Low Dose Isoflurane Exerts Opposing Effects on
Neuronal Network Excitability in Neocortex and
Klaus Becker1,2*., Matthias Eder3., Andreas Ranft4, Ludwig von Meyer5, Walter Zieglga ¨nsberger3,
Eberhard Kochs4, Hans-Ulrich Dodt1,2
1Department of Bioelectronics, FKE, Vienna University of Technology, Vienna, Austria, 2Center for Brain Research, Medical University of Vienna, Vienna, Austria, 3Max
Planck Institute of Psychiatry, Munich, Germany, 4Department of Anaesthesiology, Klinikum rechts der Isar, Munich, Germany, 5Institute for Forensic Medicine, Ludwig-
Maximilian University of Munich, Munich, Germany
The anesthetic excitement phase occurring during induction of anesthesia with volatile anesthetics is a well-known
phenomenon in clinical practice. However, the physiological mechanisms underlying anesthetic-induced excitation are still
unclear. Here we provide evidence from in vitro experiments performed on rat brain slices that the general anesthetic
isoflurane at a concentration of about 0.1 mM can enhance neuronal network excitability in the hippocampus, while
simultaneously reducing it in the neocortex. In contrast, isoflurane tissue concentrations above 0.3 mM expectedly caused
a pronounced reduction in both brain regions. Neuronal network excitability was assessed by combining simultaneous
multisite stimulation via a multielectrode array with recording intrinsic optical signals as a measure of neuronal population
Citation: Becker K, Eder M, Ranft A, von Meyer L, Zieglga ¨nsberger W, et al. (2012) Low Dose Isoflurane Exerts Opposing Effects on Neuronal Network Excitability
in Neocortex and Hippocampus. PLoS ONE 7(6): e39346. doi:10.1371/journal.pone.0039346
Editor: Christopher Mark Norris, Univ. Kentucky, United States of America
Received December 21, 2011; Accepted May 22, 2012; Published June 18, 2012
Copyright: ? 2012 Becker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Anesthetics can cause transient arousal and excitation especially
during inhalation induction of anesthesia. The excitement phase
has first been described as stage II anesthesia for diethyl ether 
but also occurs with modern volatile anesthetics, including
isoflurane [2–5]. This phenomenon is in contradiction with the
notion that neuronal excitability is dampened during general
anesthesia. In fact, one should expect an increased excitability in
distinct parts of the central nervous system (CNS). This
assumption is supported by the observation that low isoflurane
concentrations can accentuate electroencephalographic signs of
Motor excitation of rats during induction of anesthesia with
isoflurane is minimized after inactivation of the hippocampal CA1
region . This indicates that, beside the neocortex, also the
hippocampus is involved in anesthetic-induced excitation. How-
ever, the contribution of the individual subregions to this
phenomenon is still largely unknown. To address this question,
in vitro studies seem to be well-suited .
In the present study, we performed measurements of the
intrinsic optical signal (IOS) as an indicator of neuronal network
excitability (NNE). In brain slices, electrical stimulation induces
minimal changes in light scattering and optical transparency.
These changes are well-correlated with the spatial extent of
neuronal excitation and depend on action potentials [9–11].
The tiny variations in optical properties of spiking neurons can
best be detected by infrared microscopy and visualized by
computer-based image processing . For an optimal compar-
ison of different brain regions with regard to their susceptibility
to isoflurane, they should be monitored at the same time in
a single brain slice. In order to accomplish this, we used
a multielectrode array (MEA) for selective and simultaneous
stimulation of multiple anatomical targets. Since the hippocam-
pus seems important in mediating the arousal occurring during
stage II anesthesia , we chose the CA1 output subfield as one
stimulation site. Due to the fact that neocortical layer 5
pyramidal neurons significantly contribute to the EEG , we
further stimulated neocortical layer 5.
Materials and Methods
Parasagittal brain slices (300 mm thick) were prepared from 22
male Sprague-Dawley rats (postnatal days 14–21) as described by
Eder et al. . All steps of the preparation were carried out in
ice-cold artificial cerebrospinal fluid (ACSF) oxygenated with
carbogen (95% O2/5% CO2). The ACSF consisted of (in mM):
NaCl, 125; KCl, 2.5; NaH2PO4, 1.25; CaCl2, 2; MgCl2, 1;
NaHCO3, 25; glucose, 25; pH 7.4. The slices were incubated in
glass vials containing oxygenated ACSF for 60 min at 34uC and
subsequently transferred to the recording chamber. The temper-
ature of the recording chamber was maintained at 34uC by
a heating-system (Multi-Channel Systems, Reutlingen, Germany)
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during all experiments. Animal care and euthanasia was done in
accordance with the local animal care comities and with the
German animal protection law. According to the German animal
experiments act, special approval by an ethics committee or an
approval number was not required, since all experiments were
performed on isolated organs and not on living animals.
Slices were visualized by an upright microscope (Axioskop 2 FS,
Zeiss, Go ¨ttingen, Germany) with a 2.5x Neofluar objective (N.A.
0.075). To avoid disturbing reflections at the water-air interface,
a dipping cone with a transparent cover slip was mounted in front
of the objective. All images were obtained in the near infrared
spectrum by filtering the light of a 100 W halogen lamp with an
optical band-pass filter (lmax=780650 nm). To block erroneous
stray-light emitted from the room illumination, a second light filter
with an identical cutoff frequency was put into the light pathway
behind the objective (Figure 1).
For electrical stimulation, a rectangular 1066 MEA (Multi-
Channel Systems, Reutlingen, Germany) was used (200 mm
electrode spacing, 30 mm electrode diameters). The stimulation
pulses were generated by a Digitimer 2533 isolated stimulus
generator (Digitimer Limited, Herts, UK), which was triggered by
a programmable impulse generator board (DAQ PCI 6601,
National Instruments, Munich, Germany). The stimulus intensities
were varied between 3 and 10 V, and could separately be adjusted
for each electrode of the MEA by a control unit equipped with 60
potentiometers. For each electrode of the MEA, the stimulus
intensity was adjusted in a way that IOSs of comparable spatial
extent and intensity were obtained. In all experiments, the
stimulus frequency was 50 Hz and the pulse width was 200 ms.
The length of the applied pulse trains was always 2 s. Electrical
stimuli were simultaneously applied to neocortical layer 5 and area
CA1 of the hippocampus by activating corresponding electrodes of
the MEA. Due to a sufficient transparency of the brain slices,
individual electrodes of the MEA could easily be localized by
The imaging system (Figure 1) consisted of a scientific-grade
12-bit CCD-camera (CoolSnap cf, Roper Scientific, Tucson, USA)
connected to a computer. Before starting a recording sequence, 10
background images were taken and averaged. Afterwards, the
electrical stimulation was started and 10 further images were
recorded in intervals of 2 s. The first image was always taken at the
onset of stimulation. From each image, the averaged pre-stimulus
image was subtracted. To reduce noise and to enhance the
visibility of the IOS, the images were low pass filtered by a 565
Gaussian filter and subsequently converted into 8-bit pseudo color
images. Subsequently, they were computationally overlaid on
a black and white image of the brain slice taken before the onset of
For data analysis, the pixel intensities of the IOS images (12-bit
resolution) obtained 4 s after onset of stimulation were integrated
within a rectangular bounding box around the respective
stimulation electrode, leading to a numerical quantifier for each
Figure 1. Schematic diagram of the setup used for IOS recordings. The brain slice is superfused with isoflurane-enriched ACSF and
electrically stimulated by a MEA. The intensity of the electrical stimuli is separately adjustable for each electrode by a control unit, equipped with 60
potentiometers. The IOS is recorded via an infrared sensitive CCD camera, computationally processed, and subsequently overlaid with a black and
white image of the brain slice, thus allowing a correlation with anatomical structures.
Effects of Isoflurane on Neural Excitability
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evoked IOS. All IOS intensities were normalized to the mean IOS
intensity obtained during minutes 0 to 20 before onset of drug
application. To improve the signal-to-noise ratio, intensity values
below a 5% threshold were discarded.
Application of Isoflurane
Isoflurane (Forene, Abott, Wiesbaden, Germany) was dissolved
in the ACSF as follows: carbogen gas was passed through a Forene-
calibrated vapor (Vapor 19.3, Dra ¨gerwerk AG, Lu ¨beck, Germany)
at a constant flow rate of 500 ml/min and afterwards bubbled
through the ACSF reservoir. Three different dial settings were
chosen to achieve three different concentration levels. Before
delivering the isoflurane-containing ACSF to the recording
chamber via Teflon tubing, we allowed an equilibration time of
half an hour.
Determination of Isoflurane Concentrations
Isoflurane bath concentrations were determined with a Perkin
Elmer 8420 headspace gas chromatograph. The separation was
performed on a Megabore capillary column (Rtx-1701; 60 m,
0.53 mm ID, 3.0 mm) with helium as carrier gas at 5 ml/min. By
means of a switch valve system, a positive pressure was generated
in the vial, which drove the volatiles onto the capillary column that
had been preset to 40uC. The oven then was heated by 10uC/min
to 135uC. Volatiles were detected by a flame ionization detector
and peaks of the compounds were recorded.
Tertiary butanol in water served as internal standard (80 mg/
l). Headspace vials (volume: 20 ml) were filled with sodium
sulfate anhydrous (500 mg), water (500 ml), internal standard
solution (100 ml) and aqueous samples (250 ml), and were
immediately sealed with Teflon caps. The sample was equili-
brated for 40 min at 60uC. A headspace sample was applied to
the capillary column as mentioned above. From the ratios of
the peaks of tertiary butanol (retention time 7 min 56 s) and
isoflurane (retention time 7 min 17 s), isoflurane concentrations
were derived by a calibration curve. To generate the calibration
curve, known amounts of isoflurane (2 or 5 ml, respectively)
were dissolved in methanol (5 ml) in a narrow-necked volumet-
ric flask and ACSF was added to a total volume of 100 ml.
Together with sodium sulfate anhydrous, water, and internal
standard, these calibration solutions were analyzed as described
above. For each of the three vapor settings A, B, and C,
aqueous samples were withdrawn from the recording chamber
at 5 min, 15 min, and 60 min after onset of delivery of
isoflurane containing ACSF. The determined bath concentra-
tions corresponding to the three vapor settings A, B, and C were
0.08 mM (A), 0.17 mM (B), and 0.33 mM (C) after 5 min, and
0.12 mM (A), 0.24 mM (B), and 0.49 mM (C) after 15 and
60 min, respectively. We derived the following exponential
equations describing the isoflurane bath concentrations in mM
at vapor settings A, B, and C from these values (Figure 2A).
Modelling of Isoflurane Tissue Diffusion
Diffusion processes are generally described by Fick’s 2ndlaw:
(D: diffusion constant. C(x,t): concentration at location x and
It was assumed that an exchange with the bath medium only
takes place at the upper slice surface being exposed to the bath
solution. Under this assumption solving eq. (2) with the boundary
conditions C(0,t)=cB(t) and C(‘,t)=0 yields to
(t: time after onset of isoflurane administration. c(t): isoflurane bath
concentration. x: penetration depth within the slice).
We determined numerical solutions for (3) with respect to the
three functions cB12(t), cB24(t), and cB49(t) describing the measured
isoflurane bath concentrations at vapor settings A, B, and C (eq. 1).
D=1.6610210m2/s. By integration over the whole slice thickness
(300 mm) the average tissue concentrations were modelled as
depicted in Figure 2A.
All values are expressed as the mean 6 standard error of the
mean (SEM). Only a single brain slice was used from each animal,
and only a single experiment was carried out with each slice. All
statistical evaluations were performed with the single-sample
Image processing, stimulus generation, application of drugs, and
calculation of average signal intensities was done by custom-made
software written in the Igor Pro programming language (Wave-
Metrics, Oregon, USA). Statistical calculations were done with
SigmaPlot (Systat software, Germany). Modelling of isoflurane
tissue diffusion was performed via Maple (Maplesoft, Canada).
We investigated concentration-dependent actions of isoflurane
in neocortical layer 5 and in the CA1 region of the hippocampus
by quantifying the intensity of the IOS after electrical stimulation.
To this end, we simultaneously applied 2 s long electrical pulse
trains to the two brain regions via a MEA. Starting from the onset
of stimulation, we recorded temporal sequences of ten IOS images
with a time interval of 2 s. These measurements were repeated in
5 min intervals over a time span of totally 115 min. The intensity
of the evoked IOSs was quantified by a computer program. To test
the stability of control recordings, we performed six IOS
measurements (min 0–25) before onset of drug application in
each brain slice. As demonstrated in previous studies, the IOS
remains constant for up to several hours, if a stable baseline once
has been achieved and the stimulation frequency is moderate .
We confirmed this for our experimental conditions by control
experiments without any drug application (Figure 3, BL).
Figure 4A left depicts two typical IOSs in neocortex and
hippocampus. As a further control we applied 1 mM of the sodium
channel blocker tetrodotoxin (TTX). Expectedly, the IOSs were
Effects of Isoflurane on Neural Excitability
PLoS ONE | www.plosone.org3 June 2012 | Volume 7 | Issue 6 | e39346
fully abolished, confirming that the measured signal depends on
neuronal action potential firing (Figure 4A right).
Starting from min 25 of the experiments, we applied isoflurane
for a time span of 60 min. Afterwards, the slices were superfused
with isoflurane-free ACSF for further 35 min. We totally
performed 22 experiments using three different vapor settings
and, thus, three different isoflurane concentrations (vapor setting A
(lowest): n=7 brain slices; vapor setting B: n=6 brain slices, vapor
setting C (highest): n=9 brain slices).
Figure 4B and C depict IOSs in the absence (control) and
presence of 0.1 or 0.44 mM isoflurane. For quantitative analysis,
we determined average IOS intensities within rectangular regions
of interest (ROIs). The ROIs were set due to visual inspection so
that they best-possibly covered the CA1 region of the hippocam-
pus or neocortical layer 5, respectively, and least-possibly extended
into neighbored structures. From Figure 4B an increase in the
IOS intensity caused by low dose isoflurane (0.1 mM) is clearly
detectable in the hippocampus, but not in the neocortex. Figure 3
comprises the data from 22 experiments. In neocortical layer 5,
a monotonous, concentration-dependent decrease in NNE as
measured by the IOS intensity was observed for vapor settings A,
B, and C (single-sample t-test with p,0.05).
We found a different isoflurane action on NNE in the
hippocampus. Using the lowest vapor setting (A), a significant
increase in the IOS intensity occurred after 10 min isoflurane
application. Higher isoflurane concentrations applied using vapor
settings B and C, however, decreased the IOS by a similar amount
as in the neocortex (Figure 3). An initial transient increase in
NNE, which was followed by a decrease, was observed in most
experiments performed with the intermediate vapor setting B.
Using gas chromatography, we determined the isoflurane bath
concentrations corresponding to the different vapor settings (see
Figure 2. Relation between isoflurane concentration and NNE as measured by IOSs. (A) Isoflurane bath concentrations during the
experiments measured by gas chromatography (closed circles, solid lines) and isoflurane tissue concentrations modeled via Fick’s diffusion law
(dotted lines) for the three different vapor settings (A, B, and C) used. Isoflurane has to enter the brain slice submerged in the recording chamber via
diffusion, causing a delayed equilibration of bath and tissue concentrations. (B) Dose-response curves calculated using the isoflurane tissue
concentrations from (A) and the measured IOS intensities depicted in Figure 3. 0.1 mM isoflurane increases the IOS in the CA1 region of the
hippocampus, while it decreases it in neocortical layer 5 (L5) at the same time. Isoflurane concentrations .0.3 mM considerably reduce the IOS in
CA1 as well as in the neocortex.
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Methods). From the results, equations describing the rise of the
isoflurane bath concentrations with time after vapor activation
were derived (Figure 2A). Isoflurane solved in the bath solution
enters a submerged brain slice passively via diffusion mainly at the
upper surface, hence causing a delayed equilibration of isoflurane
concentrations in the neuronal tissue. Since it is not practical to
measure isoflurane concentrations directly in the brain slice, we
modeled the expected time courses of isoflurane concentrations in
the brain tissue utilizing Fick’s diffusion law (Figure 2A) (see
Using the determined isoflurane tissue concentrations, we were
able to derive dose-response curves for the measured isoflurane
effects on NNE. Figure 2B shows that a concentration of 0.1 mM
isoflurane significantly enhances NNE in the CA1 region of the
hippocampus, while it simultaneously decreases it in cortical layer
5 (p,0.05, single-sample t-test). At concentrations .0.3 mM,
a significant decrease in NNE was found for both brain regions
General anesthetics are able to produce clinical excitation. This
still enigmatic and potentially harmful anesthetic state, which can
precede and/or follow deep surgical anesthesia is marked by
a coincidence of behavioral excitation and unconsciousness [1,14].
These symptoms give reason to speculate that general anesthetics,
at slightly narcotic concentrations, may increase activity in some
part(s) of the CNS, while simultaneously decreasing it in other
CNS structures. We provide evidence for this hypothesis by
demonstrating in vitro that the volatile general anesthetic isoflurane
can concurrently increase and decrease NNE in the hippocampal
CA1 region and neocortical layer 5, respectively. Such a mecha-
nism may help to understand the symptoms of behavioral arousal,
although consciousness and the ability for targeted actions are
Previous studies clearly show that IOSs represent a valid
measure of NNE. In particular, the generation of IOSs depends on
excitatory synaptic transmission as well as spiking of neurons
[10,15]. Consistently, the GABAA receptor agonist muscimol
decreases IOSs, while the GABAAantagonist bicuculline markedly
enhances them . In addition, a high correlation between
spatial distributions of field potentials and the spatial distribution
of IOSs was proven by different groups [11,16,17].
We consciously chose the IOS technique, as we attempted to
study isoflurane effects at the neuronal network level. Neither field
potential recordings, nor patch-clamp experiments are suited to
perform that way, since they only provide a local measure of the
spiking activity of a very limited population of neurons. It has
repeatedly been shown previously that isoflurane markedly
decreases field excitatory postsynaptic potential and population
spike amplitudes in area CA1 in rodent brain slices [18,19]. The
strength of these effects, as well as their kinetics, is similar to those
we observed in our IOS recordings. MacIver and Roth  also
reported an enhancement of neuronal activity in the CA1 region
with low-dose isoflurane using classical electrophysiological
It has been confirmed in multiple studies that volatile
anesthetics increase GABAergic inhibition [20,21] and decrease
glutamatergic synaptic transmission [22,23]. Both mechanisms
presumably contribute in parallel to the reduction in brain activity
associated with deep anesthesia [8,14,23–25]. This corresponds
well to the results of our experiments performed with high
isoflurane doses. However, the enhancement of the IOS by low
dose isoflurane cannot be explained by these mechanisms and
needs further investigation to become unraveled.
We have chosen isoflurane for our experiments as test substance
because it is a commonplace general anesthetic known to induce
an excitement phase also in rats . As a reliable measure of NNE
in brain slices, we used the intensity of the IOSs that were evoked
by electrical stimulation of neocortical layer 5 and hippocampal
Figure 3. Summarized results from 22 experiments performed with three different isoflurane vapor settings. A (low dose, n=7 brain
slices), B (medium dose, n=6 brain slices), and C (high dose, n=9 brain slices). BL, baseline obtained without drug application (n=3 brain slices). Start
and end of isoflurane application are marked by the bar.
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Figure 4. Recording of the IOS in hippocampal area CA1 and neocortical layer 5 (L5) in a rat brain slice. (A) Control experiment without
isoflurane application (left). IOSs in the same brain slice after application of TTX (right). (B) IOSs in the absence and presence of 0.1 mM isoflurane. An
amplification is clearly detectable in the hippocampus, but not in the neocortex. (C) Isoflurane (0.44 mM) markedly reduced the IOS in the
hippocampus as well as the neocortex.
Effects of Isoflurane on Neural Excitability
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area CA1 [9–11,26]. These brain regions represent important
output structures of the neocortex and hippocampus.
To our knowledge, only one study exists in which sub-narcotic
doses of a volatile anesthetic have been found to increase neuronal
excitability in vitro . In this work, a transient augmentation of
population spike amplitudes, followed by suppression with in-
creasing doses of isoflurane, was found in the CA1 region of the
hippocampus. The dose applied for augmentation was 0.5 VOL%
(0.13 mM), which is almost identical to the lowest bath concen-
tration applied in our experiments.
Conceived and designed the experiments: KB ME AR. Performed the
experiments: KB ME AR LM. Analyzed the data: KB ME AR.
Contributed reagents/materials/analysis tools: WG EK HUD. Wrote the
paper: KB ME AR.
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