Brain Stimulation 14 (2021) 513e530

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Brain Stimulation
journal homepage: http://www.journals.elsevier.com/brain-stimulation

Kilohertz-frequency stimulation of the nervous system: A review of

underlying mechanisms
Clemens Neudorfer a, Clement T. Chow a, Alexandre Boutet a, Aaron Loh a,
Jürgen Germann a, Gavin JB. Elias a, William D. Hutchison b, c, Andres M. Lozano a, b, *
a
Division of Neurosurgery, Department of Surgery, Toronto Western Hospital, University of Toronto, Canada
b
Krembil Research Institute, University of Toronto, Ontario, Canada
c
Department of Physiology, Toronto Western Hospital and University of Toronto, Ontario, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Background: Electrical stimulation in the kilohertz-frequency range has gained interest in the field of
Received 16 September 2020 neuroscience. The mechanisms underlying stimulation in this frequency range, however, are poorly
Received in revised form characterized to date.
8 March 2021
Objective/hypothesis: To summarize the manifold biological effects elicited by kilohertz-frequency
Accepted 11 March 2021
Available online 20 March 2021
stimulation in the context of the currently existing literature and provide a mechanistic framework for
the neural responses observed in this frequency range.
Methods: A comprehensive search of the peer-reviewed literature was conducted across electronic da-
Keywords:
Kilohertz-frequency
tabases. Relevant computational, clinical, and mechanistic studies were selected for review.
Electrical stimulation Results: The effects of kilohertz-frequency stimulation on neural tissue are diverse and yield effects that
Facilitation are distinct from conventional stimulation. Broadly, these can be divided into 1) subthreshold, 2)
Desynchronization suprathreshold, 3) synaptic and 4) thermal effects. While facilitation is the dominating mechanism at the
Conduction block subthreshold level, desynchronization, spike-rate adaptation, conduction block, and non-monotonic
Strength-duration response activation can be observed during suprathreshold kilohertz-frequency stimulation. At the synaptic
Temperature level, kilohertz-frequency stimulation has been associated with the transient depletion of the available
Short-term plasticity
neurotransmitter pool e also known as synaptic fatigue. Finally, thermal effects associated with extrinsic
Synaptic fatigue
(environmental) and intrinsic (associated with kilohertz-frequency stimulation) temperature changes
have been suggested to alter the neural response to stimulation paradigms.
Conclusion: The diverse spectrum of neural responses to stimulation in the kilohertz-frequency range is
distinct from that associated with conventional stimulation. This offers the potential for new therapeutic
avenues across stimulation modalities. However, stimulation in the kilohertz-frequency range is asso-
ciated with distinct challenges and caveats that need to be considered in experimental paradigms.
© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction suggested that “supraphysiological” stimulation paradigms in the

Electrical stimulation of the nervous system constitutes an
established therapy in a wide range of neurological disorders and is
currently under rigorous evaluation for its applicability to psychi-
atric diseases [1]. While stimulation modalities conventionally
employ stimulation frequencies within physiological ranges i.e.,
<500 Hz (Table 1) a growing body of literature in recent years has
* Corresponding author. 399 Bathurst St., WW 4-431, Toronto Western Hospital,
Toronto, ON, M5T 2S8, Canada.
E-mail address: lozano@uhnresearch.ca (A.M. Lozano).
kilohertz-frequency range may hold distinct advantages. These
include reduction or abolishment of stimulation-induced side-ef-
fects [2,3], targeted transcranial stimulation of deep brain struc-
tures [4,5], and selective modulation of neural activity [6e8]
(Table 1). Seeking to exploit these advantages, kilohertz-frequency
stimulation has consequently been assessed across a multitude of
stimulation modalities including transcranial electrical stimulation
[4,5], deep brain stimulation [3], spinal cord stimulation [2,9], as
well as peripheral nerve stimulation modalities [10e12].
Despite growing interest and increased clinical application of
kilohertz-frequency stimulation, the mechanisms that underlie its
effects remain poorly characterized to date. A deeper

https://doi.org/10.1016/j.brs.2021.03.008
1935-861X/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
C. Neudorfer, C.T. Chow, A. Boutet et al. Brain Stimulation 14 (2021) 513e530

understanding of stimulation-induced effects in this frequency
range is, however, desirable for a better exploitation of neural
properties across stimulation modalities. In an effort to characterize
the various responses elicited by kilohertz-frequency stimulation,
we have compiled currently known hypotheses of biophysical ef-
fects on the neural membrane that differ significantly from con-
ventional stimulation (Table 2). Firstly, we review the fundamentals
of electrical stimulation and outline the frequency dependent
behavior of the neural membrane. We then discuss the effects
observed during sub- and suprathreshold kilohertz-frequency
stimulation. Finally, we examine how thermal effects e induced
by changes in ambient temperature, joule heating, or metabolic
heat generation e may alter the neural response to kilohertz-
frequency stimulation.
Methods
To gain insight into the mechanisms governing neural responses
in the kilohertz-frequency range a review of the existing literature
was conducted using MEDLINE, Embase, and Cochrane CENTRAL
databases. The search was conducted on September 12, 2019 and
December 7, 2020 using the search terms “kilohertz” AND “stim-
ulation” along with other synonymous keywords (e.g. kHz, 1000 Hz,
2000 Hz etc.). The search strategy was refined by the addition of the
search terms “adaptation”, “accommodation”, “facilitation”,
“desynchronization”, “conduction block”, “synaptic” and “thermal”
which were combined with the boolean operator “OR”. Studies
investigating mechanisms in-silico, in-vitro, and in-vivo (i.e., ani-
mal and human studies) were considered. All potentially relevant
studies were screened for eligibility in two stages. First, titles and
abstracts of identified articles were screened by three authors (CN,
AB, AL). For abstracts which met inclusion criteria the full texts
were retrieved and independently reviewed. Disagreements and
technical uncertainties were discussed and resolved in conjunction.
Additional records identified through reference lists were included
during manuscript generation.
Fundamentals of electrical stimulation
The goal of electrical stimulation of neural tissue is to drive the
generation of action potentials. By leveraging the electrochemical
gradient, electric current alters the potential across the neural
membrane thereby opening voltage-gated ionic conductance
channels that allow the influx of sodium into the cell [13]. The
square pulse constitutes the basic unit of electrical stimulation and
is defined by its pulse width (milli- or microseconds, ms/ms),
amplitude (ampere, A or voltage, V), and polarity (anodal or cath-
odal). Whether or not a single square pulse is able to sufficiently
engage voltage-gated ion channels and depolarize the neural
membrane such that an action potential is elicited depends on the
interplay of the respective parameters which is informed by two
key concepts: the strength-duration response (SDR) curve and the
charge-duration relationship (CDR) (Fig. 1).
Strength-duration response and charge-duration relationship
The SDR establishes the relationship between pulse width and
amplitude and describes the threshold amplitude as a function of
the pulse width of a single monophasic pulse (Fig. 1) [14]. Pulse
widths close to the rheobase current effectively depolarize the
neural membrane at low intensities. As the pulse duration is
decreased, however, non-linear amplitude increases are necessary
to meet the membrane’s threshold potential and maintain the
consistent entrainment of neural tissue (Fig. 1A). Similar to the SDR,
the electrical charge features a dependence on the pulse duration
that is characterized by the charge-duration relationship (CDR)
(Fig. 1B). The threshold charge (2*tch*Irh) is defined as the product
of rheobase current (Irh) and pulse duration and increases non-
linearly as a function of the pulse duration.
Both the SDR and CDR establish the conditions required to
induce a single action potential in response to a single pulse.
However, they do not take into account the membrane and channel
dynamics that arise from repetitive stimulation. This is a particularly
pertinent limitation as the effects of repetitive stimulation dictate a
neuron’s firing characteristics during prolonged stimulation and
are thus paramount to our understanding of stimulation-induced
effects. For example, individual pulses that are too weak to bias
the membrane potential towards the firing threshold may elicit
action potentials when applied in short succession (see section
‘Facilitation’) [15,16]. Similarly, repetitive entrainment within short
time windows may induce plastic changes at the synapse
strengthening or attenuating signal transmission between neurons
(see section ‘Synaptic effects’) [17,18]. During repetitive stimulation
additional factors need to be considered. First, the build-up of
charge that is determined by the CDR may cause tissue damage
during prolonged stimulation when pulses are delivered in a
monophasic mode [13]. In contrast, biphasic stimulation generally
implies a zero-net charge as the charge introduced by the

Table 1
Stimulation settings conventionally employed during electrical stimulation and kilohertz stimulation paradigms. Note that kHz-frequency paradigms are currently under-
explored and have only received wide employment in spinal cord stimulation (SCS). Other stimulation paradigms have thus far only been investigated sporadically in the
context of clinical trials. DBS, deep brain stimulation; DRG, dorsal root ganglion stimulation; PNS, peripheral nerve field stimulation; SCS, spinal cord stimulation; tACS,
transcranial alternating current stimulation; VNS, vagus nerve stimulation.

Modality Conventional Stimulation Kilohertz-frequency Stimulation

Frequency Pulse Current Frequency Pulse Current Suggested advantages over conventional paradigms
(Hz) Duration (mA) (kHz) Duration (mA)
(ms) (ms)

tACS 4e200 Sine 2 2e133 Sine/2.5 1e9 Reduced charge density and skin sensations while delivering sufficiently high current
intensities to deep brain structures by means of waveform modulation to induce action
potential generation. Targeted transcranial stimulation of deep brain structures [4,5].
DBS 130e180 60 1e6 10 30 3e6 Reduction of stimulation induced paresthesia and speech impairment [3].
SCS 50e100 300e600 4e9 10 30 1e5 Abolishment of stimulation-induced paresthesia while maintaining pain relief [2,136];
maintenance of proprioceptive feedback while providing postsynaptic excitation to motor
neurons [9].
PNS 100 100 2.5e3.5 10 Sine 0.4e10 Paresthesia-free pain relief [12]; improved control over motor responses, restoration of tactile
percepts [31]. ‘Pseudospontaneous’ activity mimicking physiological conditions [42,43]
VNS 1e30 130e500 0.5e1.5 5 90 1e8 Vagotomy-like effect [10]
DRG 20e100 200e400 0.8e1.8 10 30 1e3 Paresthesia-free pain relief [11]

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C. Neudorfer, C.T. Chow, A. Boutet et al. Brain Stimulation 14 (2021) 513e530

Table 2
Comparison of stimulation induced effects on the neuronal membrane during conventional (top) and kilohertz-frequency stimulation (bottom).

Modality Proposed Description Ref.

Mechanism

CONVENTIONAL ELECTRICAL One-to-one Each electrical stimulus is time-locked with a measurable neuronal response [33]
STIMULATION (<500 Hz) entrainment
Direct presynaptic Depletion of releasable neurotransmitter pools, synaptic inhibition of descending projections [190]
depression
Direct activation Depolarization, in an excitatory (e.g. Glutamatergic) neurotransmitter environment [35]
Direct inhibition Hyperpolarization, in an inhibitory (e.g. GABAergic) neurotransmitter environment [190]
Antidromic Decoupling of soma and dendrite leads to retrograde activation of dendrites [114]
Excitation
Orthodromic Decoupling of soma and axon causes anterograde propagation of stimulation-induced potentials [114]
Excitation
Information lesion Highly regularized firing of entrained neurons reduces the complexity of transmitted information [117]
Indirect presynaptic Synaptic depletion of the available neurotransmitter pool reduces propagation of stimulation induced [17]
depression action potentials creating a low-pass filter
KILOHERTZ-FREQUENCY Facilitation Subthreshold pulses arriving in short succession bias the membrane potential towards depolarized [23]
STIMULATION (>1 kHz) values eventually eliciting an action potential
Desynchronization Unpredictable potential fluctuations during the relative refractory period and entrainment to different [42]
kilohertz-frequency stimuli desynchronize neuronal firing
Spike-rate Prolonged exposure to a constant stimulus reduces the spike frequency over the course of stimulation [75]
adaptation
Conduction block Stimulation leads to a permanent depolarization or hyperpolarization of the neuronal membrane [30]
blocking the propagation of subsequent action potentials
Non-monotonic Both stimulation frequency and amplitude dictate a neurons response to kilohertz-frequency stimulation [29]
activation
Synaptic fatigue Synaptic depletion of the available neurotransmitter pool reduces propagation of stimulation induced [128]
action potentials creating a functional block

depolarizing phase is reverted by the subsequent hyperpolarizing
phase. This not only has implications with respect to the safety of
mono- and biphasic stimulation paradigms (charge-balanced
waveforms are safer), but also alters the membrane responses
during kilohertz-frequency stimulation, as will be discussed in
subsequent sections.
Kilohertz-frequency stimulation
Under physiological conditions a neuron’s maximum firing rate
(typically 500 Hz, although frequencies up to 1000 Hz have been
observed in distinct cell types such as cerebellar mossy fibers) [19]
is dictated by the type, density, and distribution of ion channels
across the neural membrane. In contrast, external electrical stim-
ulation is not subject to these limitations and capable of manipu-
lating the neural membrane in any given frequency range. The
application of ‘supraphysiological’ frequencies in the kilohertz-
frequency range consequently has severe implications on the
overall observed neural response.
As will be discussed in the following sections, kilohertz-
frequency stimulation has the ability to evoke a broad spectrum
of neural responses that are distinct from conventional stimulation
(Table 1). In the present review, these responses were compiled
from a total of 92 studies. The majority of these studies (n ¼ 50)
constituted animal trials conducted in amphibians (n ¼ 3) and
mammals (n ¼ 47); 28 studies were conducted in-silico using
myelinated (n ¼ 24) and unmyelinated (n ¼ 4) axon models; 9
studies described the effects of kilohertz-frequency stimulation in-
vitro. Finally, 5 of the 92 identified studies were clinical trials
conducted in humans. Supplementary Table 1 provides a detailed
description of the models used to study the effects of kilohertz-
frequency stimulation.
Generally, the responses observed during kilohertz-frequency
stimulation can be categorized into excitatory and inhibitory ef-
fects that occur at sub- and suprathra threshold intensities across
different time scales (Fig. 2). The majority of studies investigated
the mechanisms of conduction block and associated phenomena
515
including onset response, desynchronization, and spike-rate
adaptation, which occur during suprathreshold stimulation. In
addition, several studies investigated the non-monotonic rela-
tionship between stimulus frequency and amplitude that has
typically been observed in the frequency range above 30 kHz.
Studies were primarily conducted in peripheral nerves (n ¼ 45),
with a minority reporting blocking mechanisms during stimulation
of the central nervous system (n ¼ 5). In contrast, subthreshold
effects, namely facilitation, were observed during auditory nerve
stimulation experiments (n ¼ 9). Studies investigating the synaptic
effects of kilohertz-frequency stimulation (n ¼ 5) primarily
described mechanisms such as synaptic depletion and fatigue.
Finally, several studies reported how external temperature changes
may affect kilohertz-frequency stimulation (n ¼ 7). In addition, the
potential of kilohertz-frequency stimulation to locally increase
temperatures by means of joule heating and metabolic heat gen-
eration was proposed in a minority of studies (n ¼ 5).
The methods used to quantify the neural responses of kilohertz-
frequency stimulation varied considerably across studies. While in-
vitro studies determined stimulation-induced effects by directly
recording the transmembrane potential (n ¼ 28), in-vivo studies
employed a variety of direct and indirect measures such as muscle
force (n ¼ 27), urethral sphincter and bladder pressure (n ¼ 7),
compound action potentials (n ¼ 15), electromyography (EMG)
(n ¼ 2), evoked potentials (n ¼ 3), and transmembrane potentials
(n ¼ 7). In computational studies stimulus-response phenomena
were determined by recording transmembrane potentials (n ¼ 28).
An itemized description of methods and parameters unique to each
study including study type, species, waveform shape, stimulation
parameters, stimulator and electrode model, outcome measure,
calibration routines, reported temperatures, and block frequencies
can be obtained from Supplementary Table 1.
Subthreshold effects of kilohertz-frequency stimulation
Subthreshold effects of electrical stimulation are well charac-
terized at the synaptic level, but have only received little attention
C. Neudorfer, C.T. Chow, A. Boutet et al.
Fig. 1. Strength-duration response curve (A) and charge-duration response curve
(B). Entrainment of neuronal activity in response to a monophasic electrical stimulus is
dependent on the relationship between pulse width [ms] and amplitude [mA]. As pulse
width decreases, a non-linear increase of the threshold current is necessary to main-
tain effective neuronal excitation (A). The charge duration relationship (B) dictates the
threshold charge necessary to effectively entrain neuronal activity and increases with
increasing pulse width. Shorter pulse widths are consequently more charge efficient at
generating excitation.
at the neural level. Indeed, in the field of kilohertz-frequency
stimulation most of the effects associated with subthreshold
stimulation are largely derived from auditory nerve stimulation
studies (Supplementary Table 1). These studies sought to charac-
terize the frequency-dependent neural response properties
thought to be relevant for cochlear implant stimulation. By elec-
trically probing the auditory nerve, these groups identified a major
subthreshold mechanism that will be discussed here, facilitation.
Synaptic (subthreshold) effects such as temporal summation are
secondary to the neural effects evoked by kilohertz-frequency
stimulation and will not be addressed in this review as their time
scales are orders of magnitude larger and only relevant in the
context of ‘synaptic fatigue’, which will be discussed in the ‘Syn-
aptic effects’ section.
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Brain Stimulation 14 (2021) 513e530
Fig. 2. Diagram featuring the temporal course of absolute refractoriness, relative
refractoriness, facilitation, accommodation, and spike-rate adaptation. Colors
indicate whether the induced neuronal response is excitatory (green) or inhibitory
(red). During relative refractoriness (gray), the random open- and close-kinetics of
voltage-gated ion-channels introduce unpredictable fluctuations of the membrane
potential that may allow excitation of the neuronal membrane via a subsequent
stimulus if the membrane potential is biased towards more positive values. In contrast,
if the neuronal membrane features a more hyperpolarized state, the generation of an
action potential in response to a subthreshold stimulus is inhibited. (For interpretation
of the references to colour in this figure legend, the reader is referred to the Web
version of this article.)
Facilitation
Temporal summation is a well-established synaptic character-
istic that allows the generation of action potentials via integration
of successive subthreshold stimuli. Non-synaptic neural mem-
branes feature similar integrative properties, which allow the
neural membrane to generate action potentials in response to
subthreshold stimuli arriving in short sequence [20,21]. This is
attributable to the passive charging of the membrane during elec-
trical stimulation. Specifically, if subsequent pulses are separated
by a time period that is shorter than the membrane time constant,
the accumulated charge from previous stimuli will be too great to
completely discharge before the membrane begins to integrate the
subsequent pulse (Fig. 3A) [22]. This leads to a net accumulation of
charge over the course of stimulation, which in turn shifts the
membrane potential towards the firing threshold eventually elic-
iting an action potential.
Monophasic stimulation of cat and guinea pig auditory nerves
was shown to effectively induce facilitation with each subthreshold
stimulus adding to the already built-up charge [23]. In contrast, the
passive accumulation of charge across the membrane proves more
difficult during biphasic stimulation. Here the hyperpolarizing
phase reverts the built-up charge back to baseline causing an
overall faster dissipation of charge. However, experiments in
auditory nerve fibers have demonstrated that facilitation can be
achieved using biphasic pulses [24,25], which suggests active
mechanisms that e in addition to passive capacitive charging e
may be able to suppress the hyperpolarizing phase. Investigating
potential mechanisms in auditory nerve models of the Hodgkin-
Huxley (HH) model, Negm and Bruce identified the activation of
‘residual’ sodium channels as a likely mechanism underlying
facilitation (Fig. 3B) [26]. As the membrane potential was moved
toward the threshold potential during the first subthreshold stim-
ulus the authors observed that activation of an increased number of
sodium channels yielded a slower decay towards rest during the
interpulse interval. Hence, as subsequent stimuli followed, the
relative increase in depolarization and the associated higher re-
sidual membrane potential were sufficient to facilitate action po-
tential generation (Fig. 3B). Similar responses were observed by
other groups in-silico using the Frankenhaeuser-Huxley model
[27,28]. These authors demonstrated that frequencies greater than
4 kHz and 5 kHz, respectively could repetitively activate sodium
channels to the point of firing.
Seeking to exploit the time integrating properties of the neural
membrane, recent studies have devised time-multiplexing
methods for transcranial stimulation paradigms to effectively
C. Neudorfer, C.T. Chow, A. Boutet et al. Brain Stimulation 14 (2021) 513e530

Fig. 3. Subthreshold mechanisms of kilohertz-frequency stimulation (A) Subthrehsold stimuli that arrive in a time period that is shorter than the membrane time constant are
able to generate action potentials by passive capacitive charging and active ion channel opening. (B) Graphs featuring the passive and active contributions to facilitation of neural
firing in a masker-probe paradigm as simulated in a single node of Ranvier (Hodgkin-Huxley model). Top and middle panel show instances where the model spikes in response to
the probe pulse (green line), where the second pulse does not yield an action potential (magenta line), and where the number of open ion channels is fixed at resting values (passive
response; dashed line). Note the difference in sodium channel opening in response to the first pulse (middle panel) that results in a slower decay of the membrane potential
depolarization back to rest (top panel). The subsequent probe pulse builds upon this ‘residual’ membrane depolarization and facilitates action potential generation. B adapted from
Negm and Bruce (2014) [26]. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

entrain deep brain structures. Using multi-electrode arrays that
intersected in the desired target area, Vo € ro
€slakos et al. delivered
short pulses below SDR threshold (pulse width: 2.5 ms, frequency:
~133 kHz) to deep brain structures [5]. In the area of overlapping
electric fields, the subthreshold pulses integrated temporally,
yielding localized entrainment of neural activity and action po-
tential generation by means of facilitation. It is important to note,
however, that stimulation in healthy subjects was associated with
distinct side effects including phosphenes, vestibular effects, and
slight tingling and burning sensations of the skin. A similar
mechanism of action is likely attributable to temporal interference
stimulation, which generates a beating signal from two electric
fields driven at two different kilohertz frequencies (e.g. 2.0 kHz and
2.01 kHz) [4]. While neural recruitment as a result of amplitude
increase due to constructive interference cannot be excluded, a
contribution of facilitation to the overall response seems likely due
to the short interpulse intervals (IPIs). The clinical value of time-
multiplexing methods, however, remains to be established. This
holds especially true for temporal interference stimulation that has
thus far only been demonstrated in a mouse model during subcu-
taneous stimulation, thus neglecting shunting of current by skin
and soft tissue.
Suprathreshold effects of kilohertz frequency stimulation
In the field of kilohertz-frequency stimulation considerable
effort has been dedicated towards the characterization of supra-
threshold effects. Most of our current knowledge arises from
computational and peripheral nerve stimulation studies seeking to
leverage the nerve blocking abilities of kilohertz-frequencies to
disrupt information transmission along axons [29,30]. In contrast,
recent research has started to unveil the potential of desynchro-
nization and spike-rate adaptation in clinical practice [31,32]. These
mechanisms will be discussed in the following sections.
At the neural level suprathreshold stimulation at frequencies
within a neuron’s physiological range (i.e., from 0.05 Hz to 500 Hz)
typically entrains neural activity in a one-to-one relationship. In
consequence, each applied cycle or pulse is time-locked with a
measurable response that features a regular firing pattern [33,34].
Increases in stimulus amplitude within this frequency range in-
crease the distance over which axons and white matter tracts are
being entrained. Intrinsic changes in the timing of neural firing
517
(e.g., desynchronization) are not observed during amplitude in-
creases below 500 Hz.
Desynchronization of neural firing
An axon’s refractory period typically ranges from 1 to 2 ms and
imposes a frequency limit of 500e1000 Hz for external stimuli to
trigger neuronal responses in a one-to-one relationship [35]. Dur-
ing the absolute refractory period - a period of complete inexcit-
ability that immediately follows action potential generation -
pulses of higher frequencies will not entrain neural activity as ion-
channels are unavailable. However, as ion-channels become grad-
ually available in the relative refractory period, unpredictable,
macroscopic fluctuations of the membrane potential can be
observed. As demonstrated by in-silico studies, this is owing to the
random open-close kinetics of voltage-gated sodium channels that
bias the membrane potential towards more depolarized or hyper-
polarized states [36e38]. The relative refractory period conse-
quently introduces an element of uncertainty and stochasticity
(known as channel noise) capable of eliciting unpredictable neural
responses [39,40]. For example, a high-frequency stimulus may
supersede the threshold and entrain neuronal activity during the
relative refractory period if the membrane potential is biased to-
wards depolarized states (Fig. 4A and B). In contrast, an incoming
stimulus will not elicit neuronal firing while ion channels bias the
membrane potential towards hyperpolarization.
As a result of stochastic membrane dynamics during the relative
refractory period, the overall neural response observed in axons
exposed to stimulation in the kilohertz range may entail pseudo-
random, disorganized firing with each unit discharging at its own
rate and pattern (Fig. 4A and B) [41e45]. Woo and Campbell pro-
vided the first evidence of desynchronization [45]. By stimulating
cat tibial and frog sciatic nerves at 20 kHz and recording compound
action potentials, the authors noted highly irregular and asyn-
chronous firing that gradually decreased in amplitude and was
succeeded by conduction block. Bhadra et al. quantified the
magnitude of asynchronous firing by calculating the force-time
integral (area under the force curve) as a measure of frequency
and amplitude [46](Fig. 4B). Stimulating rat sciatic nerves, they
showed that amplitude and duration of desynchronization varied
inversely with frequency and amplitude with exceptions at the
lowest amplitudes and frequencies tested (Fig. 4B) [47]. Today,
C. Neudorfer, C.T. Chow, A. Boutet et al. Brain Stimulation 14 (2021) 513e530

desynchronized firing in the context of ‘onset response’ (see ‘Con-
duction block’) has been identified in both in-silico [48e54] and in-
vivo studies using single fiber recordings [45,49,55], motor cortex
recordings [56], muscle force [46,53,57e61], and urethral sphincter
pressure [62,63]. Furthermore, the ability of kilohertz-frequency
stimulation to sustain desynchronized firing has been reported in
computational models and cat auditory nerve fiber stimulations
[42,43].
Under physiological conditions, neural ensembles commonly
feature asynchronous activity that is partially driven by the sto-
chastic nature of action potential generation (channel noise) and in
part by the probabilistic nature of quantal neurotransmitter release
and the gating kinetics of ligand-binding ion channels (synaptic
noise) [64]. In consequence, highly synchronous neuronal activity
as employed during conventional pulsed stimulation may
adversely affect outcomes, especially in biomimetic neuro-
stimulation applications. For instance, the synchronicity of stimu-
lation has been hypothesized to impair natural tactile percepts in
the restoration of tactile feedback in amputees [65,66]. Similarly,
pulsed stimulation has been demonstrated to induce jerky muscle
contractions and higher muscle fatigue, which has important
ramifications in brain-machine-interfaces aimed at directly
inducing muscle contractions [67,68]. Here, alternative stimulation
paradigms such as kilohertz-frequency induced desynchronization
yield greater control over induced motor responses [31]. A similar
strategy has been proposed in auditory nerve stimulation, where
desynchronized, ‘pseudorandom’ stimulation was employed to
mimic the spontaneous activity observed in the healthy ear and
enhance the neural representation of temporal detail and dynamic
range of cochlear implants [42,43]. Psychophysical results, how-
ever, have reported inconsistent results during desynchronized
auditory stimulation [69e73], contradicting the assertion that this
paradigm produces improvements in speech perception.
Spike rate adaptation
Spike rate adaptation (SRA) alters the extent of neuronal firing
in response to suprathreshold stimuli in the kilohertz-frequency
range. When exposed to a constant stimulus, many neurons
feature high firing rates upon initiation of stimulation that gradu-
ally decay over time even though the stimulus remains constant
(Fig. 4C and D) [74,75]. The mechanism is believed to remove su-
perfluous information and conserve energy by lowering the neu-
ron’s excitability in response to prolonged neural discharge [76].

Fig. 4. Suprathreshold mechanisms of action underlying desynchronization and spike-rate adaptation of neuronal activity. (A) (top panel) During stimulation in the low-
kilohertz-frequency range neurons lose their ability to follow the quickly alternating stimuli in a one-to-one relationship. Instead, membrane potential fluctuations during the
relative refractory period dictate the neurons response to subsequent stimuli. Given that the nature of these fluctuations is stochastic variable temporal delays in neuronal firing
ensue (purple shaded areas). (middle panel) Neuron populations exposed to kilohertz-frequency stimulation entrain to different pulses of the applied stimulus train, leading to
desynchronization of neuronal firing across the stimulated population. (B) Contour plot featuring the force-time integral (area under the force curve) during exposure of rat sciatic
nerve to biphasic sinusoidal stimuli as a function of frequency and amplitude. Areas of low force-time integral indicate a rapid onset of block with minimal desynchronization. (C)
Change in spike frequency (top) of an adapting neuron during exposure to a constant stimulus (bottom). Following an initial period of increased neuronal firing (f0) spiking de-
creases exponentially as stimulation proceeds (middle). Eventually, firing reaches a steady-state rate (f∞). (D) Relationship between onset- (green) and steady-state (red) firing at
increasing current amplitudes. B and D adopted from Bhadra et al. (2005) [46] and Benda et al. (2013) [75]. (For interpretation of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)

518
C. Neudorfer, C.T. Chow, A. Boutet et al.
SRA is commonly observed during prolonged stimulation pe-
riods and may range from ~10 ms to several minutes as determined
in-vivo and in-silico [43,55,77]. The degree of observed adaptation
depends on multiple factors including stimulus frequency, ampli-
tude, and overall duration of the applied stimulus [76,78,79].
Bowman and McNeal published a comprehensive account of the
mechanisms underlying SRA following exposure of single alpha
motoneurons to electric current [41]. By stimulating alpha moto-
neurons over several minutes, the authors found that frequencies
below 1 kHz were associated with time-locked neural responses,
that often decreased in spiking over the course of stimulation
(Fig. 4C and D). As frequencies increased beyond 1 kHz, the decline
in firing became more prominent, eventually resorting to baseline
firing at frequencies of around 4 kHz. Finally, at frequencies be-
tween 4 kHz and 10 kHz, neurons gradually transitioned to com-
plete conduction block following initial bouts of firing at
frequencies of several hundred Hz. Amplitude increases at a given
frequency generally accelerated the decline in neuronal spiking and
facilitated the neurons’ transition to conduction block. Similar ob-
servations were made by Zhang et al. who noted a similar behavior
of cat auditory nerve fibers following electrical stimulation at fre-
quencies between 1 and 5 kHz [80].
Both desynchronization and spike rate adaptation can be
observed in the initial stages of conduction block, where the neural
membrane still retains some excitability. This phase is also referred
to as ‘onset response’ and will be discussed below.
Conduction block
Neural conduction block describes the ability of kilohertz-
frequency stimulation to reversibly inhibit the propagation of ac-
tion potentials through axons [30]. The probability to induce
effective block is strongly dependent on two waveform parameters,
namely frequency and amplitude (Fig. 5) [30,46]. As demonstrated
in animal studies, frequencies of at least 1 kHz are required to
induce true conduction block (Supplementary Table 1). Higher
frequencies up to 100 kHz [54,81] and 300 kHz [82,83] have been
explored in-vivo and in-vitro, however, there have been no studies
reporting a ‘maximum’ frequency beyond which no effective block
was achieved.
Apart from stimulus frequency and amplitude other factors such
as nerve fiber diameter, waveform shape, and electrode design
influence the probability of conduction block, albeit to a lesser
extent (see ‘Methodological Differences’) [84]. Across studies there
is large heterogeneity with respect to these factors, which might
explain the variability of reported minimum and optimal block
thresholds (Supplementary Table 1). For example, Bhadra et al.
reported true block at frequencies as low as 1 kHz in cat pudendal
nerve [63], while other authors determined that frequencies of at
least 6 kHz would be required to induce block in the same model
[85,86].
To date, two mechanisms have been identified by which con-
duction block may manifest in response to kilohertz-frequency
stimulation. First, exposure to successive pulses of high frequency
may deplete the readily available pool of presynaptic neurotrans-
mitters inducing a state of synaptic fatigue (see ‘Synaptic effects’)
[17]. Second, high-frequency stimulation may directly inhibit the
target axon and its ability to conduct action potentials. This tem-
porary and reversible abolishment of neural activity was first
observed in 1935 by Bugnard and Hill who noted diminished nerve
responses in the frog sciatic nerve upon exposure to high-
frequency stimulation at 2500 Hz [87]. Reboul et al. confirmed
these initial findings demonstrating inhibition of the cat popliteal
nerve at frequencies up to 40 kHz [88]. Woo and Campbell provided
the first evidence that true conduction block was preceded by an
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Brain Stimulation 14 (2021) 513e530
initial period of increased, asynchronous bursting of neural activity
in cat tibial and frog sciatic nerves [89]. This phase, termed ‘onset
response’, lasted about 1e2 s and displayed an amplitude typically
two to three-fold higher as compared to a single action potential
(Fig. 5A) [46]. Alternatively, onset response may manifest by means
of a prolonged period of strong activation that ceases over several
seconds (Fig. 5A) [8,81]. Based on their observations in rat sciatic
nerves, Bhadra and Kilgore [46] categorized the onset response into
three phases: During ‘Phase I onset’ kilohertz stimulation distorts
the transmembrane potential entraining the majority of available
and excitable ion channels. This allows the neuron to produce a
series of action potentials at a rate as fast as the refractory period of
the ion channels allows [6,51,57]. However, as stimulation pro-
gresses the cell membrane transitions into a variable period of
asynchronous and repetitive firing. During this ‘Phase II onset’ the
responsiveness of the nerve gradually decreases with the cell
membrane accommodating to subsequent stimuli [51]. Finally, all
firing activity ceases and the nerve enters the third and final phase
of partial or complete block [46]. Of note, a significant body of
literature has been published in recent years that sought to mini-
mize the onset response as it has been associated with painful
sensations and muscle contractions [30]. Proposed strategies to
reduce the onset response included ramping current intensities
[53,57] waveform modulation [60,90], thermal cooling [91],
changes in electrode geometry [59] and contact separation distance
[58], direct current stimulation [92e94], and the employment of
high-frequency, high-amplitude stimulation paradigms [59,95].
Clinical implementations of kilohertz-frequency stimulation,
however, lack reports of onset response-induced side effects. This
holds especially true for vagus nerve stimulation paradigms, so-
matic nerve stimulation therapies for treatment of post-amputee
pain, but also spinal cord stimulation paradigms where conduc-
tion block is presumed to be the underlying mechanism of action
[30]. This suggests that either the selected stimulation parameters
do not induce onset response and that conduction block is the
unlikely mechanism of action (‘Phase I onset’ is always present
when conduction block is initiated, while ‘Phase II onset’ can be
reduced or eliminated by waveform and electrode optimization
[30,58,59]), or that the onset response plays an overall minor role in
clinical practice; however, this remains to be further investigated
[29]. It is important to note that it is unclear whether conduction
block is the mechanism of action underlying the therapeutic benefit
observed in these modalities [29,96e100].
The ion channel gating kinetics of neural membranes are of
major importance during conduction block induced by kilohertz-
frequency stimulation. While sodium channels feature fast ki-
netics that are of major importance in the initiation of action po-
tentials, potassium conductances activate almost 10 times slower
[101]. Owing to these intrinsic properties, the potassium channels’
ability to follow high-frequency pulse trains is limited. Indeed,
modeling studies in unmyelinated nerve models of the Hodgkin-
Huxley (squid axon) and Frankenhaeuser-Huxley (frog axon)
model have indicated that potassium channels enter a state of
persistent activation during kilohertz-frequency stimulation in the
range from 4 to 10 kHz [50,102,103]. The ensuing efflux of potas-
sium consequently overwhelms the depolarizing sodium currents
and biases the transmembrane potential towards hyperpolar-
ization, which eventually culminates in true conduction block
[50,102,104,105]. While the sodium conductances remain intact in
this frequency range, they eventually saturate and undergo inacti-
vation as frequencies increase beyond 10 kHz [106]. As a result of
persistent sodium channel opening, the depolarizing sodium cur-
rents eventually surpass the large potassium conductance, inducing
a state of dynamic depolarization. Ultimately, the transmembrane
C. Neudorfer, C.T. Chow, A. Boutet et al. Brain Stimulation 14 (2021) 513e530

Fig. 5. Potential manifestations of conduction block and stimulus-amplitude dependent non-monotonic activation. Direct inhibition of neuronal activity by means of
conduction block may be observed during kilohertz-frequency stimulation. (A) Neuronal blockage is preceded by the onset response which may manifest as a prolonged period of
strong activation that ceases over several seconds (top) or a bout of asynchronous, irregular firing lasting 1e2 s with an amplitude two to three-fold larger than a conventional
action potential (middle). Following the onset response, the neuron enters the stage of block, that persists throughout the duration of stimulation. Upon cessation of stimulation
intrinsic neuronal firing promptly resumes. (B) Frequency- and amplitude-dependent block threshold curves of compound action potentials evoked during stimulation of rat sciatic
(top panel) and vagus (bottom panel) nerve. Note the non-monotonic relationship for slow, unmyelinated (red) fibers featuring decreases in threshold amplitude at frequencies
above 30 kHz. Shaded regions indicate stimulation settings were fiber-specific conduction block could be achieved. This is in contrast to (C) where block threshold curves obtained
during rat vagus nerve stimulation feature a monotonic relationship between frequency and amplitude across the entire frequency spectrum tested. (D) Dependence of conduction
block on stimulation frequency and amplitude as modeled in-silico using monophasic waveforms. Both increases in frequency and amplitude increase the fraction of blocked fibers
during kilohertz-frequency stimulation. B, C and D adopted from Patel et al. (2015) [8], Pelot et al. (2020) [124] and Couto et al. (2016) [34]. (For interpretation of the references to
colour in this figure legend, the reader is referred to the Web version of this article.)

potential is maintained at an average depolarized value which
prevents any further spiking [49,107].
Block associated with potassium channel activation likely plays
a role in unmyelinated axons and myelinated axons of amphibians
where potassium transients are large and play a major role in action
potential generation [30,104,108]. However, it is unlikely to be the
primary cause in mammals where potassium currents are small in
comparison [109,110]. This notion has led to the proposal of sodium
channel inactivation as the likely mechanism underlying conduc-
tion block in mammals [48]. This postulate is supported by a body
of in-silico and in-vivo studies. For example, MRG axon models
(mammalian axon model) have proposed a dynamic membrane
depolarization during kilohertz-frequency stimulation that is
driven by relative increases of inward sodium currents as compared
to outward potassium currents [48,51,52,111]. Indeed, Ackermann
et al. demonstrated that while depolarizing currents associated
with sodium influx facilitated the blocking effect, the
hyperpolarizing currents associated with potassium efflux dimin-
ished conduction block in the MRG model [107]. These computa-
tional findings are supported by in-vivo studies that clearly
demonstrate membrane depolarization in response to kilohertz-
frequency stimulation [30]. Furthermore, pharmacological
blockage of sodium channels using tetrodotoxin and ranolazine
abolished stimulation-induced depolarization [27] and was asso-
ciated with a 20% increase in block threshold during stimulation
paradigms [107].
Despite an improved understanding of the mechanism under-
lying conduction block in recent years, neither of the proposed
hypotheses are likely complete [29]. For example, recent studies
reporting increases in both the sodium and potassium conductance
suggest that the mechanisms may not be mutually exclusive with
either of the mechanisms dominating over different temporal and
spatial scales [29,51,82]. In addition, current models do not account
for post-stimulation block e the continued inactivation of axons

520
C. Neudorfer, C.T. Chow, A. Boutet et al.
following cessation of stimulation [112]. A study in frog sciatic
nerve concluded that post-stimulation block depends on stimula-
tion intensity and duration, but not frequency [113]. As a potential
mechanism, the authors suggested that the restoration of intra-
axonal ion concentrations by ion pumps may play a role. Experi-
mental validation of these findings is, however, necessary.
Preliminary studies of kilohertz-frequency stimulation in deep
brain structures suggest that conduction block and a resulting
decline in entropy constitute a potential mechanism of action in
deep brain stimulation (DBS) [34,56]. This is corroborated by cur-
rent hypotheses of conventional DBS where highly regularized
firing patterns delivered at 130 Hz are presumed to reduce the
complexity of transmitted information creating an “informational
lesion” [114e117] (Table 2). In consequence, both conventional and
kilohertz-frequency stimulation seem to induce a state of reduced
entropy, albeit by exploiting different membrane properties. This
hypothesis awaits clinical validation and verification.
Stimulus amplitude dependent non-monotonic activation
While frequency selection is crucial to induce the desired neural
response during kilohertz-frequency stimulation, the membrane
dynamics is not only contingent on the respective employed
stimulus frequency, but also the stimulus amplitude (Fig. 5C)
[41,118,119]. This relationship is often demonstrated by plotting the
block threshold e the lowest amplitude capable of inducing block
in the target nerve e as a function of tested kilohertz-frequencies
(Fig. 5C and D). The ensuing block threshold curve allows the
characterization of stimulus-response phenomena across nerve fi-
ber types and species. As a result, block threshold curves have been
shown to increase linearly as a function of stimulus frequency in
fast (myelinated, large diameter) fibers [46,52,82,120], whereas a
non-monotonic relationship is assumed in slow (unmyelinated,
small diameter) fibers (Fig. 5D). The latter was first observed by
Joseph et al. during sinusoidal stimulation of Aplysia unmyelinated
nerve fibers [7]. Specifically, as frequencies extended beyond
12 kHz during stimulation the thresholds required to effectively
induce conduction block gradually decreased. Using the same
experimental paradigms, the group was able to replicate these
findings later in frog sciatic nerve preparations [6] and in-vivo
performing rat sciatic and vagus nerve stimulations (Fig. 5D) [8].
Analogous to prior studies, the authors obtained monotonic block
threshold curves during stimulation of myelinated A-fibers, while
unmyelinated C-fibers featured non-monotonic relationships with
peak thresholds reported at 30 kHz. Studies in cat tibial nerves [45]
and retinal ganglion cells (RGCs) [121,122] further corroborate
these findings. In the latter case the non-monotonic behavior of
RGCs could be exploited to selectively activate and inhibit cell
subtypes using asymmetric stimulation paradigms [118].
Investigating the potential mechanisms underlying stimulus
amplitude dependent non-monotonic activation Zhao et al.
modeled the effects of electrical stimulation at frequencies up to
300 kHz in myelinated and unmyelinated axon models [82,123]. For
both fiber types the authors noted monotonic threshold increases
across the investigated frequency spectrum when employing
symmetric waveforms. This is in accordance with recent rat vagus
nerve stimulation studies that demonstrated a monotonic increase
of current thresholds in the frequency range between 10 and
80 kHz (Fig. 5C) [124]. The employment of non-symmetric wave-
forms, however, yielded non-monotonic block threshold curves at
frequencies above 12e40 kHz (unmyelinated) and 40e80 kHz
(myelinated), respectively, with block ensuing as a result of mem-
brane depolarization (if the negative pulse was longer) or hyper-
polarization (if the positive pulse was longer) [82,123]. Based on
these findings the authors concluded that the non-monotonic
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Brain Stimulation 14 (2021) 513e530
relationship reported in previous studies may be the result of
slightly asymmetric (<1 ms in pulse width) waveforms. Unfortu-
nately, asymmetric waveforms have not been employed in the
frequency range above 10 kHz in-vivo today to validate these
findings. However, comparative studies have investigated neural
behavior in response to stimulation at fixed pulse widths vs. pulse
withs varying as a function of frequency [85,103]. Investigating a
frequency range between 1 and 10 kHz Tai et al. described a
monotonic relationship between frequency and block thresholds in
cat pudendal nerves both, at fixed and varying pulse widths [85].
Block was most effective if pulse with changed with frequency (i.e.,
if a 50% duty cycle was maintained); duty cycles above 50%, how-
ever, were not investigated.
Taken together, the differential response characteristics of fiber
and cell subtypes suggest the possibility to selectively modulate
neural activity [29]. However, as shown in Fig. 5D, for selective
block to be feasible, the area under the block threshold curve of the
cell/fiber type of interest is required not to overlap with the area
under the block threshold curve associated with the non-target
cell/fiber type. The influence of pulse width changes has thus far
only been demonstrated in the frequency range up to 10 kHz; its
effect on non-monotonic behavior, however, awaits experimental
validation in-vivo.
Methodological considerations
Studies employing kilohertz-frequency stimulation feature
great heterogeneity with respect to the materials, methods and
outcome measures reported. For example, differences in waveform
properties, whether the waveform is current- or voltage-controlled,
the electrode type and differences in fiber type, size, and diameter
may alter experimental findings and complicate the generalization
and interpretation of findings. The accumulation of studies inves-
tigating conduction block in the past years allows a comparison of
methodological differences and their influence on conduction block
thresholds. A summary of findings is provided in the following
sections. For an itemized description of methods across all studies
please refer to Supplementary Table 1.
Charge balanced vs imbalanced stimulation
In addition to waveform asymmetries kilohertz-frequency
stimulation is susceptible to charge imbalances that have the po-
tential to confound the effects of electrical stimulation [125e127];
the mechanisms associated with charge-imbalanced stimulation
are fundamentally different from biphasic stimulation as they
maintain a net charge on the nerve membrane, thus resembling
mechanisms related to monophasic and DC stimulation [29,48]. In
this context, work by Solomonow et al. is frequently discussed
which has led to mechanistic misinterpretations in the field
[128e131]. These authors performed monophasic stimulation of
cat sciatic nerves in the frequency range between 0.2 and 16 kHz
reporting effective block at frequencies as low as 600 Hz. In
contrast, similar studies employing biphasic square pulses had
found much higher frequencies e namely 8 kHz e to be most
effective for block [41]. This discrepancy has been attributed to the
monophasic stimulation paradigm employed by Solomonow et al.
[48,132], which caused the accumulation of net charge and explains
the lower frequencies required to induce effective block. The
mechanism of this waveform is thus likely to be related to a DC
effect on the neural membrane and different from true conduction
block [126,133,134]. Furthermore, based on the experimental
methods used in these studies, it is likely that the ‘block’ observed
by the authors resulted from synaptic fatigue (see ‘Synaptic effects’)
[30,51]). Studies of the MRG model by Couto et al. corroborate the
C. Neudorfer, C.T. Chow, A. Boutet et al. Brain Stimulation 14 (2021) 513e530

effects observed during monophasic stimulation, suggesting an
inverted block threshold curve in this stimulation paradigm, where
block is achieved both as a function of frequency and amplitude
increase (Fig. 6D) [34].
Importantly, the accumulation of charge during DC and mono-
phasic stimulation not only alters neural responses, but also has
implications on long-term effects at the electrode-tissue interface
yielding potentially undesirable effects such as electrochemical
reactions and neural damage [13]. Hence, it is imperative to reduce
DC offsets to a minimum during kilohertz-frequency stimulation.
However, only a minority (~40%, Supplementary Table 1) of inves-
tigated in-vitro and in-vivo studies have reported calibration rou-
tines to account for direct current (DC) contamination. Methods
and guidelines for ensuring signal integrity and minimal DC
contamination have been reported in the literature [125,127] and
need to be considered when employing kilohertz-frequency stim-
ulation. These authors determined that DC-blocking in-line ca-
pacitors, that were frequently employed across studies, are not
always sufficient, to eliminate DC offsets. Conversely, inductor-
capacitor circuits were identified as the most ideal solution. For
an individualized description of stimulation equipment and cali-
bration routines employed by each study refer to Supplementary
Table 1.
Waveform shape
Across investigated studies, sine waves (n ¼ 52) and square
waves (n ¼ 45) constituted the most employed waveform shapes.
However, several other paradigms and waveform modulations have
been tested (n ¼ 9) (Supplementary Table 1). Qualitative compar-
isons between waveform shapes have been conducted by Tai et al.
[85,86]. Stimulating cat pudendal nerves, the group compared
block thresholds associated with sinusoidal and biphasic pulsed
waveforms; in pulsed paradigms the pulse width was either fixed at
10 ms or 30 ms, or varied as a function of the frequency maintaining a
50% (symmetric) duty cycle. Block was most effective when the
pulse width varied with respect to stimulus frequency in the range
between 6 and 10 kHz. Pen ~ a et al. confirmed and quantified these
findings in-vivo (rat tibial nerve) and in-silico at 10 and 20 kHz,
respectively, determining that sinusoidal waveforms required ~30%
higher currents than biphasic symmetric square waves [47].
Conversely, sine waves had the lowest power consumption
achieving block at a power ~10e20% lower than square waves.
Nerve diameter
Kilohertz-frequency stimulation induced conduction block has
been demonstrated across a variety of animal models
(Supplementary Table 1) with nerve diameters ranging from
approximately 1 mme6 mm (dog radial nerve) [135]. While large
diameter nerves require overall greater intensities to achieve block
as axons located at the nerve’s center are farther from the electrode
[30] it is important to note that the influence of nerve diameter on
block threshold has not been investigated systematically to date. A
deeper understanding of this relationship is, however, critical for
clinical practice where kilohertz-frequency stimulation is typically
applied to large diameter nerves, such as vagus, tibial, sciatic, and
common peroneal nerves [10,100] or the spinal cord [136]
assuming conduction block as the underlying mechanism of action
[30]. Further research is warranted and necessary.
Fiber type and size
Both, computational and animal studies reported the effects of
kilohertz-frequency stimulation across fiber types of varying
diameter (Supplementary Table 1). Tanner first described the fiber
size selectivity of peripheral nerves in response to kilohertz-
frequency stimulation [137]. By exposing frog sciatic nerves to
alternating currents at a frequency of 20 kHz and varying the
amplitude of the signal he noted that larger fibers were preferen-
tially blocked at lower intensities as compared to smaller fibers.
This is corroborated by in-vivo and in-vitro studies of frog sciatic
and rat sciatic/vagus nerves that demonstrated similar relation-
ships in the frequency range below 30 kHz [6,8]. Systematic in-
vestigations, however, were primarily conducted in-silico. In 24
studies block thresholds were determined in myelinated axon
models (investigated fiber diameters: 5e20 mm), whereas four
studies employed unmyelinated nerve models (investigated fiber
diameters: 1e20 mm) (Supplementary Table 1). Across studies,
axons with larger diameters featured overall lower block thresh-
olds as compared to small diameter axons
[50,51,98,103,105,138,139]. This finding was consistent across both
fiber types, irrespective of employed model. In addition, some

Fig. 6. Thermal effects of kilohertz-frequency stimulation. (A) Influence of thermal cooling on minimal blocking frequencies as obtained from cat pudendal nerves studies during
kilohertz -frequency stimulation. Owing to the slower potassium gating kinetics at reduced temperatures block can be effectively induced and maintained at lower frequencies. (B)
Modeled temperature increases during kilohertz-frequency spinal cord stimulation (SCS). Graphs feature temperature changes (DT) from baseline at the level of the spinal cord
owing to active (bioheat including blood perfusion and metabolic heat generation) (top) and passive (joule heating) (bottom) heating at frequencies of 0.05, 0.1, 1, 5, and 10 kHz and
varying peak amplitudes (1.0e5.0 mA). A adopted from Tai et al. (2008) [155]. Data for B obtained from Zannou et al. (2019) [159].

522
C. Neudorfer, C.T. Chow, A. Boutet et al.
studies reported a dependence of block thresholds on frequency at
a given diameter [51,52]. Indeed, block thresholds featured great
variance in small diameter fibers with intensities varying by 1 mA
in the frequency range between 4 and 40 kHz. These differences
decreased monotonically as fiber diameters were increased [52].
Because of the disparity in block thresholds between small and
large diameter fibers several authors have proposed that by tuning
stimulation frequency and amplitude it may be possible to achieve
selective, fiber-type specific blockage [29,81,138]. Indeed, the se-
lective activation of small diameter fibers has been the goal of
several studies aiming to mimic physiological recruitment in neu-
roprosthetic systems [140e143].
Electrode design, geometry, and distance
Cuff electrodes that establish intimate contact with the target
tissue constitute the gold standard in the investigation of blocking
effects today. Indeed, 73% of screened peripheral nerve stimulation
studies reported the usage of cuff electrodes (Supplementary
Table 1). This is owing to the monotonic relationship between
block threshold and perpendicular distance to the axon with
thresholds varying inversely as the second power to the distance
[51]. Computational studies of myelinated axon models in am-
phibians and mammals corroborate this finding [49,53,144,145]. Of
note, these studies demonstrated a dependence on the spatial
relationship to node and internode in-silico at electrode-to-axon
distances below 1 mm, with electrode placement over internodes
requiring much higher block currents [51]. As a result, computa-
tional studies (70%) frequently employed electrode-to-axon dis-
tances of 1 mm to account for this issue (Supplementary Table 1).
Studies employing intra-fascicular electrodes [146,147], glass suc-
tion electrodes [6,7], and electrodes placed along the nerve [30]
have demonstrated the ability to induce nerve block in peripheral
nerves; the duration of onset response and the current levels
required to induce block, however, were significantly increased in
some of these paradigms. Cuellar et al. investigated a variety of
electrode designs during electrical stimulation of rat and goat
dorsal roots [81]. Although no systematic comparison was con-
ducted, the authors noted that bipolar hook electrodes achieved the
lowest block thresholds during kilohertz-frequency stimulation.
Apart from electrode design, electrode geometry significantly
influences conduction block thresholds. Ackermann et al. first
described how electrode number and spacing may alter the block
threshold curve [138]. Investigating monopolar, bipolar, and tri-
polar cuff electrodes in rat sciatic nerves, the authors concluded
that block could be achieved with all configurations, albeit at the
cost of longer onset responses during monopolar stimulation. This
is corroborated by Gaunt and Prochazka [148], who noted similar
effects in cat pudendal nerve studies comparing mono- and bipolar
electrode configurations.
The effect of contact separation distance on block threshold was
investigated by Ackermann et al. in computational and rat sciatic
nerve studies [58,138]. The authors demonstrated a monotonic
relationship between bipolar contact separation distance and block
threshold [138]. Similarly, the extent of onset response greatly
depended on separation distance. Specifically, Ackermann et al.
demonstrated a monotonic increase in phase II onset integral and
decay time with increasing bipolar separation distance [59]; phase I
onset, however, remained unaltered. Based on this finding the au-
thors proposed that with the proper electrode design, phase II
firing could be eliminated altogether. The optimal configurations,
however, would have to be tailored to the respective target as larger
nerve diameters require greater spacing [59].
Differences in electrode geometric surface area and contact
width have been implicated in altering the neural response to
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Brain Stimulation 14 (2021) 513e530
kilohertz-frequency stimulation. Results from tibial nerve studies
in rats suggest that increases in geometric surface area are associ-
ated with block threshold reductions [149]. The extent of block
threshold reduction, in return, featured a great dependence on the
employed frequency with block thresholds decreasing two-fold
during 20 kHz stimulation and three-fold at frequencies of
40 kHz. Finally, contact width may influence conduction block ef-
ficacy [123], however, no systematic investigations into this matter
have been conducted to date.
Voltage vs current controlled stimulation
Both voltage- and current-controlled waveform generators were
employed in in-vivo and in-vitro studies of kilohertz-frequency
stimulation with the majority of studies applying current-
controlled stimulation (67%). Systematic comparisons between
both types of stimulation, however, are sparse. In their initial
studies, Kilgore and Bhadra published an account of voltage-vs.
current-controlled stimulation while demonstrating the potential
of kilohertz-frequency paradigms to effectively block nerve con-
duction in the frog sciatic nerve [48]. Maintaining electrode im-
pedances at constant values (5e10 kU) the authors demonstrated
that block thresholds were not significantly altered by either
current-controlled or voltage-controlled sinusoidal waveforms. By
performing stimulations in a total of 24 sciatic nerve preparations
effective block was achieved at frequencies between 2 and 20 kHz
and amplitudes of 0.15e0.8 mA and 1.5e2.0 V, respectively. Of note,
to account for potential DC contamination, the employed signal
generators were capacitively coupled to the stimulating electrodes.
This is of major importance as both voltage- and current-controlled
stimulation may unintentionally add DC-offsets to a kilohertz-
frequency signal [125]; controlling for this factor is thus imperative.
In voltage-controlled stimulation, where the amplitude is
described by a potential difference, the current flow will depend on
the electrode impedance. In return, the electrode impedance fea-
tures a dependence on the frequency and decays as stimulation
frequency is increased due to its capacitive nature [150]. In
consequence, the current applied to the tissue may be larger for
higher frequencies during voltage-controlled stimulation owing to
a smaller voltage drop at the interface. For example, the impedance
of DBS electrodes measured in vitro and in vivo at 10 kHz is up to
one order of magnitude smaller than 10 Hz [151,152]. This can have
clinical implications as lower electrode impedances may result in
greater volumes of tissue activated and lower block thresholds as
compared to current-controlled stimulation [153].
Thermal effects
Thermal effects have the potential to alter ion channel dynamics
and influence the neural response to kilohertz-frequency stimula-
tion [102,154,155]. Yet, the majority of studies failed to control for
local temperature changes with reported temperatures greatly
varying between 6  C and 39.0  C (Supplementary Table 1). Hence,
the following section will only consider reports that distinctly re-
ported local temperatures in proximity to the blocking electrode.
Studies reporting animal core temperatures without accounting for
local differences are not considered.
Tai et al. published a comprehensive account of the
temperature-frequency relationship during electrical stimulation
of cat pudendal nerves [155]. Investigating the effects of thermal
cooling at constant current thresholds, the authors noted a reduc-
tion of minimal blocking frequencies from 6 to 4 kHz during tem-
perature reductions from 37  C to 15  C (Fig. 6A). This is
corroborated by computational studies of myelinated nerves where
similar temperature changes were associated with drastic
C. Neudorfer, C.T. Chow, A. Boutet et al.
decreases (as low as 500 Hz) in minimal blocking frequencies [154].
Unfortunately, the authors did not investigate the contribution of
temperature to block threshold intensity; early in-vitro work by
Rosenblueth et al., however, suggests that external temperature
changes do not affect minimum blocking thresholds. Stimulating
excised cat sciatic nerves in a tempered chamber (25e35  C) at
frequencies up to 40 kHz and varying intensities the group did not
discern any notable differences across stimulation paradigms [88].
Finally, in an attempt to reduce onset response during kilohertz-
frequency stimulation, Ackermann et al. performed thermal cool-
ing of cat and rat sciatic nerves [91]. By reducing nerve temperature
to 6e14  C the authors effectively induced conduction block.
Without eliciting an onset response, the cooling block could then
be transitioned into electrical block, which was effectively main-
tained even after reheating of the nerve.
A temperature-frequency relationship was also established in
amphibian myelinated axon models, which indicated reductions of
minimum block frequencies from 6 kHz at 37  C to 3 kHz at 15  C
[102]. The change in minimum block frequency in this study was
attributed to the temperature-dependent activation of potassium
channels; namely, as temperature decreases the potassium gating
kinetics decrease as well allowing lower blocking frequencies to
keep the potassium channels in a constantly open state. Similar
thresholds were observed in-vivo by Kilgore et al. who reported
effective conduction block of isolated frog sciatic nerves at fre-
quencies as low as 2 kHz at room temperature (20e25  C) [48].
In addition to the external temperature environment, electrical
stimulation has the ability to induce temperature increases in
nervous tissue [156e160](Table 3). In the context of kilohertz-
frequency stimulation two determinants governing the degree of
heat generation have been proposed: joule heating and metabolic
heat generation [161e163]. Joule heating describes the generation
of thermal energy during the passage of electric current through a
conductor i.e. the nervous tissue [156]. The spatial distribution and
magnitude of temperature change is primarily determined by the
stimulus amplitude; in stimulation paradigms employing increased
duty cycles, however, the root mean square (RMS) power of a
rectangular waveform varies positively with the square root of its
duty cycle [159]. In consequence, the power of current flow from an
electrode may produce temperature increases (Fig. 6B). This has
implications for deep brain stimulation and spinal cord stimulation,
where kilohertz-frequencies may deposit more power in nervous
tissue than conventional stimulation paradigms [97]. Metabolic
expenditure in response to increased neural activity may also
contribute to local tissue heating during kilohertz-frequency
stimulation [163e165]. Indeed, under physiological conditions,
metabolically active states such as arousal, feeding, or social in-
teractions have been shown to induce temperature increases of up
to 2e3  C in animal models [166e170]. These temperature
Table 3
Summary of temperature changes observed during conventional and kilohertz-frequency stimulation in-silico and in-vitro.
Stim settings Modality Model
10 kHz 3.5 mA 40-10-40 ms SCS Bio-heat model (CAD) 3.42 mM/34.22 mM/154 mM NaCl
1 kHz, 3.5 mA, 100-100-100 ms
50 Hz, 3.5 mA, 200-100-200 ms
10 kHz, 2.45 mA (RMS) SCS 0.25 S/m (Agarose gel)
0.04 S/m
0.085 S/m
185 Hz, 3.0 V, 90 ms DBS Bio-heat transfer model, FEMLAB 3.2 (COMSOL)
185 Hz, 10.0 V, 210 ms
100 Hz, 2.0 V, sine DBS 34 mM NaCl
1 kHz, 2.0 V, sine
10 kHz, 2.0 V, sine
10 kHz, 3e7 mA, 30 ms DBS MRI-based joule-heat coupled bio-heat model (CAD, Solidworks)
524
Brain Stimulation 14 (2021) 513e530
elevations occurred with greater magnitude and significantly faster
than the corresponding temperature changes in arterial blood
suggesting that local cerebral heat production is the likely primary
source of the observed hyperthermia [171]. Similarly, enhanced
metabolic activity in response to kilohertz-frequency stimulation
and concomitant temperature increases have been observed in-
silico [156e160]. Along with joule heating, metabolic expenditure
consequently shapes the local temperature gradient in the vicinity
of the electrode (Table 3).
While individual studies have suggested that temperature in-
creases alone may be capable of driving neural activity [172,173]
temperature effects are more likely to augment neural information
processing instead. For example, the dissipation of heat at the
electrode-tissue interface distinctly alters neural properties (e.g.
transmembrane capacitance and ion-channel gating kinetics)
leading to changes in firing threshold [113], peak firing rate, nerve
conduction velocity [174] and conduction block threshold [175].
Tissue heating is further associated with altered synaptic trans-
mission: temperature-induced changes in ion-pump-kinetics
modify the neurotransmitter environment and alter the release
and clearance of the available neurotransmitter pool [176e178].
These changes can be observed at temperature increases as low as
0.5  C, which is within the margins determined by the majority of
bio-heat models [156,159,160] and in-vitro studies [158]. While few
studies controlled for external temperature differences in-vivo,
only one study measured temperature changes during kilohertz-
frequency stimulation. Exposing the mouse brain to 2 kHz,
biphasic alternating currents the authors noted a 0.07  C temper-
ature increase, which correlated with the magnitude of sponta-
neous deviations observed in the pre- and postoperative course [4].
Overall, the role of thermal effects induced by kilohertz-frequency
stimulation remains to be established and validated in-vivo.
Synaptic effects
The synapse is crucially involved in neuronal information pro-
cessing and features activity-dependent increases (facilitation) or
decreases (depression) in synaptic transmission that typically occur
within hundreds of milliseconds following the onset of activity. The
mechanism underlying this neuronal characteristic is referred to as
short-term plasticity (STP) and has been observed in-vitro and in-
vivo both at the pre- and postsynaptic level [17] (Fig. 7).
Mechanisms determining the throughput of a burst at the pre-
synaptic level include short-term depression and facilitation
(Fig. 7A and B). During presynaptic depression, repeated activation
at the presynaptic terminal diminishes the release of neurotrans-
mitters into the synaptic cleft either by reduction of their release
probability or by depletion of the readily available pool of vesicles
[179]. As a result, the presynaptic terminal acts like a low-pass filter
Peak Temp change in surrounding tissue [ C] Ref.
0.18e1.72 [158]
0.09e0.22
<0.05
0.84 [159]
0.74
0.39
 0.3 [156]
 0.8
0.2 [157]
0.13e1.87 [160]
C. Neudorfer, C.T. Chow, A. Boutet et al. Brain Stimulation 14 (2021) 513e530

that is not able to follow high-frequency bursts. Conversely, a

synapse may exhibit high-pass filter properties by responding to
incoming bursts with increased neurotransmitter release other-
wise known as presynaptic facilitation (Fig. 7B) [180]. This mech-
anism is implemented either via an increase of calcium
concentration at the presynaptic terminal [181e183], or saturation
of a local calcium buffer [184,185].
Similar properties can be observed at the postsynaptic mem-
brane. Here, the mechanisms governing STP underlie distinct re-
ceptor characteristics. During neurotransmitter exposure, receptors
may desensitize by transitioning into a non-responsive state and
thus be unavailable during subsequent stimuli (Fig. 7C) [186e188].
Similarly, repeated transmitter activation may result in receptor
saturation (Fig. 7D). Here, fewer receptors are available for neuro-
transmitters to bind during subsequent bouts of stimulation lead-
ing to an overall decrease in the synaptic response amplitude. The
precise response during receptor saturation, however, also features
a dependence on the channel kinetics. Consequently, currents
mediated by channels with slow kinetics (e.g. N-methyl-D-aspartate
(NMDA) channels) may still be able to elicit sufficiently large cur-
rents owing to summation with previous excitatory postsynaptic
potentials (EPSPs) (Fig. 7D) [189].
At the synaptic level, kilohertz-frequency stimulation has been
associated with the transient depletion of the available neuro-
transmitter pool e also known as synaptic fatigue or neurotrans-
mitter depletion block. Here, the high rate of generated action
potentials depletes the readily available pool of presynaptic neu-
rotransmitters [17]. Following the release of the readily available
neurotransmitter pool, the postsynaptic neuron does not receive
any further upstream input and hence enters a state of functional
“blockage”. However, despite the absence of input, the postsynaptic
neuron retains its overall excitability and may still be entrained by
external stimuli. In contrast to true conduction block, where action
potentials are arrested at frequencies greater than 1 kHz, synaptic
fatigue can be induced at much lower frequencies, typically in the
range between 0.1 and 1 kHz during endplate block [51] and as low
as 30 Hz during deep brain stimulation [190]. Increases in stimu-
lation frequency, however, accelerate the process of depletion [41].
In this respect, synaptic fatigue is a distinct mechanism and needs
to be distinguished from true conduction block.
Wedensky first described synaptic fatigue in-vivo noting a rapid
failure of transsynaptic transmission at the neuromuscular junction
following stimulation at frequencies in excess of 100 Hz [191]. A
Fig. 7. Pre- and postsynaptic mechanisms of short-term plasticity. Both pre- and
similar underlying mechanism has been attributed to several
postsynaptic mechanisms (left) shape the degree and content of information (right)
studies conducted in the 1980s and 1990s. Here, different groups that is transmitted across the synapse during short-term plasticity (STP). During pre-
described conduction block phenomena in-vivo employing fre- synaptic depression (A) the available pool of neurotransmitters is gradually depleted in
quencies as low as 200 Hz using monophasic and biphasic wave- response to stimuli arriving in short succession at the axon terminal. As the pool of
vesicles cannot be restored in time, activation of receptors at the postsynaptic mem-
forms [129e131,192]. While synaptic fatigue was not identified as
brane will decrease, which is reflected in smaller, subsequent EPSPs. Facilitation (B)
the primary underlying mechanisms in some of these studies, increases the amplitude of subsequent EPSPs. The altered response is attributable to
reevaluations have led to the conclusion that depletion of the residual elevations of intracellular calcium and the additional influx of calcium across
neurotransmitter pool likely explains the low threshold frequencies the presynaptic membrane in response to subsequent stimuli, that trigger an increased
required to induce block [30,41]. More recent studies conducted in- neurotransmitter release. Desensitization (C) is observed at the postsynaptic mem-
brane during prolonged neurotransmitter release. Receptors may transition into a state
silico and in-vivo corroborate the notion that synaptic fatigue can
of unavailability and do not contribute to biasing the membrane towards more
be achieved at lower frequencies than true conduction block [49]. depolarized values, leading to reductions in EPSP amplitude. Saturation (D) of post-
To differentiate between true conduction block and synaptic synaptic receptors may inhibit the generation of subsequent EPSPs if receptors feature
fatigue block studies typically control for this potential confound. a high affinity for a released neurotransmitter. Channels with slow kinetics that
By placing an electrode between blocking site and muscle and experience saturation may, however, produce postsynaptic currents despite the
reduced incremental amplitude of the second EPSP owing to summation with the
eliciting single suprathreshold pulses at this location, the integrity previous EPSP. (E) Graph featuring the block effectiveness as a function of stimulation
of the neuromuscular junction can be controlled for by eliciting frequency and amplitude at the neuromuscular junction. Note the high degree of block
muscle contractions. at 600 Hz, which has been attributed to the rapid depletion of the readily available
neurotransmitter pool and the subsequent ‘functional blockage’ of downstream
muscular activity. Stimulations were performed using monophasic square pulses with
Conclusion
a pulse width of 50 ms. (E) adopted from Solomonow et al. (1983) [128].

The effects of kilohertz-frequency stimulation on neural tissue

are manifold and distinct from conventional stimulation paradigms
525
C. Neudorfer, C.T. Chow, A. Boutet et al.
(Table 1) offering the potential for new therapeutic avenues. The
heterogeneous membrane properties across different neuron cell
types and differences in experimental methods, however, limit the
generalizability of findings. This holds especially true with respect
to brain stimulation where the effects of kilohertz-frequencies are
underexplored and have only recently gained attention [3,5]. In
contrast, this heterogeneity may also offer the potential for cell-
type specific entrainment of neuronal activity. By tailoring stimu-
lation paradigms towards distinct membrane properties kilohertz-
frequency stimulation may be leveraged in the future to selectively
activate or inhibit cell subtypes with the potential of improving
symptom control and reducing side-effect profiles. Finally, the
spectrum of responses elicited by kilohertz-stimulation may offer
the potential for new therapeutic avenues. Here, distinct stimulus-
response phenomena may be leveraged for more effective control
over neuronal activity. For example, desynchronization may
constitute an effective tool in the implementation of biomimetic
stimulation paradigms aiming to imitate physiological brain activ-
ity that is highly asynchronous and stochastic by nature [31,44]. In
contrast, conduction block may be desirable in implementations
seeking to functionally interrupt information transmission [117].
Funding
This work is supported by the German Research Foundation
(Deutsche Forschungsgemeinschaft, DFG NE 2276/1-1) (CN) and
the Canada Research Chair in Neuroscience (AML).
Declaration of competing interest
AML has been a consultant for Medtronic, Boston Scientific,
Abbott, and Nevro. All other authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.brs.2021.03.008.
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