Lidocaine Unmasks L-Type Ca2+ Spikes in the Thalamus

Copyright © 2016 International Anesthesia Research Society. Unauthorized reproduction of this article is prohibited.
XXX 2016 • Volume XXX • Number XXX www.anesthesia-analgesia.org 1
Copyright © 2016 International Anesthesia Research Society
DOI: 10.1213/ANE.0000000000001158
L
idocaine, when present in the systemic circulation,
produces well-known concentration-dependent cen-
tral nervous system (CNS) toxicity whose manifesta-
tion initially reflects inhibitory symptoms such as sedation,
drowsiness, and alterations in sensorium, before progress-
ing to excitatory phenomena including generalized sei-
zures, and, in the most severe cases, coma and death.1,2
That
lidocaine affects the CNS is not surprising, because it read-
ily passes the blood–brain barrier.3
However, in contrast
to lidocaine’s peripheral local anesthetic (LA) effects, the
mechanisms and sites of its central actions have received
comparatively little study because previous work has
emphasized the spinal cord, leaving supraspinal actions
relatively unexplored.4
Accordingly, there are no specific
mechanism-based therapies for LA CNS toxicity.
Exploring the thalamus as a central site implicated in sys-
temic LAactions, we previously found in rat thalamocortical
(TC) neurons that lidocaine at clinically neurotoxic concen-
trations increases excitability mediated by Na+
-independent,
high-threshold (HT) action potential spikes.5
Although the
BACKGROUND: High systemic lidocaine concentrations exert well-known toxic effects on the
central nervous system (CNS), including seizures, coma, and death. The underlying mechanisms
are still largely obscure, and the actions of lidocaine on supraspinal neurons have received com-
paratively little study. We recently found that lidocaine at clinically neurotoxic concentrations
increases excitability mediated by Na+
-independent, high-threshold (HT) action potential spikes
in rat thalamocortical neurons. Our goal in this study was to characterize these spikes and test
the hypothesis that they are generated by HT Ca2+
currents, previously implicated in neurotoxic-
ity. We also sought to identify and isolate the specific underlying subtype of Ca2+
current.
METHODS: We investigated the actions of lidocaine in the CNS-toxic concentration range
(100 μM–1 mM) on ventrobasal thalamocortical neurons in rat brain slices in vitro, using whole-cell
patch-clamp recordings aided by differential interference contrast infrared videomicroscopy. Drugs
were bath applied; action potentials were generated using current clamp protocols, and underly-
ing currents were identified and isolated with ion channel blockers and electrolyte substitution.
RESULTS: Lidocaine (100 μM–1 mM) abolished Na+
-dependent tonic firing in all neurons tested
(n = 46). However, in 39 of 46 (85%) neurons, lidocaine unmasked evoked HT action potentials with
lower amplitudes and rates of de-/repolarization compared with control. These HT action potentials
remained during the application of tetrodotoxin (600 nM), were blocked by Cd2+
(50 μM), and disap-
peared after superfusion with an extracellular solution deprived of Ca2+
. These features implied that
the unmasked potentials were generated by high-voltage–activated Ca2+
channels and not by Na+
channels. Application of the L-type Ca2+
channel blocker, nifedipine (5 μM), completely blocked the
HT potentials, whereas the N-type Ca2+
channel blocker, ω-conotoxin GVIA (1 μM), had little effect.
CONCLUSIONS: At clinically CNS-toxic concentrations, lidocaine unmasked in thalamocortical neu-
rons evoked HT action potentials mediated by the L-type Ca2+
current while substantially suppress-
ing Na+
-dependent excitability. On the basis of the known role of an increase in intracellular Ca2+
in
the pathogenesis of local anesthetic neurotoxicity, this novel action represents a plausible contrib-
uting candidate mechanism for lidocaine’s CNS toxicity in vivo. (Anesth Analg 2015;XXX:00–00)
Central Nervous System–Toxic Lidocaine Concentrations
Unmask L-Type Ca2+
Current–Mediated Action Potentials
in Rat Thalamocortical Neurons: An In Vitro Mechanism
of Action Study
Igor Putrenko, PhD,* Amer A. Ghavanini, MD, PhD, FRCPC,* Katrin S. Meyer Schöniger, MD,*†
and Stephan K. W. Schwarz, MD, PhD, FRCPC*†
From the *Department of Anesthesiology, Pharmacology & Therapeutics,
The University of British Columbia, Vancouver, British Columbia, Canada;
and †Department of Anesthesia, St. Paul’s Hospital, Vancouver, British
Columbia, Canada.
Amer A. Ghavanini, MD, PhD, FRCPC, is currently affiliated with the
Division of Neurology, University of Toronto, Ontario, Canada; and Trillium
Health Partners, Mississauga, Ontario, Canada.
Katrin S. Meyer Schöniger, MD, is currently affiliated with the Klinik
für Anästhesie, Chirurgische Intensivmedizin, Rettungsmedizin und
Schmerztherapie [KLIFAIRS], Luzerner Kantonsspital, Luzern, Switzerland.
Accepted for publication December 3, 2015.
Funding: Supported in part by the Canadian Anesthesia Research Foundation
through a Canadian Anesthesiologists’ Society Research Award and a
Canadian Anesthesiologists’ Society/Abbott Laboratories Ltd. Career
Scientist Award in Anesthesia (Toronto, Ontario, Canada); the Canada
Foundation for Innovation (Ottawa, Ontario, Canada); the British Columbia
Knowledge Development Fund (Victoria, British Columbia, Canada); a Pfizer
Neuropathic Pain ResearchAward (independently peer-reviewed public oper-
ating grant competition sponsored by Pfizer Canada Inc.; Kirkland, Quebec,
Canada); and the St. Paul’s Hospital Department of Anesthesia (Vancouver,
British Columbia, Canada). Dr. Schwarz holds the Dr. Jean Templeton Hugill
Chair in Anesthesia, supported by the Dr. Jean Templeton Hugill Endowment
for Anesthesia Memorial Fund.
The authors declare no conflicts of interest.
Reprints will not be available from the authors.
Address correspondence to Stephan K. W. Schwarz, MD, PhD, FRCPC,
Department of Anesthesiology, Pharmacology & Therapeutics, The
University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC,
Canada V6T 1Z3. Address e-mail to stephan.schwarz@ubc.ca.

Lidocaine Unmasks L-Type Ca2+
Spikes in the Thalamus
2 www.anesthesia-analgesia.org anesthesia analgesia
ionic basis of these action potentials is unknown, TC neu-
rons–central to the generation of conscious states, absence
epilepsy, as well as drug-induced sedation, anesthesia, and
analgesia6–9
–express high-voltage–activated (HVA) Ca2+
cur-
rents that generate HT potentials, similar to those in our
investigations.10–12
Hence, we conducted an in vitro mecha-
nism of action study with the goal to characterize these HT
potentials and determine their specific underlying ionic con-
ductances, testing the hypothesis that they are generated by
HT Ca2+
currents, previously implicated in neurotoxicity.13,14
We also sought to identify and isolate the specific underlying
subtype of HT Ca2+
current and investigate the mechanisms
that mediate the observed increase in excitability.
METHODS
Preparation of Brain Slices
All animal experiments were approved by the Committee
on Animal Care of the University of British Columbia. Our
experimental procedures were similar to those described
previously.5
Sprague-Dawley rats, aged P13 to P16, were
decapitated under deep isoflurane (Abbott Laboratories,
Montreal, Canada) anesthesia. The cerebrum was rapidly
removed and placed in oxygenated (5% CO2/95% O2), cold
(1°C–4°C), artificial cerebrospinal fluid (ACSF) of the fol-
lowing composition (mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4,
2 CaCl2, 2 MgCl2, 26 NaHCO3, 10 dextrose (pH, 7.3–7.4; 290
mOsm). The brain was dissected into 1 block and mounted
on a tissue slicer with cyanoacrylate adhesive. Coronal slices
(thickness, 250–300 μm) containing the ventrobasal tha-
lamic nuclei were cut on a Leica VT1200S vibratome (Leica
Biosystems, Nussloch, Germany), and the block was sub-
merged in oxygenated cold ACSF. After cutting, the slices
were incubated for 1 hour at room temperature (22°C to
24°C) in oxygenated ACSF.
Electrophysiologic Recordings
Whole-cell patch-clamp recordings were performed in
the current-clamp mode with a MultiClamp 700B ampli-
fier (Molecular Devices, LLC, Sunnyvale, CA). The patch
pipettes for recording were prepared with a Narishige
PP-83 2-stage electrode puller (Narishige Scientific
Instrument Lab, Tokyo, Japan) using thin-walled boro-
silicate glass (World Precision Instruments, Inc., Sarasota,
FL) and filled with a solution containing the following (in
mM): 139 K-gluconate, 10 ethylene glycol-bis(β-aminoethyl
ether)-N,N,N′,N′-tetraacetic acid, 6 KCl, 4 NaCl, 3 MgCl2,
10 HEPES, 0.5 CaCl2, 3 adenosine 5′-triphosphate (disodium
salt, Na2ATP), 0.3 guanosine-5′-triphosphate (sodium salt,
NaGTP), titrated to pH 7.3 to 7.4 with 10% gluconate. The
range of the measured electrode resistances was 5 to 6 MΩ,
and access resistance ranged from 10 to 25 MΩ.
For recording, the slices were transferred into a Perspex
submersion chamber (volume, 1.5 mL), where they were
immobilized with nylon mesh and continuously perfused by
gravity with oxygenated ACSF at a flow rate of 2.5 mL/min
controlled by a FR-50 flow valve (Harvard Apparatus,
Holliston, MA). Individual neurons were visualized with
the aid of differential interference contrast infrared videomi-
croscopy. For this purpose, an Axio Examiner.D1 fixed-stage
microscope (Carl Zeiss Microimaging GmbH, Göttingen,
Germany) was equipped with an apochromatically corrected
water immersion objective (W Plan-APOCHROMAT 40×/1.0
DIC M27; working distance, 2.5 mm), differential interference
as well as Dodt contrast optics, and a 0.9 condenser. Infrared
light was obtained by placing a 770-nm polarizer in the light
path. The images were recorded with an IR-1000 infrared
CCD video camera system (DAGE-MTI, Michigan City, IN)
and displayed on a Triview TBM-1503 black and white moni-
tor (Tatung Company of America, Inc., Long Beach, CA).
The patch pipettes were mounted on the headstage of the
amplifier and advanced with an MP-285 motorized precision
micromanipulation system (Sutter Instrument, Novato, CA).
Signals were low-pass filtered at a frequency of 3 kHz and
digitized at 10 kHz with a Digidata 1440A 16-bit data acqui-
sition system (Molecular Devices, LLC) controlled using
pCLAMP software version 10 (Molecular Devices, LLC).
Membrane potentials were corrected offline for a liquid junc-
tion potential of −8 mV.15
No leak subtraction was performed.
Drugs
Lidocaine HCl, Cd2+
, and nifedipine were obtained from
Sigma-Aldrich Canada Ltd. (Mississauga, ON, Canada);
ω-conotoxin GVIA and tetrodotoxin (TTX) were obtained
from Alomone Labs, Ltd. (Jerusalem, Israel). To prepare con-
centrated drug stock solutions, lidocaine and Cd2+
were dis-
solved in ACSF; TTX and ω-conotoxin GVIA were dissolved
in distilled water; and nifedipine was dissolved in dimethyl
sulfoxide. Before application, required aliquots of the stock
solutions (stored at 4°C [lidocaine, nifedipine] and −20°C
[Cd2+
, TTX, ω-conotoxin GVIA, respectively]) were dissolved
in fresh ACSF to obtain the respective concentrations. All
drugs were applied to the bath by switching from the con-
trol perfusate to ACSF containing a desired drug concentra-
tion with the aid of a VC-6 perfusion valve control system
(Warner Instruments, LLC, Hamden, CT). Recordings were
conducted after 6 minutes of perfusion of the slices with a test
solution. All results reported reflect steady-state responses.
Data Analysis
Data are presented as mean ± SD unless mentioned oth-
erwise. Multigroup dose-response comparisons were con-
ducted with the use of 1-way analysis of variance with the
Bonferroni post test as appropriate. Testing for differences
of normalized data from baseline (i.e., a hypothetical mean
of 1.0) was performed with a 1-sample t test. Comparisons
between 2 groups were conducted with the use of a paired
Student t test. We quantified the degree to which 2 variables
are related by computing the Pearson correlation coefficient.
Statistical tests were 2 tailed. Sample sizes were based on pre-
vious experiments,5,16
and, to be conservative, results of sta-
tistical comparisons were considered significant at α = 0.01.
Configuration variables (amplitudes and dV/dtmax) of indi-
vidual spikes in action potential trains are given descriptively
and expressed as ranges. Data were analyzed with the use of
ORIGIN (OriginLab Corporation, Northampton, MA) and
Prism (GraphPad, La Jolla, CA) software.
RESULTS
All neurons used in this study exhibited the characteristic
electrophysiologic properties of TC relay neurons, firing

repetitive tonic spikes when depolarized by direct current
injection from membrane potentials near rest, and a low-
threshold Ca2+
potential triggering a spike burst on depolar-
ization from hyperpolarized potentials or on rebound from
hyperpolarizing pulses.5,10,16
They were characterized by an
average resting membrane potential of −64.6 ± 4.8 mV, input
resistance of 315 ± 131 MΩ, and membrane capacitance of
188 ± 62 pF (n = 46).
Lidocaine Unmasks High-Threshold Action
Potentials
Consistentwithitswell-knownLAproperties,bathapplica-
tion of lidocaine at 100 μM to 1 mM abolished Na+
-dependent
tonic firing evoked by injection of depolarizing current pulses
into cells current-clamped at approximately −58 mV in all
neurons (Fig. 1A, left and center panels). However, in 39 of
46 (85%) neurons, lidocaine unmasked a distinct repetitive fir-
ing pattern evoked by injection of current pulses of increased
amplitude and characterized by a higher activation threshold
and a different spike configuration (Fig. 1A, right panel). The
average activation threshold for these HT potentials, defined
as the lowest membrane voltage at which these potentials
were activated after depolarizing injection of increasing step
current pulses from a holding potential of approximately −58
to −60 mV, was −21 ± 8 mV (n = 12), compared with −44 ±
3 mV for control action potentials (P 0.001). The average low-
est current pulse evoking HT potentials (“current threshold”)
exceeded that for control action potentials approximately
Figure 1. Lidocaine unmasked high-threshold (HT) action potential spikes in ventrobasal thalamocortical neurons. A, Typical fast tonic repeti-
tive action potential firing is evoked in a current-clamped neuron superfused with normal artificial cerebrospinal fluid under baseline condi-
tions (control) by injection of a 1-second depolarizing current pulse (left). Application of 100 μM lidocaine completely blocked the tonic firing
(middle), but on injection with a current pulse of increased amplitude, lidocaine unmasked a distinct pattern of repetitive HT action potential
firing (right). Note that the first 1 to 3 spikes in a train of HT action potentials unmasked by lidocaine of similar morphology to those in control.
Lidocaine’s effects were reversible after washout (data not shown). B, Illustrated are magnifications of the fifth spike in a train from (A) at
baseline (control) and after application of lidocaine. Lidocaine unmasked spikes of a different configuration, with markedly higher activation
thresholds, decreased amplitudes, slower depolarization/repolarization phases, and larger afterhyperpolarizations (AHP; arrow). C, Application
to a different neuron of the tonic Na+
channel blocker, tetrodotoxin (TTX; 600 nM), also completely blocked fast tonic “control” firing (left
and middle) and unmasked HT potentials when injected with a current pulse of increased amplitude (right). In contrast to lidocaine, neurons
treated with TTX fired no action potentials at the beginning of the HT spike trains of similar morphology as those in control.

4-fold (251 ± 158 vs 60 ± 42 pA; n = 12; P 0.001). The indi-
vidual HT action potentials themselves had lower amplitudes
(range, 25−37 vs 60−75 mV in control) but were followed by
fast afterhyperpolarizations (AHPs) characterized by higher
amplitudes (range, 8−16 vs 3−11 mV in control). The HT
potentials also exhibited markedly slower depolarization
and repolarization phases (maximum rates of increase and
decrease, dV/dtmax), which ranged between 0.23 to 0.55 and
2.5 to 4.4 mV/ms, respectively, compared with 13 to 48 and
7 to 23 mV/ms for control action potentials at baseline
(Fig. 1B). Of note, the first 1 to 3 spikes in a train of HT action
potentials unmasked by lidocaine were fast and had similar
morphology to those in control (Fig. 1A, right panel). TTX
application to neurons (600 nM) also suppressed baseline
tonic firing and unmasked the tonic firing of HT potentials
(Fig. 1C). As opposed to lidocaine, TTX completely inhib-
ited all fast action potentials preceding the HT potentials.
Compared with the 39 neurons in which lidocaine unmasked
HT potentials, the 7 neurons that did not exhibit lidocaine-
induced HT potentials had similar resting membrane poten-
tials and input resistances (P = 0.88 and 0.43, respectively); the
average membrane capacitance in the latter was 136 ± 51 vs
197 ± 60 pF in the former (P = 0.015; 95% confidence interval
of difference between means, 12−109).
Ionic Basis of High-Threshold Action Potentials
Unmasked by Lidocaine
Coapplication of TTX (600 nM) with lidocaine did not
block the unmasked HT potentials, supporting the hypothe-
sis that the underlying current was not primarily carried by
Na+
ions but mediated by Ca2+
. When neurons in which HT
potentials were evoked in the presence of lidocaine and TTX
were perfused with an extracellular solution deprived of
Ca2+
, the potentials disappeared completely in all neurons
tested (n = 4, Fig. 2A). This effect was fully reversible, with
the HT potentials reappearing instantly when the super-
fusing medium was changed back to the Ca2+
-containing
control solution. Application of the nonselective HVA Ca2+
channel blocker, Cd2+
(50 μM),12
completely blocked these
HT potentials in a reversible manner in all neurons tested
(n = 4; Fig. 2B). Collectively, these results indicated that the
HT action potentials unmasked by lidocaine in TC neurons
are driven by a HVA Ca2+
current.
The L-Type Ca2+
Current Mediates High-Threshold
Action Potentials Unmasked by Lidocaine
Ventrobasal TC neurons express a variety of HVA Ca2+
currents that potentially could underlie the HT poten-
tials, including L, N, P/Q, and R types.17
Of these, N-type,
L-type, and residual “R”-type currents have been shown to
mediate HT potentials in TC neurons in a composite man-
ner, with the latter R-type current previously reported to
represent the major component.12,18
In the present experi-
ments, the observed sustained nature of the HT tonic firing
and depolarized holding potential implied that inactivation
properties (voltage dependence and rate of inactivation) of
Ca2+
currents are critical for determining their suitability for
mediating this firing. Characterized by the slowest inactiva-
tion rate and most depolarized voltage dependence of inac-
tivation among HVA Ca2+
currents is the “long-lasting” L
type.19,20
To test the hypothesis that this current mediates the
HT tonic firing unmasked by lidocaine, we used the selec-
tive L-type Ca2+
channel blocker, nifedipine. In all neurons
tested, application of 1 μM nifedipine decreased the ampli-
tude of HT potentials (paired t test, P = 0.0001, n = 4) from
34 ± 4 to 25 ± 4 mV (mean ± SD of pairwise difference, −8 ± 1
mV) and increased (depolarized) their activation threshold
(paired t test, P = 0.009, n = 4) from −24 ± 9 to −17 ± 10 mV
Figure 2. The high-threshold
(HT) action potentials unmasked
by lidocaine are mediated by a
high-voltage–activated Ca2+
cur-
rent. A, The HT action potentials,
evoked in the presence of lido-
caine (600 μM) and coapplica-
tion of tetrodotoxin (TTX; 600
nM; to block Na+
currents and
presynaptic transmitter release),
reversibly disappeared when the
superfusing artificial cerebrospi-
nal fluid was deprived of Ca2+
. B,
The nonselective high-voltage–
activated Ca2+
channel blocker,
Cd2+
(50 μM), reversibly pro-
duced a complete blockade of
the HTSs. HTS = high threshold
spike (arrows).

(mean ± SD of pairwise difference, 7 ± 2 mV. At 5 μM, nife-
dipine completely blocked the HT potentials and produced
low-amplitude (~2–5 mV) irregular membrane potential
oscillations (Fig. 3A). Of note, the fast action potentials at
the beginning of the current pulse were not affected by
nifedipine. The above effects of nifedipine were reversible.
Application of the selective N-type Ca2+
channel blocker,
ω-conotoxin GVIA (1 μM), had little effect on the HT fir-
ing in 2 of 4 tested neurons (data not shown) and decreased
(hyperpolarized) its activation threshold without affecting
the amplitude of potentials in the other 2 neurons (ΔV, −7
and −8 mV, respectively; Fig. 3B). Collectively, these results
implied that the L-type Ca2+
current, and not N- or R-type
currents, mediates the HT action potentials unmasked by
lidocaine in TC neurons.
Na+
Channel–Independent Actions of Lidocaine
Contribute to the Unmasking of High-Threshold
Action Potentials
In addition to voltage-gated Na+
channels, a variety of
other ion channels and receptors have been implicated in
the actions of lidocaine.5,21–24
To better understand whether
such Na+
channel–independent actions contribute to the
unmasking of HT spikes in TC neurons, we investigated
lidocaine’s actions under conditions of robust Na+
channel
blockade in neurons pretreated with TTX (600 nM).
Lidocaine concentration dependently decreased the cur-
rent pulse magnitude required to trigger HT potentials in
neurons (“current threshold”; Fig. 4A). This effect corre-
lated strongly with a concomitant concentration-dependent
increase in neuronal input resistance (Fig. 4B).5
At the same
time, lidocaine did not affect HT potential spikes per se. The
voltage threshold for activation of the HT potential spikes
remained unaffected by 600 μM lidocaine in 3 of 5 neu-
rons (Fig. 4A); in 2 neurons, the threshold decreased from
approximately −19 and −30 to −29 and −37 mV, respectively
(data not shown). We observed no significant shift in the
voltage threshold of HT potentials with higher (1 mM)
and lower (100 and 300 μM) concentrations; as illustrated
in Figure 4A, lidocaine also did not significantly affect HT
potential amplitudes over the concentration range (each
concentration, n = 3−5; data not shown).
At the same time, however, lidocaine blocked the slow
AHPs after cessation of depolarizing current pulse injections,
known to be attributable to deactivation of the hyperpolar-
ization-activated mixed cationic current, Ih
25
(Fig. 5). Under
control conditions (TTX), injection in 15-second intervals of
step current pulses of a successively increased magnitude
(20–40 pA increments) into neurons produced successively
larger slow AHPs that gradually hyperpolarized the resting
potential at the onset of the next depolarizing pulse (Fig. 5, A
and C). This AHP increase was most pronounced (maximum
Figure 3. The L-type Ca2+
current mediates the high-threshold (HT) action potentials unmasked by lidocaine. A, Voltage responses of
a current-clamped neuron (upper traces) to 1-second depolarizing suprathreshold step current pulse injections. In the neuron shown,
100 μM lidocaine unmasked tonic HT action potential firing. Coapplication of the L-type Ca2+
current blocker, nifedipine, concentration-
dependently (1 μM, partially; 5 μM, completely) and reversibly blocked the HT potential trains under control conditions. B, Shown is
the example of another neuron firing HT action potentials unmasked by 600 μM lidocaine. Coapplication of the N-type Ca2+
current
blocker, ω-conotoxin GVIA (1 μM), produced no inhibiting effects on the HT potentials. In contrast, in the neuron shown, ω-conotoxin
GVIA decreased (hyperpolarized) the HT potential activation threshold, without affecting amplitude, by approximately 7 mV (note also
the higher baseline HT potential voltage threshold and current pulse magnitude required to trigger HT potentials [“current threshold”]
compared to 100 μM lidocaine; cf., Fig. 4A and body text).

increase, 10 mV) at depolarized voltages after the activation
of tonic HT potential firing and associated with a successive
decrease in their frequency (Fig. 5, B and D). Application
of lidocaine at 100 μM substantially (Fig. 5, A and B) and
at 600 μM completely (Fig. 5, C and D) inhibited the AHPs
and facilitated the HT firing, such that neurons responded
to increased current amplitudes with higher discharge fre-
quencies in a quasilinear manner (Fig. 5D).
DISCUSSION
In the present in vitro mechanism of action study, we
found that lidocaine, in the clinically CNS-toxic and con-
vulsive concentration range, reversibly unmasked evoked
HT action potentials mediated by the L-type Ca2+
current
in rat TC neurons, while blocking Na+
-dependent excit-
ability as expected. The higher thresholds, slower rates of
rise and fall, and lower amplitudes of the unmasked Ca2+
-
dependent action potentials compared with those driven by
Na+
are explained by the differences between the activation
and inactivation properties of voltage-gated Ca2+
versus
Na+
channels.19,26,27
These findings in TC neurons, implicated in the systemic
CNS actions of LAs,5,16
are in contrast to previous obser-
vations in other tissue preparations that lidocaine blocks
various Ca2+
conductances.28–31
Conversely, our results are
remarkably consistent with those of Mulle et al.,32
who
reported an unmasking of fast prepotentials in TC neurons
by intracellular injection of QX-314 (0.1 M in the pipette), a
relatively impermeable experimental quaternary lidocaine
derivative. The authors concluded that these potentials rep-
resent dendritic Ca2+
spikes. Somewhat similarly, Liu et al.33
found in dorsal root ganglion neurons from newborn rats
that ropivacaine at 10 and 30 μM markedly increases a HVA
Ca2+
current (but decreases it at ≥50 μM).
What possible functional implications do the present
results have, and how do they change our views about LA
neurotoxicity and its mechanisms? The overall physiologic
role of HVA Ca2+
currents in TC neurons, while incom-
pletely defined, includes regulation of tonic repetitive firing
by triggering Ca2+
-induced Ca2+
release from intracellular
stores with subsequent activation of Ca2+
-dependent K+
cur-
rents (IK(Ca))11,17,18,34,35
and generation of high-frequency oscil-
latory activity.36
They often complement the firing on top of
the low-threshold Ca2+
potentials mediated by T-type Ca2+
currents,37
providing a range of burst firing patterns, and
have been implicated in activity-dependent synaptic plas-
ticity.38
In addition to these putative physiologic functions,
however, HVA Ca2+
channels play an important role in
neurotoxicity and cell death in the CNS. The pathogenesis
of neurotoxicity involves several mechanisms that collec-
tively lead to an increase in intracellular Ca2+
concentra-
tions ([Ca2+
]i). HVA Ca2+
channels participate in this process
both directly and indirectly. They directly facilitate Ca2+
flux
intracellularly, which, partially because of their slow inac-
tivation, may produce substantial increases in [Ca2+
]i.11,34
Alternatively, HVA Ca2+
channels, particularly those of the
N type, may increase [Ca2+
]i indirectly by mediating excit-
atory amino acid release12,39
and subsequent excitatory
amino acid–induced excitotoxicity.40
In this regard, the pres-
ent observations that lidocaine also unmasked the HT Ca2+
spikes under conditions of presynaptic transmitter release
blockade by TTX and N-type Ca2+
channel blockade render
direct increases in [Ca2+
]i from extracellular sources more
likely to be relevant for lidocaine’s neurotoxicity than indi-
rect actions such as those mediated by N-type channels.
Although more is known in the literature about the pro-
cesses that govern excitotoxicity in general (cf. above) than
those involved in systemic LA neurotoxicity specifically,
the present findings add to the growing body of evidence
indicating that impaired Ca2+
homeostasis similarly con-
tributes to the complex and multifaceted mechanisms of
the latter. For example, a previous study found that IV lido-
caine increases [Ca2+
]i in the cortex of anesthetized rats.41
In vitro, an increase in [Ca2+
]i in cultured neurons has been
reported to play a critical role in the pathogenesis of lido-
caine toxicity.23,42
However, it is noteworthy that the effec-
tive millimolar concentrations of lidocaine in the latter in
vitro studies were higher than the micromolar concentra-
tions corresponding to systemic CNS-toxic blood plasma
levels in vivo and are, therefore, perhaps more applicable to
spinal and regional anesthesia, where such high perineural
Figure 4. Lidocaine decreased the amplitudes of injected current
pulses required to trigger high-threshold (HT) action potentials.
A, HT potential firing evoked in a neuron under conditions of robust
tonic Na+
channel blockade in a neuron pretreated with tetrodo-
toxin (TTX; left). Lidocaine had no effects on the voltage thresh-
old for activation of HT potentials (arrows) or their amplitudes but
decreased the current pulse magnitude required to trigger the
HT potentials (current threshold; 600 μM: mean, 76.4% of con-
trol [95% confidence interval, 53.1%–99.7%; P = 0.0485; n = 4]).
B, Concentration-dependent effects of lidocaine in neurons pre-
treated with TTX (600 nM) on the reductions in HT potential current
threshold (ΔI) in correlation with concomitant increases in normal-
ized neuronal input resistance (ΔRi). The 2 variables exhibited
negative correlation based on the 3 concentrations, with a Pearson
correlation coefficient of r = −0.9993. Shown are normalized means
± SEM; each concentration, n = 3−5. All effects of lidocaine showed
recovery after washout.

concentrations of lidocaine are normally encountered.43
These considerations notwithstanding, there has been com-
paratively little research on the specific molecular and cellu-
lar mechanisms that govern lidocaine-induced increases in
[Ca2+
]i; both the release of Ca2+
from internal stores and the
influx of extracellular Ca2+
have been implicated.23
Hence,
the present finding of an unmasking of an L-type channel–
mediated HVA Ca2+
current may help bridge the gap in
explaining the mechanistic basis of CNS toxicity produced
by high lidocaine concentrations in vivo.
Clustered at the somatodendritic junction, L-type Ca2+
channels in TC relay neurons are coupled to intracellular
Ca2+
stores through ryanodine receptors and modulate
Na+
-dependent tonic firing by triggering Ca2+
-induced
Ca2+
release (cf. above).35,44,45
Therefore, a lidocaine-induced
unmasking of L-type Ca2+
current-driven firing can lead
to considerable Ca2+
influx and increases in [Ca2+
]i in these
neurons. The roles of L-type Ca2+
channels in the media-
tion of neurodegeneration and cell death, on one hand, and
neuroprotective attenuation by pharmacologic L-type Ca2+
channel blockade, on the other hand, are well known from
studies in other neuronal tissues.13,14,46,47
For LA neurotox-
icity, the observation in the peripheral nervous system by
Bainton and Strichartz48
that nifedipine renders frog sciatic
nerves more resistant to lidocaine-induced degradation
supports this hypothesis. Given the previous findings on
the role of [Ca2+
]i increases in the pathogenesis of LA neuro-
toxicity (mentioned earlier),23,42
the present results are con-
sistent with the literature and, hence, represent a plausible
contributing mechanism for lidocaine’s supraspinal toxic
effects. However, the net level of [Ca2+
]i increases in ventro-
basal TC relay neurons in humans in vivo is unknown and
will also depend on whether lidocaine affects other regula-
tory mechanisms of Ca2+
homeostasis, such as Ca2+
-induced
Ca2+
release, Ca2+
-dependent inactivation, and functioning
of the Na+
–Ca2+
exchanger.35,49–51
For example, Xu et al.22
reported that lidocaine inhibits KCl- and carbachol-evoked
intracellular Ca2+
transients in a concentration range similar
to that in our study (0.1–2.3 mM), albeit in a neuronal cell
culture. Clearly, further research, including studies using
Figure 5. Lidocaine effects on afterhyperpolarizations (AHPs) following step current pulse injection and high-threshold (HT) action potential
current–frequency relationships. A and C, Tonic firing of HT action potentials in current-clamped neurons evoked by 1-second depolarizing current
step pulses injected in 15-second intervals under conditions of Na+
channel blockade with 600 nM tetrodotoxin (TTX; control) and after coap-
plications of 100 and 600 μM lidocaine, respectively. Under control conditions, the incrementally increasing step depolarizations (red traces)
were followed by successively larger slow AHPs (left: arrows), leading to gradual hyperpolarization of the baseline resting potential (in the neuron
shown, −61 and −59 mV, respectively). Lidocaine at 100 μM partially and at 600 μM completely blocked these AHPs (right: asterisks). Under
control conditions, the frequency of HT potential firing evoked by incremental injection of successive step current pulses peaked around 10 Hz;
subsequent injection with pulses of higher magnitude decreased firing frequency; red traces on the left side of (A) and (C). B and D, Number of
HT spikes triggered in the corresponding neurons (A and B, respectively) plotted against the amplitude of injected current pulses.

Ca2+
imaging, will be helpful and required to elucidate the
net effect of lidocaine on Ca2+
homeostasis in the thalamus.
Ventrobasal thalamic neurons functionally express 2
L-type Ca2+
channel isoforms that are distinguished bio-
physically and pharmacologically, Cav1.2 and Cav1.3.51–53
Cav1.3 channels, which are less sensitive to dihydropyri-
dines, are blocked by 5 to 10 μM nifedipine, whereas Cav1.2
channels are already significantly (90%) inhibited by nife-
dipine concentrations as low as 1 μM.52
Therefore, our obser-
vations imply that Cav1.3, rather than Cav1.2, likely drives
the observed HT action potentials. Furthermore, the more
hyperpolarized (~20–25 mV) activation range of Cav1.3
channels compared with Cav1.2 would render them more
suitable for mediating the tonic HT firing. The biophysi-
cal properties of a fast rate of inactivation, hyperpolarized
inactivation range, and depolarized activation threshold
collectively appear to be the key factors preventing other
HVA Ca2+
currents expressed in TC neurons from mediat-
ing the HT firing. In this regard, our findings also indicate
that an R-type Ca2+
current, contrary to what might have
been expected based on older studies12,18
(cf. Results, fourth
parapgraph) does not significantly contribute to the HT
potentials unmasked by lidocaine. This in turn is consistent
with reports that the R-type channel, Cav2.3, is not densely
expressed in TC neurons.54,55
In this study, TTX also was able to unmask tonic HT
firing while completely inhibiting Na+
-dependent action
potentials. However, the actions of lidocaine, which unlike
TTX readily crosses the blood–brain barrier,3
differed from
those of TTX in a number of ways because its unmasking
effect was more pronounced than TTX. First, the threshold
amplitudes of injected current evoking Ca2+
-dependent
action potentials were lower in lidocaine-treated neurons.
It is noteworthy in this context that the HT potentials
recorded in the presence of TTX had voltage thresholds
consistent with those previously observed in ventrobasal
thalamus by Ries and Puil (circa ≥−30 mV)56
but higher than
those reported in rat auditory thalamus (ventral partition
of the medial geniculate body; approximately ≥−40 mV)
by Tennigkeit et al.37
Although these discrepancies remain
unclear because neither of these 2 studies isolated the spe-
cific underlying HVA Ca2+
currents, it is known that Ca2+
channels, and specifically those of the L type, are subject to
differential distribution and expression in different thalamic
neurons.45
Second, as opposed to TTX, lidocaine-treated
neurons, because of the concentration-dependent inhibi-
tion of the slow AHPs, were able to respond to increased
current amplitudes by increased HT firing frequency. These
effects of lidocaine were likely because of its robust inhibi-
tion of Ih in these neurons, which we have previously shown
to result in an increase in input resistance and AHP inhibi-
tion.5
Third, the activation threshold of the HT potentials,
although not in all neurons, was higher in the presence of
TTX. A direct action of lidocaine on the L-type Ca2+
current
producing a hyperpolarization of the activation threshold
may account for this effect. In addition, we have previously
demonstrated that inhibition of Ih and a K+
conductance
(other than the inward rectifier, IKir) by lidocaine in the high
micromolar range produces depolarization of TC neurons
operating in the tonic firing mode.5
The latter is consistent
with recent evidence that LAs block various members of the
2-pore domain (“tandem”) K+
channel family that underlies
“leak” K+
currents (IKL) in a range of other tissues/prepara-
tions.57–59
Combined with a concomitant increase in input
resistance, a lidocaine-induced K+
conductance blockade
explains the reduction of the amplitudes of injected current
required for triggering Ca2+
-dependent potentials to the lev-
els necessary for evoking Na+
-dependent action potentials
under control conditions. In this regard, our findings sup-
port the 3-decade-old hypothesis of Mulle et al.32
that the
occurrence of dendritic HT spikes in TC neurons in the pres-
ence of QX-314 (cf. above) results from an increase in input
resistance because of inhibition of persistent Na+
and/or K+
conductances. Given the all-or-none nature of Ca2+
-driven
action potentials, we postulate that the above Na+
channel–
independent actions of lidocaine collectively account for
the unmasking of HT action potentials driven by the L-type
Ca2+
current.
Regarding the clinical relevance of the present lidocaine
concentrations, 100 μM lidocaine HCl converts to approxi-
mately 27 μg/mL (i.e., a 0.0027% solution). Lidocaine read-
ily passes the blood–brain barrier; according to the classic
study by Usubiaga et al.,3
CSF concentrations of lidocaine
in humans after IV injection correlate to arterial blood lev-
els with a factor between 0.73 and 0.83. In rabbits receiv-
ing a continuous IV lidocaine infusion, the correlation
factor (with a 10-minute latency between arterial and CSF
sampling) ranges between ~0.47 and 0.64.60
These data col-
lectively imply that a 100 μM CSF concentration would
correspond to arterial levels between ~33 and 57 μg/mL in
vivo, which unequivocally is in the CNS-toxic range.a
By the nature of the experimental methodology, this
study has inherent limitations and shortcomings. First,
because the investigations were conducted in juvenile rat
brain slices in vitro, the question arises as to what extent
the results can be extrapolated to the human patient in
vivo. We recognize that such extrapolations require caution.
For example, because the experiments were performed at
room temperature to facilitate neuronal viability and stable
recording conditions, it is reasonable to assume that effec-
tive concentrations at 37°C in vivo would likely be lower.
Consistent with this, Yokoyama et al.62
found the mean
lidocaine plasma concentrations in adult rats at the onset
of CNS toxicity (using convulsions as an end point) to be
10.8 μg/mL; similar observations were made by Spiegel et
al.63
Although systemic CNS toxicity in humans has been
observed at concentrations 5 μg/mL,64
plasma lidocaine
levels associated with systemic analgesia in humans are
remarkably similar to those effective in rats.65–67
Second, the
present studies focused on TC neurons in the ventrobasal
thalamus; although these neurons have characteristic elec-
trophysiologic properties,5,10,16
it remains to be determined
whether other types of neurons would exhibit similar
responses to lidocaine. Third, we did not study lidocaine
a
In contrast to the aforementioned studies are the results of 1 investigation
that found correlation factors between ~0.06 and 0.08 in humans.61
Although
lidocaine’s well-known property to readily penetrate biological membranes
at physiologic pH values raises questions about the plausibility of these
results, whole brain lidocaine concentrations in the study (1) were signifi-
cantly higher than those in the CSF or blood and (2) exceeded cortical con-
centrations, indicating that lidocaine accumulates in the cerebral tissue with
a preference for subcortical structures, e.g., thalamic nuclei.

concentrations in excess of 1 mM. However, as discussed
earlier, although LAsystemic toxicity is not normally associ-
ated with millimolar concentrations in the CSF, Mulle et al.32
found that intracellular injection of TC neurons with quater-
nary lidocaine at a pipette concentration of 0.1 M unmasked
fast dendritic Ca2+
spikes. Fourth, in part because of the
well-known difficulties in attaining adequate space clamp
of dendritically expressed currents in neurons with a large
dendritic tree, we report no data from experiments where
HVA Ca2+
currents of TC neurons in the slices were voltage
clamped. Finally, it must be emphasized that it was not the
goal of this electrophysiologic in vitro study in brain slices
to examine histologic or behavioral end points of LA CNS
toxicity or investigate lidocaine’s effects on [Ca2+
]i; clearly, a
body of such future work will be required to elucidate the
myriad of concentration-dependent effects that LAs exert
on the brain. However, despite unavoidable shortcom-
ings, experiments with slices provide a unique opportunity
to study neurons in a relatively intact physiologic tissue
environment.
In summary, at high-micromolar and clinically CNS-
toxic concentrations, lidocaine unmasked in rat TC neurons
evoked HT action potentials mediated by the L-type Ca2+
current while substantially suppressing Na+
-dependent
excitability. The present findings add to our understanding
of the complex myriad of LAactions. On the basis of the well-
known role of an increase in [Ca2+
]i in the pathogenesis of LA
neurotoxicity, this novel action in the thalamus, implicated as
a central supraspinal site for LAs to produce their systemic
CNS effects, represents a plausible contributing candidate
mechanism for lidocaine’s CNS toxicity in vivo. E
DISCLOSURES
Name: Igor Putrenko, PhD.
Contribution: This author helped conduct the study, collect the
data, analyze the data, and prepare the manuscript.
Attestation: Igor Putrenko has reviewed the original study data
and data analysis, attests to their integrity, and approved the
final manuscript.
Name: Amer A. Ghavanini, MD, PhD, FRCPC.
Contribution: This author helped conduct the study, collect the
data, analyze the data, and prepare the manuscript.
Attestation: Amer A. Ghavanini approved the final manuscript.
Name: Katrin S. Meyer Schöniger, MD.
Contribution: This author helped conduct the study and collect
the data.
Attestation: Katrin S. Meyer Schöniger approved the final
manuscript.
Name: Stephan K. W. Schwarz, MD, PhD, FRCPC.
Contribution: This author developed the study rationale and
design; helped conduct the study, collect the data, analyze the
data, and prepare the manuscript; and is the archival author.
Attestation: Stephan K. W. Schwarz has reviewed the origi-
nal study data and data analysis, attests to their integrity, and
approved the final manuscript.
This manuscript was handled by: Markus W. Hollmann, MD,
PhD, DEAA.
ACKNOWLEDGMENTS
The authors thank Ernest Puil, PhD, for valuable discussions
and insightful comments.
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