Toxicology 234 (2007) 216–226

Low concentrations of ketamine initiate dendritic atrophy of

differentiated GABAergic neurons in culture
Laszlo Vutskits a,∗ , Eduardo Gascon b , Gael Potter b ,
Edomer Tassonyi c , Jozsef Z. Kiss b
aDepartment of Anesthesiology, Pharmacology and Intensive Care, University Hospital of Geneva,
24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland
b Department of Neuroscience, University of Geneva Medical School, Geneva, Switzerland
c Department of Anesthesiology, Pharmacology and Intensive Care, University Hospital of Geneva, Geneva, Switzerland
Received 15 February 2007; received in revised form 28 February 2007; accepted 1 March 2007
Available online 12 March 2007

Abstract
Administration of subanesthetic concentrations of ketamine, a noncompetitive antagonist of the N-methyl-d-aspartate (NMDA)
type of glutamate receptors, is a widely accepted therapeutic modality in perioperative and chronic pain management. Although
extensive clinical use has demonstrated its safety, recent human histopathological observations as well as laboratory data suggest
that ketamine can exert adverse effects on central nervous system neurons. To further investigate this issue, the present study was
designed to evaluate the effects of ketamine on the survival and dendritic arbor architecture of differentiated ␥-aminobutyric acidergic
(GABAergic) interneurons in vitro. We show that short-term exposure of cultures to ketamine at concentrations of ≥20 ␮g/ml leads
to a significant cell loss of differentiated cells and that non-cell death-inducing concentrations of ketamine (10 ␮g/ml) can still initiate
long-term alterations of dendritic arbor in differentiated neurons, including dendritic retraction and branching point elimination.
Most importantly, we also demonstrate that chronic (>24 h) administration of ketamine at concentrations as low as 0.01 ␮g/ml
can interfere with the maintenance of dendritic arbor architecture. These results raise the possibility that chronic exposure to low,
subanesthetic concentrations of ketamine, while not affecting cell survival, could still impair neuronal morphology and thus might
lead to dysfunctions of neural networks.
© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Dendrite; GABA; Ketamine; Pain; Neurotoxicity

1. Introduction venous anaesthetic alternative to phencyclidine (Reich

Ketamine is a phencyclidine derivative acting
primarily as a noncompetitive antagonist of N-methyl-d-
aspartate (NMDA) excitatory glutamate receptors (Oye
et al., 1992; Orser et al., 1997). While this drug was
first synthesized in an attempt to provide a safer intra-
∗ Corresponding author. Tel.: +41 22 3723311; fax: +41 22 3727511.
E-mail address: laszlo.vutskits@hcuge.ch (L. Vutskits).
and Silvay, 1989), the important psychomimetic side
effects observed following exposure to anaesthetic con-
centrations of ketamine (Freuchen et al., 1976) has
been importantly curtailed its use as an anesthetics
in current anaesthesia practice. Since the recogni-
tion of the important role of NMDA receptors in
nociceptive neurotransmission (Petrenko et al., 2003),
renewed interest, however, has focused on the role
of ketamine in pain management (Himmelseher and
Durieux, 2005). In fact, at subanesthetic doses, ketamine

0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2007.03.004
 L. Vutskits et al. / Toxicology 234 (2007) 216–226
has been shown to reduce pain perception without
provoking deranging psychomimetic reactions (Oye et
al., 1992; Arendt-Nielsen et al., 1996) and adminis-
tration of low concentrations of ketamine has now
gained widespread clinical use in perioperative pain
management (Bell et al., 2006). Importantly, long-
term administration of subanesthetic doses of ketamine
has been shown to be effective in the pharmaco-
logical treatment of complex regional pain syndrome
(Rowbotham, 2006), intractable cancer pain (Lossignol
et al., 2005) and central post-stroke pain (Vick and
Lamer, 2001).
Recent neuropathological findings following contin-
uous intrathecal administration of ketamine to relieve
neuropathic cancer pain demonstrate severe histologi-
cal abnormalities in the spinal cord, including neuronal
shrinkage and death as well as reactive gliosis (Vranken
et al., 2005). In line with these human observations,
repeated exposure to phencyclidine, ketamine or to
the noncompetitive NMDA antagonist MK801 led to
quite similar histopathological alterations in the cere-
bral cortex of adult rats (Olney et al., 1989), raising
concern about the potential neurotoxic effects of these
drugs. Indeed, although highly debated (Anand and
Soriano, 2004; Olney et al., 2004), extensive experi-
mental evidence indicate that even short-term exposure
to ketamine and other NMDA antagonists can induce
widespread apoptosis in the developing central nervous
system (CNS) during the brain growth spurt period
(Ikonomidou et al., 1999; Young et al., 2005). While
the extent of acute ketamine administration-induced cell
death is significantly less in the adult CNS (Ikonomidou
et al., 1999; Olney, 2002), little is known about how
chronic exposure to subanesthetic concentrations of
this agent can affect the adult brain. In this con-
text, it is important to note that neuronal apoptosis is
not the only parameter to be considered in evaluat-
ing potential adverse effects of drug exposure on the
nervous system. It is now well established that inter-
ference with the finely tuned molecular mechanisms
orchestrating the maintenance of dendritic arbors in dif-
ferentiated neurons can induce important reorganization
of synaptic connections, leading to persistent changes in
behaviour and psychological functions (Robinson and
Kolb, 2004).
In our previous study, we demonstrated that ketamine
can interfere with dendritic arbor development of imma-
ture GABAergic neurons (Vutskits et al., 2006). It is,
however, currently unknown whether chronic exposure
to low concentrations of ketamine can also alter the main-
tenance of dendritic arbor architecture in differentiated
neurons. The aim of the present study was to evalu-
217
ate this possibility using our previously described cell
culture model of subventricular zone-derived GABAer-
gic interneurons (Gascon et al., 2005; Vutskits et al.,
2005,2006).
2. Methods
2.1. Cell cultures and reagents
After obtaining approval from the Animal Care Committee
of the University Medical Center, cell cultures were prepared
from newborn (postnatal day 0) Sprague-Dawley rats. Animals
were sacrificed by decapitation and the brain was carefully
removed and transferred into an ice-cold Hank’s magnesium-
and calcium-free solution. Two coronal cuts were then made to
expose the anterior horn of the lateral ventricles and the sub-
ventricular zone was microdissected. The small tissue pieces
obtained were dissociated mechanically and digested with
trypsin (Invitrogen, Life technologies, Paisley, UK) for 15 min
at 37 ◦ C. The trypsin reaction was stopped with 1 ml of cold
fetal calf serum and cells recovered after 10 min centrifu-
gation at 300 × g. To eliminate cell debris, the pellet was
resuspended into 1 ml of PBS and layered onto a 22% Per-
coll gradient (Amershman Pharmacia, Little Chalfont, UK)
in phosphate-based saline (PBS) and centrifuged 10 min at
500 × g. Cells were washed three times with culture medium
before plating onto poly-ornithin (Sigma, Saint Louis, MO,
USA) coated coverslips in 35 mm Petri dishes (Falcon, Ply-
mouth, UK). Seeding density was 5000 cells/cm2 . Cells were
cultured in Neurobasal medium (Invitrogen, Life technologies,
Paisley, UK) supplemented with 2% B27 (Invitrogen, Life tech-
nologies, Paisley, UK), 200 mM l-Glutamine (Invitrogen, Life
technologies, Paisley, UK) and 1 mM Na pyruvate (Sigma,
Saint Louis, MO, USA). Under these conditions, cells read-
ily survived and developed as GABAergic neurons for up to 12
days in culture.
To test the effects of ketamine on neuronal survival and den-
dritic architecture, cultures were exposed to ketamine (Ketalar,
Parke-Davis, Berlin, Germany) and the NMDA receptor antag-
onist MK 801 [C16 H15 N·C4 H4 O4 ] (100 ␮M; Tocris, Bristol,
UK). The amount and the duration of ketamine treatment are
indicated in detail in each experiment in the Section 3. To
remove drugs from the culture medium, cultures (control and
treated) were washed 3 times with warm (37 ◦ C) Neurobasal
medium and then continued to be cultured in the presence of
Neurobasal medium before analysis according to experimental
protocols.
2.2. Immunocytochemistry
Cells were fixed with cold (4 ◦ C) paraformaldehyde 4%
in phosphate buffer (pH 7.4). Then, they were rinsed 3 times
in PBS and incubated overnight at 4 ◦ C with the primary anti-
body diluted in PBS containing bovine serum albumine (0.5%)
and Triton X-100 (0.3%). The mouse monoclonal Ab directed
against ␤-tubulin isotype III (Sigma, Saint Louis, MO, USA
218 L. Vutskits et al. / Toxicology 234 (2007) 216–226
1:400 dilution) was used to identify neurons. Bound Abs were
revealed with rhodamine- or fluorescein-conjugated sheep anti-
mouse IgG (Boehringer, Mannheim, Germany; dilution 1:40
for rhodamine and 1:80 for fluorescein) diluted in PBS con-
taining 0.5% BSA.
2.3. Cell counts, statistical analysis, image acquisition
and processing
Immunostained cultures were examined with a Nikon
Eclipse TE2000-U microscope (Nikon Corp, Tokyo, Japan).
For cell counts, a 10× Nikon objective and a digital cam-
era (Retiga EX, Qimaging, Burnaby, Canada) controlled by
the Openlab software (Version 3.1.2. Improvision, Coven-
try, England) was used. On each coverslip, 30 samples were
randomly taken and then samples were pooled (i.e., total sur-
face measured per coverslip was approximately 5 mm2 ). Cell
counts were made using the Image J (NIH) software (data are
expressed as the number of neurons per mm2 ± standard error
of mean, and reflect results obtained form at least 3 independent
experiments).
For quantitative analysis of dendritic arbours, ␤-tubulin iso-
type III positive cells were photographed using a 40× Nikon
objective linked through the digital camera to the Openlab soft-
ware. Before analysis, brightness and contrast were optimized
with Adobe Photoshop program (Adobe Systems Incorpo-
rated, San Jose, California, USA). The following parameters
of dendritic shape and extent were then determined: number of
primary dendrites, length of dendrites and the number of den-
dritic branches. Total dendritic length was measured drawing
all visible processes with Image J. The remaining parameters
were manually scored on the image. Processes shorter than
5 ␮m were excluded from the analysis.
Through the whole study, values were expressed as
means ± standard error of mean and analyzed for statistical
significance. Differences between groups were first discrim-
inated by one-way ANOVA and then the unpaired t-test was
performed, where t was corrected for multiple comparisons
against the untreated group using the Bonferroni test. *P < 0.05
compared with the untreated control group.
3. Results
3.1. Subventricular zone-derived neurons display a
differentiated phenotype after 6 days in culture
We have previously shown that subventricular
zone-derived neuronal precursors differentiate into
GABAergic neurons under serum-free conditions in vitro
(Gascon et al., 2005). These neurons rapidly elaborate
their dendritic arbor during the first few days in culture
(Fig. 1), thus providing a useful model system to study
the effects of anesthetics on dendritic development and
survival of these cells (Vutskits et al., 2005,2006). By the
6th day in vitro (DIV), cells reach a plateau phase in terms
of arborisation pattern: while dendrites still continue to
discretely sprout on poly-ornithine-covered coverslips,
no additional increase in the number of primary dendrites
(PD) and branching points (BP) can be observed by time
(Fig. 1E and F). These findings are consistent with our
recent results, indicating functional maturation of these
cells by the 6th DIV that corresponds to the functional
shift from the depolarizing to the hyperpolarizing action
of the neurotransmitter GABA on these cells (Gascon et
al., 2006).
3.2. Effect of short-term ketamine exposure on the
survival of SVZ-derived differentiated GABAergic
neurons
In our recent study, we showed that short-term expo-
sure of immature SVZ-derived neurons to ketamine
induces apoptosis in a dose-dependent manner (Vutskits
et al., 2006). As the effect of ketamine on neuronal sur-
vival has been reported to be age dependent (Ikonomidou
et al., 1999), we first assessed whether short-term expo-
sure to ketamine can induce cell loss in 6 DIV cultures
containing differentiated neurons and, if yes, whether
cell-death-inducing concentrations of ketamine in these
cells are different from those observed in immature
neurons (Vutskits et al., 2006). To this end, we first
exposed 6 DIV cultures to ketamine for 1 h in concen-
trations ranging from 1 to 100 ␮g/ml and cell survival
was evaluated up to 72 h following drug exposure (for
experimental setup see Fig. 2A). We found no cell
loss at any of these concentrations when cell survival
was assessed immediately (time 0) after this treatment
(Fig. 2B). In contrast to immature neurons, where a
1-hour-long exposure to ketamine induced cell death
at concentrations of 10 ␮g/ml as early as 24 h follow-
ing treatment (Vutskits et al., 2006), this experimental
paradigm did not influence survival of 6 DIV differ-
entiated neurons at any of the time points examined
(Fig. 2B). We found, however, a significant cell loss of
differentiated neurons 24 h after ketamine treatment at
concentrations ≥ 40 ␮g/ml and also at later time points
using concentrations ≥ 20 ␮g/ml (Fig. 2B). An 8-hour-
long-exposure to ketamine (for experimental setup see
Fig. 2A) did not induce apparent cell loss following
the end of this treatment (time 0) up to drug concen-
trations of 20 ␮g/ml (Fig. 2C). However, a significant
(P < 0.05) cell loss was detected from the 1st day
post-exposure in cultures receiving ketamine at con-
centrations of ≥ 10 ␮g/ml (Fig. 2C). Since one major
action of ketamine is the blockade of the NMDA-type
glutamate receptors, we also examined cell survival in
the presence of the noncompetitive NMDA receptor
 L. Vutskits et al. / Toxicology 234 (2007) 216–226 219

Fig. 1. Dendritic arbor architecture development of subventricular zone-derived GABAergic neurons. (A) 24 h after seeding, isolated neuroblasts
exhibit an immature morphology under serum-free conditions. (B) Representative photomicrograph of a differentiating neuron by the end of the
3rd day in culture. (C) Neurons develop a complex dendritic architecture by the 6th day in vitro. (D) While dendrites still continue to discretely
sprout on poly-ornithine-coated coverslips, no further increase in dendritic complexity can be observed at later time points (9th day in culture).
(E–F) Quantitative assessment of dendritic arbor development in terms of the number of primary dendrites and branching points, respectively. In
photomicrographs (A–D), cells were stained with the neuron specific marker tubulin-␤-III antibody. Correction bar (A–D): 25 ␮m. In figures E–F,
values on the Y-axis are expressed as the number of primary dendrites and branching points per cell. Results are presented as mean ± S.E.M.; n = 3
independent experiments for each time point expressed.

blocker MK801 (100 ␮M). The presence of MK801 up
to 8 h in the culture medium did not induce cell death
(Fig. 2B and C). Altogether, these results indicate that
sensitivity to ketamine-induced cytotoxicity decreases
along with neuronal maturation and that these effects
appear to be independent of the blockade of NMDA
receptor.
3.3. Dose-dependent effect of short-term ketamine
exposure on dendritic arbor architecture of
differentiated GABAergic neurons
We next examined whether short-term exposure to
non-cell-death-inducing concentrations of ketamine can
still alter dendritic tree architecture in differentiated
220 L. Vutskits et al. / Toxicology 234 (2007) 216–226

total dendritic length (TDL); (2) the number of primary

dendrites (PD), defined as those arising from the cell
body; and (3) the number of branching points (BP). As
seen in Fig. 3, ketamine treatment up to 8 h, at concentra-
tions ≤ 5 ␮g/ml, did not affect any aspects of dendritic
architecture. In contrast, even a 1-hour-long exposure to
this drug at concentrations of 10 ␮g/ml induced a signif-
icant remodelling of dendritic arbor, including retraction
of dendrites as well as elimination of branching points.
These effects could be observed as early as 24 h follow-
ing drug exposure (not shown) and became significant
(P < 0.05) by the 3rd day post exposure (Fig. 3 H). An 8-
hour-long exposure to 10 ␮g/ml of ketamine had an even
more robust effect (Fig. 3I). In contrast, the presence of
MK801 up to 8 h in the culture medium did not influ-
ence dendritic morphology (Fig. 3H and I). These results
thus demonstrate that even a single short episode of
ketamine treatment at non-cell-death-inducing concen-
trations can lead to persistent changes of dendritic arbor
architecture.

3.4. Effect of chronic ketamine exposure on the

survival of differentiated GABAergic neurons

Long-term administration of low, subanesthetic doses

Fig. 2. Effect of short-term ketamine treatment on the survival of
subventricular zone-derived differentiated neurons. (A) Experimental
protocol. (B) Quantitative analysis of cell survival following a 1-
hour-long treatment to ketamine (5–40 ␮g/ml) and MK801 (100 ␮M).
(C) Quantitative analysis of cell survival following an 8-hour-long
treatment to ketamine (2–20 ␮g/ml) and MK801 (100 ␮M). In fig-
ures (B–C), results are presented as mean ± S.E.M.; n = 3 independent
experiments for each time point and each treatment expressed. Values
are expressed as the number of neurons/mm2 . *P < 0.05 compared with
the untreated control group.
GABAergic neurons. Using the abovementioned exper-
imental paradigms (Fig. 3A), cultures were exposed to
ketamine at concentrations ranging from 1 to 10 ␮g/ml
and the impact of this treatment on neuronal dendritic
architecture, was measured using three parameters: (1)
of ketamine has become an accepted therapeutical
modality in postoperative sedation and pain control as
well as in chronic pain treatment (Himmelseher and
Durieux, 2005; Rowbotham, 2006). To study the effects
of long-term exposure of differentiated neurons to sub-
anesthetic concentrations of ketamine, 6 DIV cultures
were exposed to the continuous presence of this drug
(0.01–5 ␮g/ml) for 72 h and cell loss was assessed in
corresponding sister cultures every 24 h (for experimen-
tal protocol see Fig. 4A). Using this protocol, we found a
significant (P < 0.05) decrease in cell number after 48 h
at concentrations ≥ 2 ␮g/ml and cell loss became also
significant by the end of the 3rd day of continuous treat-
ment at concentrations of ≥ 1 ␮g/ml. Similarly, chronic
application of MK 801 (100 ␮M) led to a significant

Fig. 3. Effects of short-term ketamine treatment on dendritic arbor architecture of subventricular zone-derived differentiated neurons. (A) Exper-
imental protocol. (B) Representative epifluorescent micrograph of a 6-day-old neuron, showing that no effect on dendritic architecture was found
at any doses of ketamine applied (up to 40 ␮g/ml) when morphology was immediately (time 0) assessed following drug exposure. (C) In control,
placebo treated cultures, neuronal arbor architecture remained stable up to 72 h following treatment. Similarly, (D) neither exposure to ketamine
at concentrations of ≤ 5 ␮g/ml nor (E) treatment with MK801 (100 ␮M) up to 8 h had any long-term effect on neuronal morphology. In contrast,
(F) as short as a 1-hour-long treatment with ketamine at concentrations of 10 ␮g/ml induced dendritic pruning of differentiated neurons and (G)
these effects were even more robust when the length of the treatment period reached 8 h. (H–I) Quantitative assessment of dendritic morphology
72 h after a (H) 1-hour-long and an (I) 8-hour-long ketamine (1–20 ␮g/ml) or MK801 (100 ␮M) treatment. In photomicrographs (A–D), cells were
stained with the neuron specific marker tubulin-␤-III antibody. Correction bar (A–D): 20 ␮m. In figures (H–I), values on the Y-axis are expressed as
the number of primary dendrites and branching points per cell. Results are presented as mean ± S.E.M.; n = 3 independent experiments (3 culture
dish per experiment, 30 neurons per culture dish) for each time point and each treatment expressed. *P < 0.05 compared with the untreated control
group.
L. Vutskits et al. / Toxicology 234 (2007) 216–226 221
222 L. Vutskits et al. / Toxicology 234 (2007) 216–226
Fig. 4. Quantitative assessment of cell survival following chronic
application of ketamine (0.01–5 ␮g/ml) and MK801 (100 ␮M). Results
are presented as mean ± S.E.M.; n = 3 independent experiments for
each time point and each treatment expressed. Values are expressed as
the number of neurons/mm2 . *P < 0.05 compared with the untreated
control group.
decrease in the number of neurons in culture, suggesting
that NMDA-dependent mechanisms are involved in the
survival of GABAergic neurons (Fig. 4B). It is impor-
tant to note that, while application of ketamine 1 ␮g/ml
as well as MK801 induced death of both immature and
differentiated neurons, the extent of cell death was more
important in immature cells (52 ± 6% in immature ver-
sus 19 ± 4% in differentiated neurons), further arguing
for the differential sensitivity of young versus adult-like
neurons to ketamine.
3.5. Effect of chronic (>24 h) ketamine exposure on
dendritic arbor architecture of differentiated
GABAergic neurons
Studying the effects of long-term ketamine treatment
as well as the impact of NMDA receptor blockade on
dendritic architecture of mature GABAergic neurons
(for experimental protocol see Fig. 4A), we found that
dendritic morphology was severely impaired following
chronic exposure even to low concentrations of this drug
(Fig. 5). During the first 24 h in the presence of ketamine
(0.01–1 ␮g/ml) or MK801 (100 ␮M), no significant dif-
ference could be detected between control and treated
cultures in any aspects of dendritic morphology analyzed
(Fig. 5). In contrast, by the end of 2nd day, 1 ␮g/ml
of ketamine as well as MK801 induced a significant
decrease in total dendritic length and the number of
branch points compared to the beginning of treatment
(time 0) and these perturbations further accentuated by
the end of the 3rd day of drug exposure. Importantly,
following 48 h of treatment, the number of branch points
was also significantly decreased in cultures treated with
ketamine at a concentration of 0.1 ␮g/ml. Finally, elimi-
nation of branch points could also be observed in groups
receiving ketamine at a concentration of 0.01 ␮g/ml,
although it did not reach significance. Taken together,
these data indicate that chronic exposure to low doses
of ketamine, while not affecting cell survival, results
in clear atrophy of dendritic arbors of differentiated
GABAergic neurons.
4. Discussion
The NMDA-type of excitatory glutamate receptors
play an important role in central sensitization following
painful stimuli (Petrenko et al., 2003) and pharmaco-
logical blockade of these receptors, using subanesthetic
concentrations of ketamine, has been show to be an
effective treatment modality in both perioperative and
chronic pain management (Himmelseher and Durieux,
2005; Rowbotham, 2006). Based on previous laboratory
and human histopathological observations, suggesting
that ketamine can exert potential adverse effects on CNS
neurons (Olney et al., 1989; Vranken et al., 2005), the
present study was designed to evaluate the dose- and
exposure-time dependent effects of ketamine on differ-
entiated GABAergic neurons. We show that short-term
exposure to ketamine induces cell death and dendritic
arbor retraction of surviving neurons only when this drug
is administered at relatively high doses (≥10 ␮g/ml).
Most importantly, we also demonstrate that chronic
administration of ketamine at concentrations as low as
0.01 ␮g/ml can induce an important atrophy of neuronal
dendritic architecture. These results suggest that long-
term administration of subanesthetic concentrations of
ketamine, while not affecting survival, may still impair
neuronal network functions by interfering with funda-
mental mechanisms governing maintenance of dendritic
arbor in differentiated neurons.
In vitro models provide a useful complementary
approach to in vivo animal experiments in the evaluation
of potential drug-induced adverse effects on an organism
(Spielmann and Liebsch, 2001). In the present study, we
used our previously validated in vitro model, that allowed
us to study the effects of anesthetics on the survival and
dendritic arborization of developing GABAergic neu-
rons in culture (Gascon et al., 2005; Vutskits et al.,
2005,2006). Here we show that our model is also a
valuable tool to assess the potentially toxic effects of
these agents on more differentiated cells, presenting
functional characteristics of mature neurons. In these
 L. Vutskits et al. / Toxicology 234 (2007) 216–226 223

Fig. 5. Effect of long-term ketamine (0.01–1 ␮g/ml) and MK 801 (100 ␮M) treatment on dendritic architecture of differentiated GABAergic
neurons. 6 days after seeding (time 0), ketamine was applied to the culture medium and left there for up to 72 h. Representative examples of dendritic
arbor expansion in (A) time 0; (B) control; (C) ketamine (0.1 ␮g/ml); (D) ketamine (1 ␮g/ml); and (E) MK801 (100 ␮M)-treated neurons. (F–H)
Quantitative analysis of the effects of continuous exposure to low concentrations of ketamine (0.01–1 ␮g/ml) and MK801 (100 ␮M), on dendritic
arbor as expressed by the total dendritic length (F), the number of primary dendrites (G) and the number of branching points (H). Cells were stained
with the neuron specific marker tubulin-␤-III antibody. Correction bar (A–E): 35 ␮m. In figures F–H, values on the Y-axis are expressed as the
number of primary dendrites and branching points per cell. Results are presented as mean ± S.E.M.; n = 3 independent experiments (3 culture dish
per experiment, 30 neurons per culture dish) for each time point and each treatment expressed. *P < 0.05 compared with the untreated control group.
224 L. Vutskits et al. / Toxicology 234 (2007) 216–226
cultures, we recently demonstrated a GABAA receptor-
mediated functional shift from the depolarizing toward
the hyperpolarizing cellular responses to GABA by the
6th DIV and this is correlated with a significant increase
in the expression pattern of K+ /Cl− co-transporter KCC2
(Gascon et al., 2006). Parallel to these functional changes
in neuronal phenotype, there is an important decrease in
the expression of PSA-NCAM, a cell surface marker of
immature neurons, from the end of the 1st week in vitro
(Gascon et al., 2005). Finally, results presented in this
study show that differentiating neurons reach steady-
state in terms of dendritic arborization pattern by the
6th DIV. Altogether, these results thus strongly suggest
that neuronal precursors in these preparations undergo
morphofunctional maturation and acquire, by the 6th
DIV, functional characteristics similar to those of mature
neurons.
Using 6 DIV cultures to evaluate the effect of short-
term ketamine exposure on cell survival, we demonstrate
that higher concentrations and exposure times of this
drug are needed to induce cell death in differentiated
GABAergic cells, compared to their immature coun-
terparts (Vutskits et al., 2006). These results are in
agreement with previous in vivo studies (Ikonomidou
et al., 1999; Jevtovic-Todorovic et al., 2001), and sug-
gest that sensitivity to ketamine-induced cytotoxicity
decreases along with neuronal maturation. Although the
precise molecular mechanisms responsible for the dif-
ferential sensitivity of immature versus mature neurons
to ketamine remains to be determined, one plausi-
ble explanation of these observations could be the
developmental switch in the subunit composition of
NMDA receptors that occurs during neuronal differenti-
ation both in vivo and in vitro (Williams et al., 1993;
Feldmeyer and Cull-Candy, 1996; Kew et al., 1998).
It is also important to underline that, in contrast to
high-dose regimens of ketamine, we did not find any
effect of short-term exposure of cells to the noncom-
petitive NMDA receptor antagonist MK801 either on
survival or dendritic morphology. These results suggest
that ketamine-induced neurotoxicity following short-
term exposure to relatively high concentrations of this
drug is, at least partially, independent of NMDA recep-
tor blockade. As ketamine is known to interact with
a multitude of signaling pathways mediating neuro-
transmission in the CNS (Adams, 1998), it is possible
that, in the presence of important concentrations of
this anesthetics, additive or synergistic effects between
these signaling mechanisms could rapidly initiate den-
dritic remodeling and/or apoptosis. Alternatively, high
doses of ketamine could exert a nonspecific neurotoxic
effect.
One of the most intriguing finding of the present
study was that long-term exposure of differentiated neu-
rons to low, non-cell-death-inducing concentrations of
ketamine can still induce a significant atrophy of den-
drites, including retraction of branches and elimination
of branching points. Such atrophy and debranching of
dendritic arbors could have important long-term func-
tional consequences, as even minor changes in neuronal
morphology have been shown to exert a significant
impact on neuronal responses to stimuli (van Ooyen et
al., 2002; Robinson and Kolb, 2004). Little is known
about the mechanisms that underlie pruning of dendritic
arbors in the CNS (Williams and Truman, 2005) and,
to our knowledge, the role of chronic NMDA-mediated
neurotransmission blockade in this context has not been
established. Our data, showing that chronic admin-
istration of the NMDA receptor antagonist MK801,
similarly to low doses of ketamine, also results in reduced
dendritic arbor suggests that NMDA-dependent mech-
anisms are involved in the maintenance of dendritic
structures.
In an attempt to draw parallel between our data and
those obtained in previous in vivo animal studies (Olney
et al., 1989; Ikonomidou et al., 1999; Scallet et al.,
2004; Young et al., 2005), an important issue to con-
sider is whether ketamine concentrations administered
in the present study are comparable to those used in ear-
lier in vivo experiments. It has been previously reported
that following a single dose intraperitoneal injection of
20 mg/kg ketamine, known to induce apoptosis in the
cerebral cortex of young mice (Young et al., 2005),
plasma concentrations of this drug are approximately
5–6 ␮g/ml (Scallet et al., 2004). Importantly, ketamine
rapidly penetrates the blood-brain barrier and brain con-
centrations of this drug are up to six times higher than
those in the blood (Edwards et al., 2002). It is thus con-
ceivable that the abovementioned treatment paradigm
could lead to a transient accumulation of ketamine in
the brain parenchyma at concentrations of ≥ 20 ␮g/ml,
a scenario quite similar to our “short-term exposure”
protocol where the presence of ketamine for 1-hour in
the culture medium, representing conceptually the brain
rather than the plasma environment, at concentrations
of ≥ 20 ␮g/ml also induced cell loss. To our knowledge,
no in vivo data exists on the effects of chronic low-
dose ketamine exposure on the CNS. It is important to
emphasize that the present observations were made on
isolated neurons without synaptic contacts. It will be,
therefore, essential to validate these data by determining
how neuronal dendritic arbor architecture is influenced
by ketamine in a more complex and physiological envi-
ronment.
 L. Vutskits et al. / Toxicology 234 (2007) 216–226
Whereas anaesthesia-inducing plasma concentrations
of ketamine in humans have been reported to be above
1 ␮g/ml (Malinovsky et al., 1996; Weber et al., 2004),
adequate pain relief-associated plasma levels of this
anesthetics are several fold lower, situating around
20 ng/ml (Suzuki et al., 2005). Although it is ethically
impossible to determine blood-brain distribution coef-
ficient of ketamine in humans, based on experimental
studies of ketamine pharmacokinetics (Edwards et al.,
2002), it is thus strongly possible, as discussed above,
that plasma concentrations of 20 ng/ml of ketamine result
in several fold higher brain concentrations. In this con-
text, chronic exposure to subanesthetic concentrations of
ketamine would potentially result in drug levels between
0.01 and 0.1 ␮g/ml in the human brain parenchyma.
This clinical situation could be similar to our “long-term
treatment” paradigm, where exposure of GABAergic
neurons to ketamine in this concentration range induced
dendritic retraction. It is, however, important to note
that extrapolation of these laboratory results to clinical
practice requires extreme caution. Indeed, due to inter-
species differences in terms of drug effects (Berde and
Cairns, 2000), animal data provide an imprecise basis
for evaluating human risk. On the other hand, given
that dendrites are important morphofunctional substrates
underlying complex cognitive functions, our results
demonstrating the effects of ketamine on neuronal den-
dritic arbour should give further arguments to promote
clinical research on this topic. While the effect of short-
term ketamine exposure has been extensively studied in
a wide variety of neuropsychological tests (Adler et al.,
1998; Krystal et al., 1994; Rowland et al., 2005), to our
knowledge, there are currently no data on detailed neu-
ropsychological outcome in patients receiving ketamine
for extended periods. Results of these future studies
would be necessary to initiate discussion about the
risk/benefit evaluation of chronic ketamine treatment.
Acknowledgements
This work was supported by Swiss National Foun-
dation Grants (Bern, Switzerland) 310000 113555/1 to
L.V.; 3100A0-104059/1 to J.Z.K. and the Anesthesi-
ology Department Fund of the University Hospital of
Geneva to L.V. We wish to thank Sylvie Chliate (techni-
cian, Department of Neuroscience, University of Geneva
Medical School, Switzerland) for technical assistance.
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