Download propofol alters vesicular transport in rat cortical neuronal cultures

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2011, 62, 1, 119-124
www.jpp.krakow.pl
D. TURINA1,3, K. BJORNSTROM1,3, T. SUNDQVIST2, C. EINTREI1,3
PROPOFOL ALTERS VESICULAR TRANSPORT IN RAT CORTICAL NEURONAL CULTURES
1
Department of Medical and Health Sciences, Division of Anaesthesiology, Linkoping University, Linkoping, Sweden; 2Department
of Clinical and Experimental Medicine, Division of Medical Microbiology, Linkoping University, Linkoping, Sweden; 3Faculty
of Health Sciences, Linkoping University, and the Anaesthetic Department, Linkoping University Hospital, Linkoping, Sweden
Neuronal intracellular transport is performed by motor proteins, which deliver vesicles, organelles and proteins along
cytoskeletal tracks inside the neuron. We have previously shown that the anesthetic propofol causes dose- and timedependent, reversible retraction of neuronal neurites. We hypothesize that propofol alters the vesicular transport of cortical
neurons due to this neurite retraction. Primary cultures of co-cultivated rat cortical neurons and glial cells were exposed to
either 2 µM propofol, control medium or the lipid vehicle, in time-response experiments. Reversibility was tested by washing
propofol off the cells. The role of the GABAA receptor (GABAAR) was assessed with the GABAAR antagonist gabazine.
Vesicles were tracked using differential interference contrast video microscopy. Propofol caused a retrograde movement in
83.4±5.2% (mean±S.E.M.) of vesicles, which accelerated over the observed time course (0.025±0.012 µm.s-1). In control
medium, vesicles moved predominantly anterograde (84.6±11.1%) with lower velocity (0.011±0.004 µm.s-1). Cells exposed
to the lipid vehicle showed the same dynamic characteristics as cells in control medium. The propofol-induced effect on
vesicle transport was reversible and blocked by the GABAAR antagonist gabazine in low concentration. Our results show
that propofol causes a reversible, accelerating vesicle movement toward the neuronal cell body that is mediated via synaptic
GABAAR. We have previously reported that propofol initiates neurite retraction, and we propose that propofol causes vesicle
movement by retrograde flow of cytoplasm from the narrowed neurite.
K e y w o r d s : anesthetics intravenously, brain, cellular mechanisms, cerebral cortex, neurotransmission effects, pharmacology,
propofol, theories of anesthetic action, vesicular transport
INTRODUCTION
Neurons are characterized by a complex transport mechanism
that provides the cell periphery with organelles and
macromolecular complexes and also return break-down products
to the cell body. This transport is essential for communication,
growth and survival of axons and dendrites (collectively referred
to as neurites). Neuronal transport is driven by motor proteins,
which move vesicles, organelles and macromolecular complexes
bi-directionally along the neuron (1). Microtubules are
cytoskeletal elements that the motor proteins use like trails for
long-distance delivery of cellular cargos and actin filaments are
used as trails for the local movement and positioning of organelles
and vesicles (2). The motor protein kinesin drives transport in the
anterograde direction (3, 4), whereas the motor protein dynein
drives mainly retrograde transport (5, 6). Membranous organelles
are the carrier structures for fast axonal transport at average
velocities of 50-400 mm day-1 (~0.5-5 µm s-1) and, in contrast, the
slow components of axonal transport consist of the movement of
cytoskeletal and cytosolic proteins at average velocities of 0.3-8
mm day-1 (~0.004-0.09 µm s-1) (7).
Synaptic cargo trafficking is essential for synapse formation,
function and plasticity (8). General anesthetics preferentially
affect synaptic transmission rather than axonal conduction (9) by
agent-specific postsynaptic and/or presynaptic mechanisms (10-
12). The molecular mechanisms by which general anesthetics
produce their effects are still being elucidated (13). Intracellular
transport is essential for proper neuronal function and
communication between neurons, and interaction of anesthetics
with vesicle transport could be part of their cellular actions. We
have previously demonstrated that propofol (a general anesthetic)
causes a dose- and time-dependent retraction of cultured cortical
neuronal neurites and this retraction is GABAAR mediated,
reversible, and dependent on actin and myosin II. Retracted
neurites leave a retraction bulb and a thin trailing remnant on the
retracted path (14). The effects of propofol on neuronal vesicle
transport have not been studied yet and to further elucidate the
molecular mechanisms of anesthesia, the aim of this study was to
investigate if the neural retraction caused by propofol alters
vesicular transport and if the thin trailing remnant prevents
vesicle transport to the neurite periphery.
MATERIALS AND METHODS
Reagents
Reagents were purchased from the following sources:
propofol in the commercially available solution Lipuro® (10
mg·mL-1) and its lipid vehicle as the commercially available
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solution Vasolipid (20 mg·mL-1), (Braun, Melsungen, Germany);
cell culture equipment (Sarstedt, Numbrecht, Germany);
penicillin, streptomycin, glutamine and foetal calf serum (FCS)
(Invitrogen, Paisley, UK); gabazine, basic chemicals and cell
culture media (Sigma Chemical Co., St. Louis MO, USA).
Cell culture
The study was approved by the Ethics Committee for
Animal Research at Linkoping University. Primary cultures of
rat neurons were obtained essentially as described by Hansson
& Rönnbäck (15). In short, newborn Sprauge-Dawley rats
(Scanbur A/S, Stockholm, Sweden or Taconic Europe, Lilla
Skensved, Denmark) were decapitated and the cortex
dissected. The cortex tissue was sieved through a nylon mesh
(80 µm) into Dulbecco's modified Eagle's medium (high
glucose, 4500 mg·L-1) supplemented with glucose (30 mM),
insulin (5 µg·mL-1), glutamine (2 mM), penicillin (50 U·mL-1),
streptomycin (50 µg·mL-1), and 20% FCS. The cells were
cultured on poly-L-lysine-coated 25 mm φ sterile cover-slips in
Petri dishes at 37°C in a humidified atmosphere of 95% air and
5% CO2. After 24 hours, the FCS concentration of the medium
was changed to 10% and maintained at that level for 6 days. 10
µM cytosine-1-β-D arabinofuranoside was added to fresh
medium containing 10% FCS on the sixth day for 24 hours, to
suppress glial cell growth. Thereafter, the cultures received
new medium containing 5% FCS every second day. The cells
were used during day 10-30, when the cultures comprised 7580% neurons (15).
Video microscopy
Before use, each cover slip was rinsed twice in a buffer- Ca2+containing medium (CCM)- composed of (mM) CaCl2 (1.1),
NaCl (136), KCl (4.7), KH2PO4 (1.2), MgSO4 (1.2), CaCl2 (1.1),
NaHCO3 (5.0), Hepes (20), EGTA (0.1), glucose (5.5); pH
adjusted to 7.40 with NaOH (16). Cover slips with cell cultures
were mounted in a closed bath imaging chamber and observed by
light microscopy using a Zeiss Axiovert 135M (Gottingen,
Germany) equipped with a 40x 1.3 numerical aperture (N.A.) oil
immersion objective (Carl Zeiss, Microimaging GmbH,
Gottingen, Germany). To obtain 37°C in the buffer covering the
cells, they were placed in a heated stage. Differential interference
contrast images of cells were captured and transformed into video
images using a ProgRes C10plus CCD camera (Jenoptik, Jena,
Germany) and a video image processor (Hammamatsu Photonics,
Tokyo, Japan). Video images were displayed on a video monitor
and stored on tape using a video recorder. We analyzed only
superficial cells that grew on a glial cell layer. Final processing of
all images was done using Adobe PhotoShop 6.0 (Adobe
Systems, San Jose, CA, USA) and ImageJ (Rasband, W.S.,
ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA,
http://rsb.info.nih.gov/ij/, 1997-2005).
Tracking and data quantification
Time-lapse series were captured from video images and the
interval between measurements was 1 min. The velocity of
individual vesicles was determined using the Manual Tracking
plug-in for ImageJ software. Vesicle movement was analyzed by
tracking of clearly visible vesicles (not clustered) in cultured
cortical neurone neurites before and during pharmacological
manipulation. The distance between two consecutive frames or
between the initial and the final tracking points was used to
determine speed and average speed. The mean velocity for each
vesicle is calculated and then the mean velocity for all tracked
vesicles in one cell was used for further analysis. Anterograde
vesicle movement in neurites was defined as vesicles moving
toward the cell periphery, and retrograde movement as vesicles
moving toward the cell body. Bi-directional vesicle movement
was defined as equal number of movements in both directions.
For each cell, the percentage of vesicles in each direction
(anterograde, retrograde or bidirectional) was calculated. Cells
were exposed to propofol, CCM or the lipid vehicle. The
dynamics of vesicle movement were observed each min, starting
with 5 min in CCM to determine normal vesicle movement.
Thereafter either propofol (2 µM (propofol-group)), or CCM
buffer (control group) was added, and the vesicles were followed
for 10 min. Propofol was added 10 sec before time zero when
images were captured. To observe reversibility, the medium
containing propofol was removed after a 2 min exposure and
replenished by CCM and the cells were observed for further 18
min. In the GABAAR blocking experiments, the cell culture was
obtained from rats bred by Taconic Europe. The vesicles were
studied after first 5 min in CCM, followed by gabazine (7 µM)
for 5 min prior to a 10 min exposure of propofol (2 µM),
(gabazine-propofol-group). The propofol group (P2) was treated
as described above, and control cells were exposed to gabazine
(7 µM) for a total of 15 min (gabazine-group).
Statistical analysis
All measurements obtained from a single neuron is defined
as an experiment (n=1) and the neurons were obtained from six
different rat litters. Overall significant differences between
conditions were determined by analysis of variance for repeated
measurements (ANOVA). Our decision to use ANOVA is due to
multiple comparisons of time and treatment to avoid false
significance. Post hoc analyses were performed using the
Bonferroni's post hoc test for multiple comparisons. A p value of
<0.05 was considered statistically significant. The values were
expressed as the mean ± standard error of the mean (S.E.M.),
since the measures are mean of means, then the calculated S.D.
is actually the same as S.E.M. All statistical analyses and
graphing were done using Prism 4.0 software (GraphPad
Software, San Diego, USA).
RESULTS
Propofol causes retrograde vesicle movement
A comparison of vesicle movement characteristics in
propofol and control group (CCM) showed significant
differences for percentage of anterograde and retrograde moving
vesicles and average velocity. In cells only exposed to CCMbuffer, vesicles moved predominately (84.6±11.1% (mean ±S.E.
M.), n=5, 46 vesicles) outward to the cell periphery
(anterograde) for the studied time period, as compared to
propofol-treated cells where 5.5±2.8% (n=8, 74 vesicles) moved
anterograde (p<0.0001). After addition of 2 µM propofol,
83.4±5.2% (n=8, 74 vesicles) of vesicles changed direction and
moved inwardly to the cell body (retrograde), vs. 11.2±8.3%
vesicles in cells only exposed to CCM-buffer, (n=5, 46 vesicles;
p<0.0001, Table 1). The vesicles exposed to propofol continued
to move retrograde for 10 min (Fig. 1A and B). Bi-directional
movement was seen in 11.1±5.6% of vesicles in propofol treated
neurons, vs. 4.2±2.8% (n.s) in untreated cells. Propofol treatment
initially decelerated the anterograde moving vesicles, then
increased the average retrograde velocity of vesicles
(0.025±0.012 µm.s-1, n=8, 74 vesicles). This was significant
higher then in CCM-treated neurons (0.011±0.004 µm.s-1 n=5,
46 vesicles; p<0.01, Table 1)). The highest recorded velocity of
a retrograde vesicle in the propofol group was 0.3 µm·s-1. There
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Table 1. Propofol causes retrograde vesicular transport and increase vesicular velocity in neurons. Velocities and percentage of vesicles
for each direction, in neurons treated with propofol (2 µM) or with calcium containing medium (CCM) (control group). Data based
on n=8 cells, 74 vesicles tracked in the propofol group and n=5 cells, 46 vesicles tracked in the control group. For each cell, the
percentage of vesicles moving in a certain direction was calculated. Values presented as mean ±S.E.M. Statistical analysis was done
with 2-way ANOVA with Bonferroni post hoc correction, with individual p values as shown. n.s=non significant.
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Fig. 1. Propofol induces retrograde vesicle movement and increase vesicular velocity in neurons.
(A) A neuron in Ca2+-containing medium (CCM) is shown 5 minutes (time -5) before exposure to propofol in a differential interference
contrast image, with the cell body to the left and a branched neurite stretching out to the right. Cortical cell cultures in CCM were observed
for 5 min, then exposed to 2 µM propofol 10 seconds before time 0 and further observed for 10 min. Part of the neurite (within the inset
box) is shown as time-lapse images to the left of the main image. The dynamics of vesicle transport is shown at time -5, -1, 0, 2, 4, 8 and
10 min (with their corresponding numbers in each image) after the start of application of propofol. Arrows indicate individual vesicles.
Vesicles move anterograde (towards the right of the picture) between time -5 and -1, and change to retrograde direction (towards the left
of the picture) immediately after propofol application at time 0, which continues throughout the experiment. Scale bar =10 µm.
(B) Time-lapse data of the vesicular velocity obtained from the experiments described above, with data obtained each minute, from time
-5 min to time 10 min, where propofol (2 µM) was added 10 seconds before time 0, as shown by the arrow. The propofol treated cells are
shown with filled symbols. Cells only treated with CCM (control group, unfilled symbols) were observed for a total of 15 min. The mean
velocity for each vesicle was calculated and then the mean velocity for all tracked vesicles in one cell at a given time-point was used for
the analysis. At each time-point, the values are shown as mean, and because raw data are mean values of the vesicle velocity of each cell,
the error bars show standard error of the mean (S.E.M.). Control cells in CCM continued to move anterograde at a steady velocity
throughout the experiment (n=5, 46 vesicles tracked), whereas propofol-treated cells increased their velocity and changed to a retrograde
direction (n=8,74 vesicles tracked). The horizontal line is drawn at zero velocity. The propofol-treated cells showed a significant difference
in velocity compared to control cells (p<0.001 for the overall 2-way ANOVA), with significant (p<0.05) differences between groups seen
from time 3 min.
was no significant difference in the average anterograde or bidirectional velocity between the two groups. Intralipid-exposed
neurites showed the same vesicle movement as vesicles in
untreated cells (n=16, data not shown).
after removing propofol from the cells (n=6, 45 vesicles; Fig.
2A and 2B).
Propofol-induced retrograde vesicle movement is reversible
To study if the changes in vesicular dynamics were mediated
via the GABAA-receptor, the antagonist gabazine (7 µM) was
added 5 min prior to exposure to propofol. When propofol was
added after gabazine pretreatment, no retrograde vesicle
movement was observed. The average vesicle velocity in
gabazine-pretreated cells after propofol addition was
0.001±0.0005 µm·s-1, (n=6, 46 vesicles), compared to only
propofol-treated cells where vesicles had an average velocity of
-0.009±0.0009 µm·s-1 (n=6; 38 vesicles; p<0.001), i.e. a
retrograde transport. Gabazine itself did not alter vesicle
movement (0.002±0.0007 µm·s-1 (n=5, 39 vesicles, Fig. 3).
To determine whether propofol-induced retrograde vesicle
movement is reversible, we observed vesicle movements first
for 5 min in untreated cells, during which vesicles moved at low
anterograde velocity. After exposure to propofol (2 µM),
vesicles first decelerated and then accelerated in retrograde
direction, as described above. Propofol was washed off two min
after the exposure and the cells replenished with CCM. Initially
vesicles continued to move retrograde with the same velocity,
decelerated after 9 min and began to move anterograde 12 min
Gabazine inhibits propofol-induced retrograde vesicle movement
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Fig. 2. The propofol-induced retrograde vesicle movement is reversible. (A) A neuron in Ca2+-containing medium (CCM) is shown 5 min
(time -5) before exposure to propofol in a differential interference contrast image, with the cell body in the center. Part of the neurite pointing
at 12 o'clock (within the inset box) is shown as time-lapse images to the below the main image. Cortical cell cultures in CCM were observed
for 5 min, then exposed to 2 µM propofol 10 seconds before time 0. Propofol was washed away with CCM after 2 minutes. The dynamics
of vesicle transport is shown at time -5, -1, 0, 2, 5, 10, 15 and 20 min (with their corresponding numbers in each image) after the start of
application of propofol. Arrows indicate individual vesicles. Vesicles initially move anterograde, and retraction starts direct after propofol
addition at time 0. Thereafter the vesicles retract up to 10 min when some of the vesicles have started to move anterograde again. At time
20 min, the majority of vesicles move anterograde. Scale bar =10 µm.
(B) Time-lapse data of the vesicular velocity obtained from the experiments described above, with data obtained each minute, from time 5 min to time 20 min. Propofol (2 µM - filled squares) was added 10 seconds before time 0 (solid arrow) for 2 min and then washed off
the cells with CCM (dashed arrow) and the cells assessed for further 18 min. The mean velocity for each vesicle was calculated and then
the mean velocity for all tracked vesicles in one cell at a given time-point was used for the analysis. At each time-point, the values are shown
as mean, and because raw data are mean values of the vesicle velocity of each cell, the error bars show standard error of the mean (S.E.M).
The horizontal line is drawn at zero velocity. Propofol caused retrograde vesicle movement, and when propofol was washed off the cells
after 2 min treatment, the vesicles initially continued to move retrograde with the same velocity. They decelerated at time 11 min, and began
to move anterograde 12 min after removing propofol from the cells (at time 14 min). Data from n=6 neurons (45 vesicles tracked).
Fig. 3. The GABAAreceptor antagonist gabazine inhibits propofol-induced retrograde vesicle movement. Time-lapse data of the vesicular
velocity, with data obtained each minute, from time -5 min to time 15 min. Cell cultures obtained from Taconic rats were observed in CCM
for 5 min and then incubated with 7 µM gabazine (dashed arrow) at time 0 for 5 min (gabazine (unfilled rings)) and gabazine+propofol
group (solid triangle)). The gabazine+propofol group then received 2 µM propofol 10 sec before time 5 min (solid arrow). Cells were
subsequently assessed for 10 min. The cells in the propofol group (P2, - filled squares) were first observed in CCM for 5 min (starting at
time 0), and were then treated with 2 µM propofol at 10 seconds before time 5 min, and subsequently assessed for 10 min. The mean
velocity for each vesicle was calculated and then the mean velocity for all tracked vesicles in one cell at a given time-point was used for
the analysis. At each time-point, the values are shown as mean, and because raw data are mean values of the vesicle velocity of each cell,
the error bars show standard error of the mean (S.E.M.). The horizontal line is drawn at zero velocity. Data based on n=8 cells, 46 vesicles
tracked in the gabazine+propofol group, n=6 cells, 38 vesicles tracked in the propofol group and n=6 cells, 39 vesicles tracked in the
gabazine group. Gabazine did not have any intrinsic effect on vesicular movement (no changes compared to baseline velocity in CCM only)
and pre-treatment with gabazine reversed the effect of 2 µM propofol, causing the vesicles to continue their anterograde movement at low
velocity (0.001±0.0005 µm·s-1 vs. retrograde movement at 0.009±0.0009 µm·s-1 for the P2 group); p<0.001 with 2 way ANOVA.
123
DISCUSSION
We have studied propofol's effects on neurite vesicle
transport in vitro using rat cortical mixed neuronal cultures.
Live-cell imaging demonstrates distinct differences in the
dynamics of vesicle movement between the propofol group and
the control group. The concentration of propofol (2 µM) used in
the cell cultures is within a clinically relevant range (17).
Propofol causes loss of consciousness (LOC) in patients even at
sub-micromolar concentrations (0.4 µM) and there is an
excellent correlation between LOC and loss of the righting reflex
(LORR) in animals (18). After fifteen min of intravenous
infusion of propofol, the concentration of propofol in whole
brain tissue was 8.5 times that in plasma in adult male SpragueDawley rats (19). This suggests that the propofol concentration
that causes LOC would be 3.4 µM in brain tissue.
We show that following propofol treatment, neurite
vesicles move retrograde (Fig. 1), and as previously reported
(14), the neurites retract. Vesicles in the control group move
anterograde over the same time course with no retraction. The
response to propofol is prompt and already ten seconds after
the addition of propofol (time 0), the vesicles decelerate and
start to move toward the cell body. The propofol-induced effect
on vesicle transport is reversible and dependent on synaptic
GABAAR, since transport is blocked by a low dose gabazine, a
competitive GABAAR antagonist (20), (Fig. 3). A small
proportion of cells showed bi-directional movement, but there
was no significant difference between the propofol and the
control-treated group.
We also demonstrated that vesicles increase their retrograde
velocity in neurons exposed to propofol. There was no difference
in the anterograde and bi-directional velocity between the
propofol and the control group. This shows that the increasing
velocity is a direction-specific effect of propofol. The vesicles in
our cell cultures moved at an average speed of 0.025 µm·s-1 that
corresponds to the movement of cytoskeletal and cytosolic
proteins as slow components of axonal transport. In our cultures
we did not observe fast vesicular transport (>0.5 µm·s-1) (7) that
is characteristic of the transport of membranous organelles. The
highest recorded speed of retrograde moving vesicle was 0.3
µm·s-1. Our study is performed in vitro, which may not fully
correlate with in vivo conditions. The cell culture consists of a
mixture of 80% neurons and about 20% glia. For methodological
reasons, in our live cell recordings we used only superficial cells
with neurites that contained clearly visible, non-clustered
vesicles, suitable for tracking, which may not fully represent the
in vivo cellular organization.
Mechanisms of pre-synaptic actions of general anesthetics
are unclear but it is known that anesthetics have inhibitory
effects on synaptic transmission (11). In addition to propofol's
postsynaptic effects (21) on the GABAAR, it has been shown that
propofol enhances presynaptic inhibitory synaptic transmission
(12). In mice, propofol anesthesia is reversed by the GABAAR
antagonist bicuculline (22), and gabazine antagonized the
immobilizing effect of propofol in rats (23). At low
concentrations (7 µM) gabazine abolishes synaptic currents (20).
It was therefore used in this study over bicuculline to test
whether it could block propofol mediated retrograde vesicle
transport. As shown in Fig. 3, the propofol effect on vesicle
transport was completely blocked by 7 µM gabazine. This
suggests that the retrograde vesicle transport caused by propofol
is mediated via synaptic GABAA receptors without affecting
extrasynaptic GABAA receptors. We have previously shown that
the propofol induced retraction of neurites is blocked by
bicuculline (14). This demonstrates that in propofol's cell
signaling, the GABAAR is involved both in regulation of neurite
retraction and vesicle transport. Our results support a model of
co-ordinated neurite retraction and regulation of vesicle
transport caused by propofol.
We have previously demonstrated that propofol mediates
actin reorganization. This reorganization is calcium-dependent,
reversible, GABAAR-mediated and concentration-dependent
(24, 25). Propofol also causes tyrosine phosphorylation of actin
in the cell membrane and cytoskeletal fraction (26), and
increases the content of F-actin in the neurons (27). We have also
shown that propofol causes a dose- and time-dependent,
reversible retraction of neuronal neurites. The retracted neurites
leave a thin trailing remnant and a retraction bulb (14). A natural
occurring retraction is seen in hibernating squirrel's neurons.
These neurons exhibit a dramatic form of plasticity during
torpor, with dendritic arbors retracting as body temperature
cools, and then re-growing rapidly as body temperature rises
(28). The retraction caused by propofol in our model-system is
dependent on the GABAAR, actin and myosin II (14). In this
study, the propofol-induced effect on vesicle transport was
reversible (Fig. 2). After washing propofol off the cells, vesicles
continued to move retrograde, decelerated after 9 min, and began
to move anterograde again 12 min after replacing propofol with
CCM. The observed divergence in vesicular transport caused by
propofol correlates well with the time frame of propofol induced
neurite retraction (14). Both the neurite retraction and the
retrograde vesicle transport are reversible, suggesting that the
processes are based on disassembling and reassembling of
cytoskeletal proteins. Together, these findings lead us to suggest
a new model for the anesthetic mechanism of propofol. Neurite
retraction is caused by the contractility of actin/myosin and reorganization of actin due to the actions of propofol via the
GABAAR. This retraction initiates movement of the neurite
cytoplasm towards the cell body, which thereby leads to
retrograde vesicle movement. Such a mechanism can explain the
time-course to restore vesicle transport direction, since the
restoration occurs between 10 and 20 min after propofol
replacement and correlates well with recovery after propofolinduced neurite retraction (14). We cannot distinguish between
GABAergic and glutaminergic neurons in our model system, but
most neurons have GABAAR. Therefore our proposed cellular
signal model is plausible for both excitatory and inhibitory
neurons. Considering two possible mechanisms whereby
propofol can interact with neuronal transport, (I) active vesicle
transport driven by molecular motors, or (II) passive movement
of the neurite cytoplasm towards the cell body, which drives
passive retrograde vesicle transport; our studies support the
second mechanism.
In conclusion, we have demonstrated that in rat brain cortical
mixed cultures, propofol caused a rapid change in direction of
vesicle movement from anterograde to retrograde, and increased
their velocity. This alteration of vesicle transport was reversible
and blocked by the GABAAR antagonist gabazine. Based on
findings in our previous work, we propose that this is due to the
narrowing of the neurite caused by propofol, where propofol
increases the contractility of myosin via the GABAAR, induces a
reorganization of actin causing neurite retraction, and initiates
movement of the neurite cytoplasm towards the cell body. This
chain of events causes the observed retrograde vesicle
movement. This is a step in the chain to understand molecular
mechanisms of anesthesia; however details of such a proposed
mechanism need further investigation.
Acknowledgements: The authors wish to thank Hank
Schmidt for linguistic help. This work was supported by funding
from the Östergötland County Council and Linköping Society of
Medicine, Sweden.
Conflict of interests: None declared.
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R e c e i v e d : November 22, 2010
A c c e p t e d : February 28, 2011
Author's address: Prof. Karin Björnström, Department of
Medical and Health Sciences, Division of Anaesthesiology,
Faculty of Health Sciences, Linköping University, 58185
Linköping, Sweden; Phone + 46 10 103 0000; Fax +46 10 103
2836; E-mail: [email protected]