Chronic ketamine administration modulates midbrain dopamine system in mice.
ABSTRACT Ketamine is an anesthetic and a popular abusive drug. As an anesthetic, effects of ketamine on glutamate and GABA transmission have been well documented but little is known about its long-term effects on the dopamine system. In the present study, the effects of ketamine on dopamine were studied in vitro and in vivo. In pheochromocytoma (PC 12) cells and NGF differentiated-PC 12 cells, ketamine decreased the cell viability while increasing dopamine (DA) concentrations in a dose-related manner. However, ketamine did not affect the expression of genes involved in DA synthesis. In the long-term (3 months) ketamine treated mice, significant increases of DA contents were found in the midbrain. Increased DA concentrations were further supported by up-regulation of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Activation of midbrain dopaminergic neurons could be related to ketamine modulated cortical-subcortical glutamate connections. Using western blotting, significant increases in BDNF protein levels were found in the midbrain, suggesting that perhaps BDNF pathways in the cortical-subcortical connections might contribute to the long-term ketamine induced TH upregulation. These data suggest that long-term ketamine abuse caused a delayed and persistent upregulation of subcortical DA systems, which may contribute to the altered mental status in ketamine abusers.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Ketamine, a known antagonist of N-methyl-D-aspartic (NMDA) glutamate receptors, had been used as an anesthetic particularly for pediatric or for cardiac patients. Unfortunately, ketamine has become an abusive drug in many parts of the world while chronic and prolonged usage led to damages of many organs including the brain. However, no studies on possible damages in the brains induced by chronic ketamine abuse have been documented in the human via neuroimaging. This paper described for the first time via employing magnetic resonance imaging (MRI) the changes in ketamine addicts of 0.5-12 years and illustrated the possible brain regions susceptible to ketamine abuse. Twenty-one ketamine addicts were recruited and the results showed that the lesions in the brains of ketamine addicts were located in many regions which appeared 2-4 years after ketamine addiction. Cortical atrophy was usually evident in the frontal, parietal or occipital cortices of addicts. Such study confirmed that many brain regions in the human were susceptible to chronic ketamine injury and presented a diffuse effect of ketamine on the brain which might differ from other central nervous system (CNS) drugs, such as cocaine, heroin, and methamphetamine.Frontiers in Neuroanatomy 01/2013; 7:23. · 4.18 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Depression is a common chronic psychiatric disorder that is also often co-morbid with numerous neurological and immune diseases. Accumulating evidence indicates that disturbances of neuroplasticity occur with depression, including reductions of hippocampal neurogenesis and cortical synaptogenesis. Improper trophic support stemming from stressor-induced reductions of growth factors, most notably brain derived neurotrophic factor (BDNF), likely drives such aberrant neuroplasticity. We posit that psychological and immune stressors can interact upon a vulnerable genetic background to promote depression by disturbing BDNF and neuroplastic processes. Furthermore, the chronic and commonly relapsing nature of depression is suggested to stem from "faulty wiring" of emotional circuits driven by neuroplastic aberrations. The present review considers depression in such terms and attempts to integrate the available evidence indicating that the efficacy of current and "next wave" antidepressant treatments, whether used alone or in combination, is at least partially tied to their ability to modulate neuroplasticity. We particularly focus on the N-methyl-D-aspartate (NMDA) antagonist, ketamine, which already has well documented rapid antidepressant effects, and the trophic cytokine, erythropoietin (EPO), which we propose as a potential adjunctive antidepressant agent.Frontiers in Cellular Neuroscience 01/2013; 7:218. · 4.18 Impact Factor
Article: A word to the wise about ketamine.American Journal of Psychiatry 03/2014; 171(3):262-4. · 14.72 Impact Factor
Chronic Ketamine Administration Modulates Midbrain
Dopamine System in Mice
Sijie Tan1, Wai Ping Lam1, Maria S. M. Wai1, Wan-Hua Amy Yu2, David T. Yew1*
1Brain Research Center, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong S.A.R., P.R. China, 2Department of Cell Biology and
Anatomy Sciences, The Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, New York, United States of America
Ketamine is an anesthetic and a popular abusive drug. As an anesthetic, effects of ketamine on glutamate and GABA
transmission have been well documented but little is known about its long-term effects on the dopamine system. In the
present study, the effects of ketamine on dopamine were studied in vitro and in vivo. In pheochromocytoma (PC 12) cells
and NGF differentiated-PC 12 cells, ketamine decreased the cell viability while increasing dopamine (DA) concentrations in a
dose-related manner. However, ketamine did not affect the expression of genes involved in DA synthesis. In the long-term
(3 months) ketamine treated mice, significant increases of DA contents were found in the midbrain. Increased DA
concentrations were further supported by up-regulation of tyrosine hydroxylase (TH), the rate limiting enzyme in
catecholamine synthesis. Activation of midbrain dopaminergic neurons could be related to ketamine modulated cortical-
subcortical glutamate connections. Using western blotting, significant increases in BDNF protein levels were found in the
midbrain, suggesting that perhaps BDNF pathways in the cortical-subcortical connections might contribute to the long-
term ketamine induced TH upregulation. These data suggest that long-term ketamine abuse caused a delayed and
persistent upregulation of subcortical DA systems, which may contribute to the altered mental status in ketamine abusers.
Citation: Tan S, Lam WP, Wai MSM, Yu W-HA, Yew DT (2012) Chronic Ketamine Administration Modulates Midbrain Dopamine System in Mice. PLoS ONE 7(8):
Editor: Olivier Jacques Manzoni, Institut National de la Sante ´ et de la Recherche Me ´dicale, France
Received April 24, 2012; Accepted July 27, 2012; Published August 24, 2012
Copyright: ? 2012 Tan 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: This work was supported by the Beat Drugs Fund Association (100052), Hong Kong Government. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Ketamine has become a popular recreational drug in many
parts of the world in recent years due to its psychosis-like effects
and cheap prices . In Hong Kong, ketamine abuse has
increased rapidly over the last decade and is the most abused
psychotropic substance. The most recent data from the Central
Registry of Drug Abuse (CRDA) indicated that 31.5% of all the
abused drugs are ketamine in Hong Kong in 2011 . Abusive use
of ketamine mainly cause mental problems, including anxiety,
confusion and memory loss , . In the United Kingdom, even
unexplained deaths related to ketamine use were reported and the
number of cases increased 10 folds from 1999 to 2008 .
Pharmacologically, ketamine modulates neurotransmission at
postsynaptic receptors such as N-methyl-D-aspartate (NMDA)
glutamate receptors and gamma-aminobutryic acid (GABA)
receptors. As an uncompetitive antagonist, ketamine blocks
NMDA receptor and induces a dissociative anesthesia .
Ketamine-induced anesthesia was also thought to work via
enhancing the GABA inhibitory neurotransmission in the central
nervous system (CNS) . Effects of ketamine on GABA inhibition
were further verified by the finding that ketamine potentiated
GABA(A) receptors expressed in Xenopus Oocyte at anesthetically
relevant or higher concentrations . In our previous study,
upregulated GABA(A) receptors were found in prolonged keta-
mine treated mice brain, suggesting that long-term ketamine
administration increased the GABA inhibitory transmission in the
Besides the gluatmate and GABA systems, Kapur and Seeman
 reported that ketamine exhibited a strong affinity with
dopamine (DA) D2receptors, indicating that DA system was also
modulated by ketamine. Several studies have demonstrated that a
single subanaesthetic dose of ketamine rapidly increased DA
release in the prefrontal cortex of rats while repeated ketamine
administration increased the basal DA levels, suggesting that there
could be a longer duration effects of repeated ketamine on DA
concentrations ,. Since DA neurotransmission was a part
of brain reward system, it was a main target of abused drugs .
However, little is known about the long-term effects of ketamine
on the DA system in the CNS.
In the CNS, DA concentration was largely determined by the
DA synthesis rate in dopaminergic neurons. DA was synthesized
by hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (L-
DOPA) by tyrosine hydroxylase (TH) and then L-DOPA was
decarboxylated to DA by dopa decarboxylase (DDC). Thereafter,
cytosolic DA was transported to synaptic vesicle by vesicular
monoamine transporter 2 (VMAT2). Inside the DA vesicles, some
of the DA was converted to norepinephrine by dopamine b-
hydroxylase (DBH). When the action potential was triggered,
catecholamine vesicles fused with cell membrane in the presence
of synaptosomal-associated protein 25 (SNAP25) and DA trans-
mission was initiated by release of DA into synaptic cleft . The
present study used dopaminergic PC 12 cells and chronic
ketamine treated mice to investigate the long-term effects of
ketamine on the DA system in vitro and in vivo. To this end, a fuller
PLOS ONE | www.plosone.org1 August 2012 | Volume 7 | Issue 8 | e43947
elucidation of gene expressions in the enzymes mentioned above is
clearly desirable in delineating the molecular changes of the DA
system following chronic ketamine use.
Materials and Methods
Cell culture and ketamine treatment
Rat pheochromocytoma 12 (PC12) cells were obtained from the
American Tissue Culture Collection (ATCC) and were grown in
Ham’s F-12K (Kaighn’s modifications, Invitrogen) medium
supplement with 10% heat-inactivated horse serum and 5% fetal
bovine serum. Cells were cultured in dishes precoated with 5%
poly-L-lysine and incubated in a humidified atmosphere at 37uC
and 5% CO2. Culture medium was changed every other day.
PC12 cells were treated with NGF (50 ng/ml) and allowed to
differentiate for 6 days . The differentiation medium was
changed every day. Both undifferentiated and differentiated PC12
cells were exposed to ketamine from 10 to 1000 mg/ml diluted in
medium for 24 hours. After treatment, the medium was collected
for dopamine determination while the cells were subjected to
MTT assays for cell viability or used for RNA extraction.
MTT assay was performed as previously reported . After
cells were treated as above mentioned, 100 ml of culture medium
containing 0.5 mg/ml Thiazoyl blue tetra-zolium bromide (MTT)
was added and incubating at 37uC for 4 hours. Then the medium
was aspirated off carefully without disturbing the crystal and
100 ul DMSO were added to each well. After 15 min incubation,
optical density (OD) was determined using an automatic microtiter
plate reader (Epson LX-800, Molecular Devices) at wavelength of
Animals and ketamine administration
All animal experiments were approved by the Animal Exper-
imentation Ethics Committee (AEEC) of the Chinese University of
Hong Kong (CUHK) and were performed under license of the
Department of Health, the Government of the Hong Kong SAR,
according to the Animals (Control of Experiments) Ordinance
Chapter 340(Animal License ID: (10–297) in DH/HA&P/8/2/1
Pt.13). ICR mice were housed in the Laboratory Animal Services
Center (LASEC) of the CUHK, with temperature at 22u,24uC
and humidity level of 45%,55% under 12-hour alternating light-
dark cycles. Standard diet (PicoLab Rodent Diet 20, PMI
Nutrition Inc., Henderson, USA) and water were given ad
libitum. Ketamine (Alfasan Inc., Utrecht, Holland) was given
through intraperitoneal injection daily at a sub-anesthetic dose of
30 mg/kg while controls received same volume of normal saline.
Body weights of the mice were measured weekly for dose
adjustment. After 3 months, mice were sacrificed by cervical
dislocation and the brain tissues were collected for subsequent
Dopamine concentrations in the brain tissues and cell culture
were evaluated using Dopamine Research ELISATM(BA E-5300,
Nordhorn, Germany) according to manufacturer’s instructions.
Following ketamine treatment, cell culture medium was measured
directly. For brain tissues, 50 mg of tissue were homogenized in
1 ml HCl (0.01 N) with EDTA (1 mM) and sodium metabisulfite
(4 mM). Under this condition, DA is positively charged and has the
optimizedsolubility.Thehomogenatewascentrifuged at15000 gat
4uC for 15 min and the supernatant was collected for measuring.
20 ml of standards and diluted sample were used for measurements.
The process included Cis-diol-specific affinity gel extraction,
acylation, derivatization enzymatically. Finally, DA was detected
by competitive ELISA. Results were obtained from microplate
reader set to 450 nm and a reference wavelength between 620 nm
and 650 nm. The concentrations of DA in the sample were
calculated according to the six standards from 0 to 90 ng. By using
the ELISA method, this kit provided very sensitive (lower limits at
0.7 ng/mL) and high throughput measurements of DA.
Quantitative real-time PCR (qRT-PCR) was performed as
previously described . Briefly, total RNA was isolated using
RNeasy Lipid Tissue Mini Kits (Qiagen Inc., Valencia, CA, USA)
according to the manufacturer’s manual. First strand cDNA
synthesis was performed by using a RT2First Strand Kit from
Qiagen in a 20 ml reaction volume. 50 ml of PCR volumes were
composed of 25 ml Power SYBRH Green PCR Master Mix kit
(Applied Biosystems, Foster City, CA, USA), 1 ml cDNA and 1 ul
primers (500 mmol/l). The primers of TH (59GAG TTT GAC
CCT GAC CTG GAC 39, 59CTC ACC CTG CTT GTA TTG
GAA39), DDC (59 CGC AAG TGA ATT CCG AAG GA 39, 59
ACC TGG CGT CCC TCA AT 39), SNAP25(59 CCA TCT CCC
TGT GGT TTG TCA 39, 59CAG CAA TTT GGT TGT GCA
TAG C 39), VMAT2(59 CGA GCA TCT CTT ATC TCA TTG
GA 39, 59 ATA GCC ACC TTC CCA TTT TGT G 39),
DBH(59ACC GGC TAC TGC ACA GAC AAG 39, 59 TCC TGC
CCG TCA GGT GTG T39) and b-Actin(59 AGG CCA ACC
GTG AAA AGA TG 39, 59 ACC AGA GGC ATA CAG GGA
CAA 39) were designed in Primer Premier 5.0 (Premier Biosoft
International, Palo Alto, California, USA). The amplifications were
performed in 96 well plates in a 7900HT Fast Real-Time PCR
System (Applied Biosystems, Foster City, CA, USA). Specificity of
amplification was confirmed by the melting curve with one peak in
PCR reactions.Gene expressionchanges between the ketamine and
saline groups were determined according to 2-Delta Delta Ctmethod
using b-Actin as endogenous control .
Brain tissues were homogenized in 300 ml RIPA lysis buffer
containing protease inhibitor cocktail (Millipore Inc., Billerica,
MA, USA). The homogenate was centrifuged at 14,000 g for
30 min at 4uC and the supernatants were collected for following
WB experiments. Protein concentration of the extracts was
determined by the Bio-Rad DC protein assay (Bio-Rad Labora-
tories Inc., Hercules, CA, USA). 50 mg of protein were loaded on
10% SDS-PAGE gel. Electrophoresis was performed at 100 Volt
(V) for 2 hours and followed by transferring onto PVDF
membrane at 200 mA for 60 min. The membrane was blocked
with 5% non-fat dry milk for 1 h at room temperature and then
incubated overnight at 4uC with f\primary antibodies: anti-BDNF
(1:1,000) (AB1779SP), anti-b-actin (1:20,000) (MAB1501). Both
antibodies were obtained from Millipore (Millipore, Billerica,
MA). In the following day, the membrane was washed with 0.05%
Tween-20 and phosphate buffered saline three times and then
incubated with the corresponding horseradish peroxidase (HRP)
conjugated secondary antibody (Dako Corporation) at room
temperature for 1 hour. Blots were then developed using ECL
Plus Kit (Millipore) on Fuji Medical X-ray film and scanned in a
Bio-Rad 6500 scanner. Optical density was obtained with
Quantity One software (Bio-Rad).
For immunostainning, all samples were fixed in 4% parafor-
maldehyde. The samples were then dehydrated in graded
Ketamine and Midbrain Dopaminergic Neurons
PLOS ONE | www.plosone.org2August 2012 | Volume 7 | Issue 8 | e43947
concentrations of ethanol, cleared in xylene, embedded in
paraffinwax and sectioned at 5 mm. To detect TH protein, brain
sections were deparaffinized, rehydrated, and endogenous perox-
idase were blocked with 10% hydrogen peroxide (H2O2) in
absolute methanol for 30 minutes. Sections were permeabilized
(antigen retrieval) with 1x phosphate buffered saline (PBS)
supplemented with 0.1% Triton-X and 0.05% Tween 20 for
10 min. After three rinses in 1 x PBS, non-specific binding was
suppressed by 1.5% normal blocking serum for 30 min. There-
after, the sections were incubated at 4uC overnight with rabbit
anti-Tyrosine Hydroxylase (AB152, Millipore, Billerica, MA)
diluted 1:2000 in blocking solution. On the next day, sections
were washed three times with 1 x PBS (5 min each) and incubated
with biotinylated secondary antibody (1:1000 diluted in blocking
solution) for 1 hour at room temperature. Subsequently, sections
were washed three times in 1 x PBS (5 min each) again and the
sections was incubated with streptavidin-HRP(1:1000 diluted in
PBS) for 1 hours. After three times of rinse with 1 x PBS, the
colors were developed by 0.05% 3939-diaminobenzidine tetrahy-
drochloride (DAB) in PBS containing 0.01% H2O2and finally the
sections were dehydrated and mounted.
Significance of differences between control and treatment
groups were compared by student’s t-test. Differences were
considered significant when p,0.05. For real-time PCR results,
the p values were calculated based on the replicates 2ˆ(- Delta Ct)
values for each gene in the control group and treatment group.
Calculations were done using SPSS software (version 15.0).
Dose-related effects of ketamine on dopamine
concentrations in PC12 and NGF differentiated PC12 (d-
PC 12 cells (Figure 1A) are dopaminergic cells and it can be
differentiated into neuronal cells with nerve growth factor (NGF),
which make it a useful model for neurobiological and neuro-
chemical studies . After 6 days of NGF treatment, PC12 cells
were differentiated into neuronal like cells, with round cell bodies
and abundant neurites (Figure 1B). Both the PC12 cells and d-
PC12 cells were used for testing the effect of ketamine. After
24 hours treatment, ketamine at 10 mg/ml or higher caused
significant decrease in cell viability in PC12 cells (p,0.05 as
compared with the control); Ketamine at 1000 mg/ml decreased
the cell viability to 34%63.5% of their control (p,0.005)
(Figure 1C). In d-PC12 cells, significant cell viability decreases
were found at 500 mg/ml or higher (p,0.05); Ketamine at
1000 mg/ml decreased the cell viability to 54%61.6%(p,0.005)
(Figure 1D). These results indicated that d-PC12 cells were less
susceptible to ketamine’s toxicity than undifferentiated PC12 cells.
To explore the dosage effects of ketamine on dopamine,
extracellular dopamine concentrations following 24 hours expo-
sure to different concentrations of ketamine were measured using
ELISA. It was found that ketamine increased dopamine levels in
PC12 cells in a dose-related pattern, with dopamine increase from
20.862.0 ng/ml (control) to 39.963.7 ng/ml (p,0.05) for10 mg/
ml ketamine, 42.664.4 ng/ml (p,0.05) for 100 mg/ml ketamine
and 67.965.6 ng/ml (p,0.005) for 500 mg/ml ketamine respec-
tively. Similar dose-related effects were also found in d-PC12 cells
(Figure 2). Dopamine concentrations reversely correlated with the
cell viability indicated that toxic effects of ketamine to the cells
might be related partially to dopamine-induced reactive oxygen
Long-term effects of ketamine on dopamine in mouse
To investigate the long-term effects of ketamine on dopamine
concentrations, dopamine levels were determined in 4 brain
regions (prefrontal cortex, striatum, midbrain and cerebellum) of
mice receiving ketamine for 3 months. In the saline (control)
group, the highest concentration of dopamine was found in
striatum (192.4625.0 ng/ml) and lowest dopamine in the
cerebellum (18.7610.8 ng/ml) (Figure 3). When compared with
the saline group, dopamine levels were found significantly
91.9610.5 ng/ml; keamine, 260.9610.5 ng/ml, p,0.05). In-
creased dopamine levels in brain suggested dopaminergic hyper-
activity following long-term ketamine treatment.
Effects of ketamine on mRNA levels of dopamine related
genes in vitro and in vivo
Expression of dopamine-relate genes (TH, DDC, SNAP25,
VMAT2 and DBH) were evaluated in ketamine treated PC12 cells
and d-PC12 cell, but no significant change was found (Figure 4A).
Tofurtherinvestigatethe long-termeffectsofketamine ondopamine
system, mRNA levels of dopmaine metabolism-related genes were
measured by real-time PCR in the PFC and midbrain. In the PFC,
measurements of VMAT2 and SNAP25, which were molecules
involved in dopamine release, were upregulated to1.960.5 folds
(p.0.05) and 1.260.03 folds (p,0.05) when compared with the
control respectively; TH was upregulated 1.860.9 folds (p.0.05) of
the control. In the midbrain, DA synthesis enzymes TH and DDC
were upregulated to 2.46 0.3 folds (p,0.05) and 1.860.4 folds
(p.0.05) while VMAT2 and SNAP25 were 1.260.3folds (p.0.05)
and 0.860.2 folds (p.0.05) respectively (Figure 4B).
Long-term effects of ketamine on BDNF protein levels in
Brain-derived neurotrophic factor (BDNF) is a widely distrib-
uted neurotrophin in the central nervous system. A recent study
showed that tyrosine hydroxylase (TH) gene transcription was
activated by BDNF through TrkB and the ERK/MAP kinase
pathway . To explore the mechanism of ketamine induced TH
up-regulation, BDNF protein levels were determined by western
blotting in PFC and midbrain. Representative immunoblots and
quantification results were shown in Figure 5. It was found that
BDNF protein levels were up-regulated in both brain regions
(PFC, 1.3860.12 folds, p=0.12; midbrain, 2.6360.24 folds,
p=0.005) after 3 months of ketamine treatment. These results
suggested that BDNF related pathway might play a role in the up-
regulation of TH in ketamine treated mice.
Increase of TH inmmureactive neurons in midbrain
following 3 months ketamine treatment
In TH immunostaining of sections from the midbrain of mice,
TH positive neurons were observed in retrorubral field (RRF) and
median raphe nucleus (MRN) (Fig.6 left). The section of midbrain
from ketamine treated mouse revealed increased numbers of TH
neurons in MRN and RRF (Figure 6A). Moreover, scattered
distributions of TH positive neurons were also observed in the
regions where TH inmmureactively was not present in the control
Ketamine, a glutamate receptor antagonist, is an anesthetic and
also a drug of abuse. Recent studies suggested that ketamine
Ketamine and Midbrain Dopaminergic Neurons
PLOS ONE | www.plosone.org3August 2012 | Volume 7 | Issue 8 | e43947
affected not only the glutamate and GABA system but also may
influence the dopamine system. In the present study, we examined
the effects of ketamine on the dopamine system in vitro and in vivo.
Employing dopaminergic PC12 cells, we found that acute
exposure to ketamine decreased cell viability and increased DA
efflux without altering related gene expressions. The increased
Figure 1. Cell viability of pheochromocytoma 12 (PC12) and differentiated PC12 cells following 24 hours ketamine treatment. A)
Phase contrast image of undifferentiated PC12 cells. B) Phase contrast image of PC12 cells differentiated with nerve growth factor (NGF) (50 ng/ml)
for 6 days. C) Dosage effects of ketamine on cell viability in undifferentiated PC12 cells. D) Dosage effects of ketamine on cell viability in differentiated
PC12 cells. Data are presented as Mean6 S.E.M. * p,0.05 as compared with control; **p,0.005 as compared with control.
Figure 2. Dosage effects of ketamine on dopamine in
pheochromocytoma 12 (PC12) cells and differentiated PC12
cells (d-PC12). PC12 cells and d-PC12 cells were exposed to ketamine
from 10 to 500 mg/ml for 24 hours. After the treatment, dopamine
concentrations were measured using ELISA. Data were presented as
Mean6 SEM (n=3). * p,0.05 as compared with control; **p,0.005 as
compared with control.
Figure 3. Different dopamine concentrations in mouse brain
following 3 months ketamine treatment. Ketamine (30 mg/kg)
were given to the mice for 3 months. Dopamine contents were
measured by ELISA. Significant dopamine level change was found in the
midbrain. PFC, prefrontal cortex. Sal, saline group; Ket, ketamine group;
Data were presented as Mean6 SEM (n=6). * p,0.05 as compared with
Ketamine and Midbrain Dopaminergic Neurons
PLOS ONE | www.plosone.org4 August 2012 | Volume 7 | Issue 8 | e43947
total DA levels were correlated with up-regulation of the DA
synthesis related enzymes in brain following long-term (3 months)
ketamine administration. These results lend support to the
hypothesis that long-term ketamine abuse lead to DA dysregula-
tion in the CNS.
In this study, DA concentrations in PC12 cells were found to be
reversely related with cell viability after ketamine treatment. As an
abusive drug, the toxic effects of ketamine have been well
documented in vitro , , . It was reported that ketamine
activated mitochondria apoptotic pathway and induced cell
apoptosis in cultured cortical neurons through NMDA receptor
NR1 subunit . Increased NMDA receptors were known to alter
the ion and water homeostasis in the cell and thus caused cell
membrane damage , which was the hallmark of necrosis. Cell
necrosis following ketamine treatment was confirmed by a marked
well as human hepatoma Hepg2 cells , .Thus, cell necrosis
might also contribute to ketamine’s toxic effects. PC12 cells were
dopminergic cells and loss of integrity in the plasma membrane
would lead to DA leakage without activation of Ca2+dependent DA
release. Besides, ketamine has also been reported to promote
spontaneous DA release, a process without extracellular Ca2+.
Ketamine at 10 ug/ml or higher in concentration caused significant
cell death in PC12 cells and corresponding increases of DA.
However, ketamine at lower concentration (10 ug/ml and 100 ug/
ml) did not cause significant cell death but still increased DA levels
in d-PC12 cells, indicating that ketamine promoting spontaneous
DA efflux might play a role. These results suggested that ketamine
induced DA efflux in vitro was caused, at least in part, by its
cytotoxicity which lead to damages to plasma membranes.
Figure 4. Effects of ketamine on dopamine-related genes in vitro and in vivo. A) Changes in dopamine-related genes in pheochromocytoma
12 (PC12) and differentiated PC12 (d-PC12) cells. Cells were exposed to ketamine (100 mg/ml) for 24 hours. mRNA levels were measured by real-time
PCR and fold changes were calculated using mRNA levels in ketamine group over saline group. B) Changes in dopamine-related genes in mouse brain
following 3 months ketamine treatment. DBH, dopamine beta-hydroxylase; DDC, dopa decarboxylase; SNAP25, synaptosomal-associated protein 25;
TH, Tyrosine hydroxylase; VMAT2, vesicular monoamine transporter 2; PFC, prefrontal cortex. Significant gene expression changes were found for
SNAP25 in PFC and TH in midbrain. Data were presented as Mean6 SEM (n=4). * p,0.05 as compared with control.
Ketamine and Midbrain Dopaminergic Neurons
PLOS ONE | www.plosone.org5 August 2012 | Volume 7 | Issue 8 | e43947
Although in vitro studies showed that ketamine promotes DA
release without activation of the dopaminergic transmitter
machinery, in vivo effects of ketamine on the DA system may be
more complex when taking neural circuit system into consider-
ation. Acute effects of ketamine on DA system mainly depend on
cortical-subcortical circuit connections. As is well known, most of
the DA in CNS is derived from projections of dopaminergic
neurons in the ventral tegmental area/substantia nigra (VTA/SN)
and raphe nucleus (RN), which are localized in the midbrain .
In vivo, activation of dopaminergic neurons in midbrain are
regulated by cortical glutamatergic projections through a facilita-
tory pathway and an inhibitory pathway. The facilitatory pathway
is mediated by NMDA and AMPA/kainate receptors while the
inhibitory pathway is controlled by NMDA receptor via GABA
interneurons . As an NMDA receptor antagonist, ketamine
not only down-regulates the inhibitory pathway but also activated
glutamate neurotransmission at AMPA/kainate receptors through
inducing glutamate efflux . Following even a single subanes-
thetic ketamine injection, midbrain DA neurons were activated by
increased net excitatory inputs and rapid DA efflux was evoked in
the PFC , . Moreover, recent study showed that these
cortical-subcortical glutamate connections were indispensable to
midbrain DA neuron activation since ketamine did not evoke DA
release in cortical slices when this connection was severed . As
an abusive drug, ketamine is used repeatedly by addicts and
whether there are long-lasting effects of ketamine on the DA
system or not need to be elucidated in vivo. We measured DA
content in brain tissues from long-term ketamine abused mice ,
 and noted a significant increase of DA concentration in the
midbrain and slightly (but not significantly) increases of DA
contents in the striatum and cerebellum after 3 months of
ketamine administration (Figure 3).
It is noteworthy that increased DA contents depended on up-
regulation of mRNA levels in DA related genes (Figure 4). TH is
Figure 5. Increase in Brain-derived neurotrophic factor (BDNF) protein expression in mouse brain following 3-month ketamine
administration. Sal, saline group; Ket, ketamine group; A) Representative immunoblots of BDNF and Actin. B) Quantification of the increase in BDNF
in ketamine treated mice brain. Data are mean 6 SEM (n=6). *P,0.05 as compared with control group.
Figure 6. Tyrosine hydroxylase (TH) immunostaining of sections from the midbrain of mice receiving ketamine for 3 months and
control mice receiving no ketamine. TH positive dopaminergic neurons were observed in the median ventral tegmental area (VTA) and raphe
nucleus (RN). In ketamine treated mouse, increased TH positive neurons were found in RN and other regions (right figure). Scale bars: 200 mm.
Ketamine and Midbrain Dopaminergic Neurons
PLOS ONE | www.plosone.org6 August 2012 | Volume 7 | Issue 8 | e43947
the rate limiting enzyme in DA biosynthesis and its activity plays
an important role in determining dopamine concentrations .
We observed that there was significant up-regulation of TH
expression (2.4 folds) in the midbrain following a period of 3
months of ketamine treatment. Up-regulation of TH expression
was also found in animals acutely or chronically treated with other
abusive drugs such as morphine and phencyclidine , .
However, our results in PC12 cells showed that there was no
difference of TH expression following ketamine treatment. Given
the difference in the results between in vitro/in vivo experiments,
repeated and intact neuronal circuit inputs modulating the activity
of midbrain DA neurons had to be considered to explain the long-
term effects of ketamine on TH expression in the animal model.
Subanesthetic doses of ketamine activated midbrain DA neurons
through evoking glutamate efflux and stimulating AMPA/kainate
receptors. Several studies have demonstrated that AMPA/kainate
receptor increased BDNF expression via either Ca2+singnaling
pathway or mitogen-activated protein kinase (MAPK) pathway
, . Available evidences indicate that neurotrophic factors
including glial cell-derived neurotrophic factor (GDNF) and
BDNF increase TH activity and DA synthesis , . In
western blotting study, we observed significant increases of BDNF
protein levels in midbrain, suggesting that BDNF pathways may
contribute to long-term ketamine induced TH upregulation.
In summary, besides the well-described transient effects of
ketamine on DA release, the present study indicated that long-
term ketamine abuse caused a more delayed and persistent
upregulation of subcortical DA system. Our data suggested the
possibility that upregulation of TH expression represented a
common molecular adaptation in the DA system. A better
understanding of these ketamine-mediated modifications of DA
transmission may lead to the direction of pharmacotherapies for
ketamine intoxications, and targeting the DA pathways could be
taken into consideration in devising therapeutic approaches for
chronic ketamine abusers.
Conceived and designed the experiments: DTY ST. Performed the
experiments: ST WPL. Analyzed the data: WHY MW. Contributed
reagents/materials/analysis tools: WPL. Wrote the paper: DTY ST.
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Ketamine and Midbrain Dopaminergic Neurons
PLOS ONE | www.plosone.org7August 2012 | Volume 7 | Issue 8 | e43947