,
Xie RG, Xing JL, Xu H, Song XJ, Luo C & Hu SJ (2012).
Reduced conduction failure of the main axon of polymodal
nociceptive C-bres contributes to painful diabetic neuro-
pathy in rats. Brain 135, 359–375.
Thalhammer JG, Raymond SA, Popitz Bergez FA & Strichartz
GR (1994). Modality-dependent modulation of conduction
by impulse activity in functionally characterized single
cutaneous aerents in the rat. Somatosens Mot Res 11,
243–257.
Tigerholm J, Petersson ME, Obreja O, Lampert A, Carr
R, Schmelz M & Fransen E (2014). Modeling activity-
dependent changes of axonal spike conduction in primary
aerent C-nociceptors. JNeurophysiol111, 1721–1735.
Tillman DB, Treede RD, Meyer RA & Campbell JN (1995).
Response of C bre nociceptors in the anaesthetized
monkey to heat stimuli: correlation with pain threshold
in humans. JPhysiol485, 767–774.
Torebjörk HE (1985). Nociceptor activation and pain. Philos
Trans R Soc Lond B Biol Sci 308, 227–234.
Torebjörk HE & Hallin RG (1974). Responses in human A
and C bres to repeated electrical intradermal stimulation. J
Neurol Neurosurg Psychiatry 37, 653–664.
Torebjörk HE, LaMotte RH & Robinson CJ (1984a). Peri-
pheral neural correlates of magnitude of cutaneous pain and
hyperalgesia: simultaneous recordings in humans of sensory
judgments of pain and evoked responses in nociceptors
with C-bers. JNeurophysiol51, 325–339.
Torebjörk HE, Schady W & Ochoa JL (1984b).Anewmethod
for demonstration of central eects of analgesic agents in
man. J Neurol Neurosurg Psychiat 47, 862–869.
Turnquist B, Leverentz M & Swanson E (2004). Neural spike
classication using parallel selection of all algorithm
parameters. JNeurosciMethods137, 291–298.
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

1610 F. Werland and others J Physiol 599.5
Usoskin D, Furlan A, Islam S, Abdo H, Lonnerberg P,
Lou D, Hjerling-Leer J, Haeggstrom J, Kharchenko
O, Kharchenko PV, Linnarsson S & Ernfors P (2015).
Unbiased classication of sensory neuron types by
large-scale single-cell RNA sequencing. Nat Neurosci 18,
145–153.
Weidner C, Schmelz M, Schmidt R, Hammarberg B,
Orstavik K, Hilliges M, Torebjörk HE & Handwerker
HO (2002). Neural signal processing: the underestimated
contribution of peripheral human C-bers. JNeurosci22,
6704–6712.
Weidner C, Schmelz M, Schmidt R, Hansson B,
Handwerker HO & Torebjörk HE (1999). Functional
attributes discriminating mechano-insensitive and
mechano-responsive C nociceptors in human skin. JNeuro-
sci 19, 10184–10190.
Wooten M, Weng H J , H a r t k e T V , B o r z a n J , K l e i n A H ,
Turnquist B, Dong X, Meyer RA & Ringkamp M (2014).
Three functionally distinct classes of C-bre nociceptors in
primates. Nat Commun 5, 4122.
Zeisel A, Hochgerner H, Lonnerberg P, Johnsson A, Memic F,
van der Zwan J, Haring M, Braun E, Borm LE, La Manno
G, Codeluppi S, Furlan A, Lee K, Skene N, Harris KD,
Hjerling-Leer J, Arenas E, Ernfors P, Marklund U &
Linnarsson S (2018). Molecular architecture of the mouse
nervous system. Cell 174, 999-1014.e22 e1022.
Zheng Y, Liu P, Bai L, Trimmer JS, Bean BP & Ginty DD
(2019). Deep sequencing of somatosensory neurons
reveals molecular determinants of intrinsic physiological
properties. Neuron 103, 598-616.e7 e597.
Zimmermann K, Leer A, Babes A, Cendan CM, Carr RW,
Kobayashi J, Nau C, Wood JN & Reeh PW (2007). Sensory
neuron sodium channel Nav1.8 is essential for pain at low
temperatures. Nature 447, 856-859.
Additional information
Data availability statement
Microneurography data are provided in Table S1 and data from
single bre recordings in pigs are available upon request from
the corresponding author.
Competing interests
None declared.
Author contributions
Recordings were obtained by F.W., M.H. and O.O.; analyses
were performed by F.W., M.H., O.O. and M.S.; the concept and
experimental protocols were charted by O.O. and M.S. The rst
draftwaswrittenbyF.W.andO.O.Allauthorscontributedto
critically in revising the manuscript. All authors approved the
nal version and agree to be accountable for all aspects of the
work. All persons designated as authors qualify for authorship,
and all those who qualify for authorship are listed.
Funding
Supported by the Deutsche Forschungsgemeinschaft SFB1158
(M.S.), FOR2690 (M.S., R.R.) and a grant from the Inter-
disciplinary Center for Clinical Research Faculty of Medicine
RWTH Aachen University (B.N.) and by the DFG NA 970 3-1
(B.N.).
Acknowledgements
We thank A. Bistron and E. Forsch for excellent technical
assistance.
Open access funding enabled and organized by Projekt
DEAL.
Keywords
axon, C-bre, discharge frequency, excitability, nerve growth
factor, pain, sensitization
Supporting information
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
Statistical Summary Document
Table S1. Microneurography data
© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.