GABA Increases Electrical Excitability in a Subset of Human Unmyelinated Peripheral Axons
Citation: Carr RW, Sittl R, Fleckenstein J, Grafe P (
GABA Increases Electrical Excitability in a Subset of Human Unmyelinated Peripheral Axons
Richard W. Carr 0
Ruth Sittl 0
Johannes Fleckenstein 0
Peter Grafe 0
Fabien Tell, The Research Center of Neurobiology-Neurophysiology of Marseille, France
0 1 Institute of Physiology, Ludwig-Maximilians University , Munich, Germany , 2 Department of Anaesthesiology, Ludwig-Maximilians University , Munich , Germany
Background: A proportion of small diameter primary sensory neurones innervating human skin are chemosensitive. They respond in a receptor dependent manner to chemical mediators of inflammation as well as naturally occurring algogens, thermogens and pruritogens. The neurotransmitter GABA is interesting in this respect because in animal models of neuropathic pain GABA pre-synaptically regulates nociceptive input to the spinal cord. However, the effect of GABA on human peripheral unmyelinated axons has not been established. Methodology/Principal Findings: Electrical stimulation was used to assess the effect of GABA on the electrical excitability of unmyelinated axons in isolated fascicles of human sural nerve. GABA (0.1-100 mM) increased electrical excitability in a subset (ca. 40%) of C-fibres in human sural nerve fascicles suggesting that axonal GABA sensitivity is selectively restricted to a sub-population of human unmyelinated axons. The effects of GABA were mediated by GABAA receptors, being mimicked by bath application of the GABAA agonist muscimol (0.1-30 mM) while the GABAB agonist baclofen (10-30 mM) was without effect. Increases in excitability produced by GABA (10-30 mM) were blocked by the GABAA antagonists gabazine (1020 mM), bicuculline (10-20 mM) and picrotoxin (10-20 mM). Conclusions/Significance: Functional GABAA receptors are present on a subset of unmyelinated primary afferents in humans and their activation depolarizes these axons, an effect likely due to an elevated intra-axonal chloride concentration. GABAA receptor modulation may therefore regulate segmental and peripheral components of nociception.
Funding: This work was supported by the Deutsche Forschungsgemeinschaft (GR 801/3-1). The funding body had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Gamma-aminobutyric acid (GABA) is the predominant
inhibitory neurotransmitter in the mammalian central nervous
system. In addition to its role at synapses, GABA can also exert
effects extra-synaptically via GABAA receptors . Adult
primary afferent sensory neurons provide an interesting
example in this respect. The cell bodies of dorsal root ganglion
(DRG) neurones, which are devoid of synaptic contact, express
functional GABAA receptors (human: [4,5], rat: [6,7], cat: ,
rabbit: , chick: ). Functional GABAA receptor mediated
responses in the somata of DRG neurones is also evident in
their unmyelinated axons (rat: ) where application of
GABA results in depolarization (for review see [12,13]). The
GABAA mediated depolarization is attributed to an elevated
intracellular concentration of chloride in dorsal root ganglion
neurones , a condition established by the predominance of
NKCC1-mediated chloride uptake  over KCC2-mediated
For neurones in the central nervous system, extra-synaptic
axonal GABAA receptors are often composed of sub-units with a
high sensitivity to GABA allowing ambient concentrations of
GABA to modulate neuronal excitability . Similarly, in
peripheral nerve, extra-synaptic axonal GABAA receptors can
modulate excitability and may be involved in GABAergic
signalling between axons and neighbouring cells such as dermal
fibroblasts  and subtypes of peripheral glia [18,19]. Peripheral
glia can take up GABA  and reversal of the GABA transporter
 may result in release of the amino acid.
The effect of GABA on peripheral axons is dependent upon
the intracellular chloride concentration which is reported to
change following peripheral nerve injury in dorsal spinal horn
neurones  as well as DRG neurones . Changes in
intercellular chloride concentration will inevitably alter the
effects of GABA on primary sensory afferents both within the
spinal cord as well as in the periphery and such changes may
contribute to symptoms of neuropathic pain . The presence
of GABAA receptors on the unmyelinated axons of primary
sensory neurones is central to this concept. However it has not
been established whether the axons of human primary sensory
neurones express functional GABAA receptors. The
chemosensitivity of unmyelinated axons can be examined by tracking the
electrical threshold of the compound C-fibre action potential
generated in short isolated segments of isolated sural nerve 
and in the present study this method has been used to
characterize the effect of GABA on unmyelinated axons from
Experiments were carried out on 53 isolated nerve fascicles from
16 human sural nerve segments.
GABA increases C-fibre axonal excitability via GABAA
In 21 of the 53 (ca. 40%) fascicles examined, bath application of
GABA (0.1100 mM) increased the electrical excitability of
Cfibres. An increase in excitability indicates that less current is
required to evoke a compound C-fibre response of 40% maximum
amplitude. Relative changes in the calculated excitability index
(see Methods) reflect changes in membrane potential  with an
increase in excitability index representing axonal
depolarization. GABA (0.130 mM) evoked increases in C-fibre electrical
excitability are illustrated by example in Figure 1B. GABA
produces a concentration dependent increase in the excitability
index (Figure 1B & C, upper panel). This increase in excitability
index is consistent with GABA depolarizing some of the
unmyelinated axons within the fascicle.
The increase in C-fibre excitability index in response to GABA
had a rapid-onset, was transient and increased in magnitude
in a concentration dependent manner (Figure 1B and C). The
EC50 of GABAs effect on excitability index was estimated as
6.8860.01 mM from a sigmoid fit of normalised GABA responses
on concentration (Figure 1C). For this analysis, responses to
GABA were normalised to the change in excitability index
produced by 30 mM GABA. Increases in the excitability index to
repeat applications of GABA (1030 mM for 90 s) at 15 minutes
intervals were found to be reproducible and consistent across time.
The effect of GABA on peak amplitude and the latency of the
compound C-fibre response to supra-maximal electrical
stimulation varied. The unmyelinated axons in some fascicles showed no
appreciable change in either parameter (0.1100 mM; Figures 1B,
2BC & 5B) while in others (see Figures 2A & 3A) the peak
amplitude typically increased while the latency decreased.
C-fibre responses to bath applied GABA (10 mM) were
mediated by GABAA receptors, being mimicked by muscimol
(10 mM, n = 5, data not shown) and substantially reduced by prior
application of the GABAA antagonists bicuculline (1020 mM,
reduced to 30.767.92%, n = 6, p,0.05 Students paired t-test),
gabazine (1020 mM, reduced to 29.2617.14%, n = 7, p,0.01
Students paired t-test) and picrotoxin (1030 mM, reduced to
29.56618.13%, n = 3, Figure 2C). In contrast, 90 s bath
application of the GABAB agonist baclofen (100 mM, n = 5, data
not shown) was without effect on any of the parameters used to
assess the electrical excitability of human C-fibres.
Relationship between activity-induced and
GABA-induced changes in C-fibre excitability
The magnitude of GABA evoked increases in C-fibre
excitability index varied within individual fascicles according to
the prevailing value of excitability index (see Figure 3). At low rates
of stimulation (#0.33 Hz), absolute values of C-fibre excitability
index varied between fascicles (Figure 3B), i.e. C-fibre responses in
some fascicles were sub-excitable (index positive) while in others
they were super-excitable (index negative). In most fascicles
however a decrease in the excitability index of Cfibres can be
induced by increasing stimulus rate (Figure 3B, 4 & 5). This in turn
increases the magnitude of the change in excitability index
produced by bath application of GABA (30 mM, Figure 3A & C).
Figure 2. GABAA receptors mediate responses to GABA in human C-fibres. Increases in the electrical excitability index of unmyelinated
axons in human sural nerve fascicles following bath application of GABA (1030 mM) are blocked by prior application of the GABAA receptor
antagonists bicuculline (20 mM, grey bar A), picrotoxin (10 mM, grey bar B) and gabazine (10 mM, grey bar C). In contrast to both bicuculline and
gabazine, the blocking effect of picrotoxin is not reversed upon wash-out (B). The pooled effect of each compound on the change in excitability
index following bath application of GABA (10 mM) is shown in panel D. A significant reduction in the response to GABA (10 mM) was observed in the
presence of gabazine (p,0.05, Students paired t-test) and bicuculline (p,0.05, Students paired t-test). Owing to the limited availability of human
nerve fascicles a statistical comparison was not made for three fascicles exposed to picrotoxin.
As shown by example in Figure 3A this effect can be considerable.
In this example, GABA (10 mM) has no appreciable effect on
excitability index at a stimulus rate of 0.33 Hz. However, an
increase in the stimulus rate to 0.5 Hz reduced the prevailing
excitability index and resulted in a corresponding increase in the
magnitude of excitability index change observed in response to
GABA (10 mM). Increasing the stimulus rate to 1 Hz, further
reduces the prevailing excitability index and thereby increases the
magnitude of change in excitability index evoked by GABA
(10 mM). In general, the magnitude of the excitability index
change produced by GABA (30 mM) or muscimol (30 mM)
increases with increasing basal stimulus rate, i.e. as the prevailing
excitability index decreases (Figure 3C & 5D).
GABA increases axonal excitability in a subset of human
Unmyelinated axons in twenty-one of the 53 nerve fascicles
exhibited a change in electrical excitability index in the presence of
GABA (1030 mM). This restriction of GABA sensitivity to a
subset of fascicles may reflect a selective expression of GABAA
receptors in a sub-population of human unmyelinated axons. To
examine this premise an attempt was made to determine whether
a particular axonal sub-type was sensitive to GABA. The global
behaviour of C-fibres within individual nerve fascicles was
therefore determined according to their excitability index at
0.33 Hz and the profile of change in excitability index and
response latency exhibited during stimulation at 2 Hz (see
Seventeen fascicles from five nerves (each taken at biopsy) were
subject to the electrical stimulus protocol and two general response
patterns were observed. We have designated the two response
profiles Type A and Type B and the incidence of response profile
subtype in fascicles arising from individual nerves is summarized in
Table 1. Nine of the 17 fascicles contained Cfibres with a global
Type B profile. During stimulation at 2 Hz this profile comprised
an initial increase in the latency to half-maximum of the
compound C-fibre response that subsequently reversed, i.e. the
velocity of conduction in C-fibres initially decreased before
increasing slightly and approaching steady-state (Figure 4B &
5B). At low stimulus rates (,0.33 Hz) the Type B response profile
was super-excitable (negative excitability index, Figure 5C) and
during stimulation at 2 Hz became more super-excitable
(Figure 4B & 5B). Only one of the nine Type B fascicles showed
an appreciable increase in C-fibre excitability index in response to
bath application of GABA (30 mM, Figure 5D). C-fibre responses
in eight fascicles had a Type A profile indicating that during
stimulation at 2 Hz a monotonic slowing of latency and a parallel
decrease in excitability index were observed (Figure 4A & 5A).
CFigure 3. Higher rates of electrical stimulation render human C-fibres less excitable but enhance responses to GABA. The magnitude
of the excitability index increase in response to GABA increases with the rate of electrical stimulation. An increase in the rate of electrical stimulation
reduces the excitability index of C-fibres (A & B). The absolute magnitude of stimulus rate-induced decreases in excitability index varies (B). The
reduction in excitability index produced by increased electrical stimulation rate always increases the magnitude of the change in excitability index
observed in response to bath application of GABA or muscimol (1030 mM, A & C).
fibre responses in seven of the eight Type A fascicles were
subexcitable (positive excitability index, Figure 5C) at low stimulus
frequencies but became super-excitable at higher stimulus rates
(Figure 4A & 5A). The excitability index of C-fibres in all eight
Type A fascicles increased following bath application of GABA
(30 mM, Figure 5A, B & D). For individual nerve fascicles the
excitability index of C-fibres determined at 0.33 Hz ranged from
227.9% to 40.7% and was predictive for the absolute magnitude
of the excitability index change upon increasing the rate of
electrical stimulation from 0.33 Hz to 1 Hz (Figure 5C) as well as
for the magnitude of the change in excitability index observed in
response to bath application of GABA (30 mM, Figure 5D).
The application of conventional intracellular
electrophysiological recording techniques to unmyelinated axons in peripheral
nerves is precluded by their small size. Accordingly the effects of
GABA on C-fibres in single fascicles of human sural nerve can
only be examined indirectly. We have used electrical threshold
tracking  to determine an excitability index that serves as an
indirect means of assessing relative changes in membrane potential
. Exposure of human sural nerve segments to GABA (1
100 mM) increased the excitability index of a largely nociceptive
(see below) population of unmyelinated axons via GABAA receptor
activation. The increase in electrical excitability of human
unmyelinated axons in response to GABA is consistent with
axonal depolarization and suggests that human unmyelinated
axons have an elevated intracellular chloride concentration. The
findings indicate further that unmyelinated axons in peripheral
human nerve exhibit a selective chemosensitivity. The
demonstration of functional GABAA responses in human unmyelinated
axons supplements the body of work showing that GABAA
receptors are functionally expressed in axons in the mammalian
peripheral and central nervous system [1,13,28] and may have
implications for the pathogenesis of some forms of neuropathy.
The increase in human C-fibre electrical excitability seen in
response to GABA was mediated by GABAA receptors. The effect
of GABA was blocked by bicuculline, picrotoxin, and gabazine
(Figure 2). While this profile is consistent with that observed for
GABAA mediated currents in isolated human embryonic DRG
neurones , it contrasts with GABA responses evoked in cultured
human adult DRG neurones which are insensitive to both
bicuculline and picrotoxin . The basis of this apparent
discrepancy is not clear. It may reflect differences in GABA
receptor subunit composition or may be due to differences in
sampling between unmyelinated axons and a heterogeneous
population of DRG somata with potentially either myelinated or
unmyelinated axons. To what extent, if any, an underlying
neuropathology may contribute to this discrepancy between
samples of human sural nerve taken at biopsy and isolated human
DRG-somata is also not known. Baclofen did not affect the
excitability of peripheral human axons which is consistent with
reports from isolated human DRG neurones . However both
GABAA and GABAB receptors have been immunohistochemically
identified in cultured Schwann cells from rat  and segments of
rat sciatic nerve  and their activation by GABA agonists has
been suggested to influence the expression of myelin proteins P0
and PMP22 .
Changes in the electrical threshold of human C-fibres in
response to GABA are thought to reflect axonal depolarization.
This view is supported by previous evidence from rat axons  as
well as the observation that rat sympathetic  and frog DRG
 neurones have an elevated intracellular chloride activity, a
feature likely to extend to primary sensory neurones in mammals.
Consistent with this idea is the observation that GABA evokes an
The collective population of unmyelinated axons within individual nerve
fascicles was characterized by a period of electrical stimulation at 2 Hz (see
Figure 4). C-fibre responses were classified as either Type A or B (see Results).
The incidence of each type of response is shown as a function of the nerve of
origin (Nerve ID). The values in parentheses indicate the incidence of C-fibre
responses to GABA (30 mM).
inward depolarizing current in isolated human DRG neurones .
GABA also increases the excitability index determined in
unmyelinated axons traversing human nerve fascicles (Figure 5)
and a similar increase in the excitability index of C-fibres can be
induced with depolarizing current applied extracellularly to
human nerve fascicles . Further support for the notion that
GABA depolarises unmyelinated axons is the observation that
Cfibre responses to GABA increase with stimulus rate (Figure 3).
Peripheral  as well as central  unmyelinated axons
hyperpolarize upon repetitive activation, an effect attributed to
an increase in Na-K-ATPase activity . Activity-induced
hyperpolarization in unmyelinated axons would be expected to
increase the difference between membrane potential and the
reversal potential of the GABAA receptor, itself dominated by ECl,
and thereby enhance the magnitude of GABA-mediated
depolarization of peripheral unmyelinated axons.
C-fibre responses to GABA were restricted to a subset of human
nerve fascicles which is consistent with the notion that only a
subpopulation of human unmyelinated axons expresses functional
GABAA receptors. Indeed, immunohistochemical labelling of cat
skin indicates that b2/b3 and a1 GABAA receptor subunits are
restricted to approximately 12% of unmyelinated axons .
Following initial observations in single axons in rat , it has also
been possible to correlate the receptive class of individual
unmyelinated axons in humans on the basis of the change in
axonal conduction velocity they exhibit during a period of
stimulation at 2 Hz . In particular, when normalised for
differences in their basal conduction velocity, axons from
nociceptors exhibit a more pronounced slowing of axonal
conduction velocity than do non-nociceptive sensory afferents
[39,40] and sympathetic efferents  during repetitive activation.
We show here that compound C-fibre action potential responses in
isolated human nerve fascicles fall broadly into two populations on
the basis of the changes in conduction latency and excitability they
exhibit during stimulation at 2 Hz (Figure 4). This result was
somewhat surprising given that human microneurographic studies
suggest a somatotopic rather than a functional grouping of
unmyelinated axons  and that human sural nerve fascicles may
contain upward of one thousand unmyelinated axons  within
which even individual Remak bundles may contain heterogeneous
axonal sub-types . Nevertheless, based on functional
characteristics, it appears that the characteristic compound electrical
Cfibre response profile of individual human nerve fascicles is largely
consistent with either nociceptive or non-nociceptive
unmyelinated axons (Figure 4). For instance, fascicles classified as Type A
exhibited properties consistent with their containing
predominantly nociceptive unmyelinated axons, they were sub-excitable at low
stimulus rates and showed pronounced activity-dependent slowing.
Moreover, the C-fibre excitability index determined in all eight
Type A increased in response to GABA (Figure 5D). In contrast,
only one of the nine Type B fascicles deemed to be comprised
largely of non-nociceptive unmyelinated axons responded to
GABA (Figure 5D). In rat sciatic nerve, GABA responses in
myelinated nerve fibres are also restricted to sensory axons, motor
axons do not respond to GABA [13,45]. In this context, while
GABA has been shown to modify formalin-induced behavioural
responses in cats  it would be interesting to examine which
sensations, if any, GABA might evoke in human subjects.
Functionally, the activation of axonal GABAA receptors has
been associated with a reduction in the safety factor of action
potential initiation  and conduction  as well as the
regulation of neurotransmitter release . Verdier et al. (2003)
propose that axo-axonic GABAergic synapses exert an inhibitory
effect on axonal discharge such that tonic activity is periodically
interrupted resulting in a bursting pattern. This is consistent with
the dependence of GABAs efficacy upon preceding activity
observed here in human C-fibres (Figure 3) and suggests that
GABA may exert a more pronounced action on active C-fibres.
Another possible role of axonal GABAA receptors involves the
increase in the intracellular chloride concentration of DRG
neurones observed in response to both inflammatory mediators
 and following peripheral nerve injury . Speculatively,
under such conditions, it is possible that GABAA receptor
activation produces exaggerated depolarizing responses that may
acutely initiate action potentials or open voltage-gated Ca2+
channels that are present in unmyelinated human axons .
The data establishes the functional expression of GABAA
receptors in the axonal membrane of a sub-population of
unmyelinated afferent human nerve fibres that most probably
comprises nociceptors. It has been postulated that a loss of
interneuron mediated GABAergic inhibition within the spinal
dorsal horn may contribute to the establishment and maintenance
of some neuropathic pain states (see [49,50]). Indeed current
therapeutic strategies currently being developed in mice are aimed
at reversing this GABAergic disinhibition through sub-type
selective activation of GABAA receptors expressed in the spinal
dorsal horn . If the functional GABAA receptor expression in
peripheral human unmyelinated axons extends further to the
centrally projecting axons of sensory neurones, this observation
provides considerable impetus for the translation of GABAA
receptor targeted strategies to humans.
Materials and Methods
Approval for the experimental use of human tissue was granted
by the Ethics Committee of the Medical Faculty of the University
of Munich (Project Number 348/00). Patients were informed
about the biopsy procedure by an anaesthetist one day prior to
surgery at which time the patients written consent to the removal
of an additional portion of nerve for research purposes was
Experiments were carried out on isolated fascicles of human
sural nerve obtained from 16 patients (9 male, 7 female) previously
scheduled for either sural nerve biopsy or lower limb amputation.
Segments of sural nerve were obtained from patients ranging in
age from 20 to 89 years with a median age of 68 years. The
underlying diagnosis precipitating biopsy was either
polyneuropathy of unknown aetiology or peripheral artery occlusive disease.
Neither the profiles of electrical excitability nor the
pharmacological responses of C-fibres within individual fascicles were
correlated with the prevailing pathological classification.
Segments of human sural nerve obtained at biopsy were
typically 1525 mm long. Individual nerve fascicles were carefully
extracted from isolated segments of sural nerve by gently pulling
them free with jewellers forceps. Isolated fascicles were mounted
between suction electrodes in an organ bath. Each end of the
nerve fascicle was drawn into a glass suction electrode and
embedded in Vaseline to establish both a mechanical and a high
resistance electrical seal. The organ bath (volume ca. 1 ml) was
perfused continuously at a rate of 68 ml.min21 with physiological
solution of the following composition (in mM) NaCl 117, KCl 3.6,
CaCl2 2.5, MgCl2 1.2, D-glucose 11.0, NaHCO3 25, NaH2PO4
1.2, bubbled with 95% O2/5% CO2 to pH 7.4. The temperature
of the solution perfusing the bath was held constant at 34uC.
Axonal excitability was determined in C-fibres by stimulating
the nerve fascicle electrically with constant current pulses (A395,
WPI, Sarasota, USA). A silver wire inside the stimulating suction
electrode served as the anode and a second silver wire wound
around the suction pipette in the organ bath served as the cathode.
Extracellular signals were recorded over the sealing resistance of
the second suction electrode with a differential amplifier (NPI,
Tamm, Germany). The distance between stimulating and
recording electrodes was typically 48 mm. A window
discriminator allowed the C-fibre response to be monitored in isolation.
An electrical stimulus protocol was used to examine the effect of
GABA on the excitability of C-fibres in sural nerve fascicles.
Constant current pulses of fixed duration (1 ms) and varying
amplitude were used to track changes in C-fibre excitability with
QTRAC software ( Institute of Neurology, London, UK). Three
interleaved stimulus parameters were monitored sequentially.
Firstly, a supra-maximal current intensity was established at
which the amplitude of the compound C-fibre response was
maximal and this was designated a 100% response (100%,
Figure 1A). Electrical threshold was the second stimulus parameter
monitored. This is defined as the current required to elicit a
compound C-fibre response with an amplitude 40% that of the
response to supra-maximal stimulation (40%, Figure 1A). The
stimulus current required to evoke a 40% amplitude C-fibre
compound action potential was continuously adjusted by the
QTRAC software. The third parameter was post-spike electrical
threshold and this is the stimulus current required to maintain a
conditioned C-fibre compound action potential response of 40%
amplitude, that is 30 ms after a conditioning supra-maximal
electrical stimulus (40% cond., Figure 1A). The difference between
the conditioned and unconditioned current intensities normalised
to the stimulus intensity of the conditioned response is defined as
the excitability index and is expressed as a percentage.
Gabazine (Biotrend, Cologne, Germany), baclofen, bicuculline,
gamma-aminobutyric acid, muscimol and picrotoxin (Sigma,
USA) were stored as stock solutions in distilled water. The desired
concentration of each substance was achieved by dilution from
stock into the solution perfusing the bath on the day of each
Data are expressed as mean and standard deviation for
comparisons between groups while for population descriptors
mean and standard error of the mean are indicated. Students
ttest was used for statistical comparisons of paired datasets. Curve
fitting was performed in Igor Pro (Wavemetrics, Lake Oswego,
USA) which uses the Levenberg-Marquadt algorithm for
We would like to thank the anonymous patients who generously provided
material for the experiments, Christina M uller for technical assistance, Drs.
Philip Lang and Dominik Irnich for administrative support and the
surgeons of the Department of Hand and Plastic Surgery who performed
the sural nerve biopsies.
Conceived and designed the experiments: RWC PG. Performed the
experiments: RWC RS PG. Analyzed the data: RWC. Wrote the paper:
RWC RS PG. Informed patients and collected material from surgery: RS.
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