13-cis-Retinoic Acid Alters Intracellular
Serotonin, Increases 5-HT1AReceptor, and
Serotonin Reuptake Transporter Levels
KALLY C. O’REILLY,* SIMON TRENT,? SARAH J. BAILEY,?,1AND MICHELLE A. LANE?,1,2
*Institute of Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712;
?Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY UK; and
?Department of Human Ecology, Division of Nutritional Sciences, The University of Texas at Austin,
Austin, Texas 78712
In addition to their established role in nervous system develop-
ment, vitamin A and related retinoids are emerging as regulators
of adult brain function. Accutane (13-cis-retinoic acid, isotreti-
noin) treatment has been reported to increase depression in
humans. Recently, we showed that chronic administration of 13-
cis-retinoic acid (13-cis-RA) to adolescent male mice increased
depression-related behaviors. Here, we have examined whether
13-cis-RA regulates components involved in serotonergic
neurotransmission in vitro. We used the RN46A-B14 cell line,
derived from rat embryonic raphe nuclei. This cell line
synthesizes serotonin (5-hydroxytryptamine, 5-HT) and ex-
presses the 5-HT1Areceptor and the serotonin reuptake trans-
porter (SERT). Cells were treated with 0, 2.5, or 10 lM 13-cis-RA
for 48 or 96 hrs, and the levels of 5-HT; its metabolite, 5-
hydroxyindoleacetic acid (5HIAA); 5-HT1Areceptor; and SERT
were determined. Treatment with 13-cis-RA for 96 hrs increased
the intracellular levels of 5-HT and tended to increase intra-
cellular 5HIAA levels. Furthermore, 48 hrs of treatment with 2.5
and 10 lM 13-cis-RA significantly increased 5-HT1Aprotein to
168.5 6 20.0% and 148.7 6 2.2% of control respectively. SERT
protein levels were significantly increased to 142.5 6 11.1% and
119.2 6 3.6% of control by 48 hrs of treatment with 2.5 and 10 lM
of 13-cis-RA respectively. Increases in both 5-HT1Areceptor and
SERT proteins may lead to decreased serotonin availability at
synapses. Such an effect of 13-cis-RA could contribute to the
increased depression-related behaviors we have shown in mice.
Exp Biol Med 232:1195–1203, 2007
Key words: 13-cis-retinoic acid; depression; serotonin; 5-HT1A
13-cis-Retinoic acid (13-cis-RA, isotretinoin), is the
active ingredient in Accutane, a medication prescribed for
severe acne. Administration of Accutane coincides with the
or not 13-cis-RA induces depression in human patients (2–5),
some case studies have reported that the onset of depressive
symptoms occurred after commencement of treatment and
were relieved following cessation of drug treatment (1).
Additionally,there are casereports ofrecurrenceofdepressive
symptoms after rechallenge with 13-cis-RA (1).
All-trans-retinoic acid (ATRA) is the endogenous
ligand for retinoic acid receptors (RAR) and is synthesized
from dietary vitamin A (retinol) (6). 13-cis-RA is a synthetic
retinoid capable of either binding to RAR itself (7) or being
isomerized to ATRA before RAR binding (8). RAR
heterodimerize with retinoid X receptors (RXR), and
together they bind to specific DNA sequences termed
retinoic acid response elements (RARE) (6). Binding of
ligand to the RAR initiates transcription (6), and this has led
to the view that retinoids are primarily regulators of gene
transcription, either directly or via intermediate genes. The
expression of many neuronal genes is affected by ATRA,
and a few have been reported to have functional RARE,
This work was supported by grant ES07784 from the National Institute of
Environmental Health Sciences (NIEHS) for neurotransmitter work performed by
Herng-Hsiang Lo at the University of Texas at Austin, Center for Research on
Environmental Diseases Analytical Instrumentation Facility Core. The work was
supported in part by grant ES09145 from the NIEHS-National Institutes of Health, by
training grant T32 ES007247 from the NIEHS to K.C.O., and by a grant for Fiscal
Year 2004–2005 from the University of Texas at Austin.
1These authors contributed equally to this work.
2To whom correspondence should be addressed at Department of Human Ecology,
Division of Nutritional Sciences, Gearing Hall Room 115, Mail Stop A2700, The
University of Texas at Austin, Austin, TX 78712. E-mail: firstname.lastname@example.org
Received March 30, 2007.
Accepted June 14, 2007.
Copyright ? 2007 by the Society for Experimental Biology and Medicine
suggesting that retinoids act as transcriptional regulators in
neuronal cells [for review see (9)]. However, retinoids can
also influence cellular function by altering protein levels via
nontranscriptional mechanisms, such as increasing mRNA
(10, 11) or protein stability (12).
Although the role of retinoids in development is widely
studied, over the past few years, reports of 13-cis-RA
actions in the adult brain have emerged. In human patients
treated with 13-cis-RA, brain imaging studies have shown a
decrease in orbital frontal cortex metabolism (13). In
addition, in mice, 13-cis-RA administration has been shown
to impair learning and memory (14) and increase depres-
sion-related behaviors (15). In the latter study, conducted by
our research group, adolescent, DBA/2J mice that were
treated with 1 mg/kg/day of 13-cis-RA, the same dose
prescribed to human patients, exhibited significantly more
immobility than vehicle-treated controls in both the tail
suspension test and the forced swim test (15). Further
analysis of data in the forced swim test revealed that the
increase in immobility was accompanied by a decrease in
swimming, but not climbing, behavior (15). For rodents, in
the forced swim test, antidepressants that target the
serotonergic system decrease immobility because of an
increase in swimming time (16). In contrast, antidepressants
that target the noradrenergic system, decrease immobility
and increase time spent climbing (16).
Although the etiology of depression is still unknown,
the serotonergic system has long been a target of
antidepressant drugs (17). Serotonin (5-hydroxytryptamine,
5-HT) reuptake and metabolism are targets of antidepres-
sants, with the net result being increased levels of 5-HT in
the synaptic cleft. Intracellular levels of 5-HT are controlled
by 5-HT synthesis from tryptophan, 5-HT degradation to 5-
hydroxyindole acetic acid (5HIAA), vesicular packaging
and release of 5-HT into the synapse, and reuptake of 5-HT
into the cell by the serotonin reuptake transporter (SERT or
5-HTT). An additional regulator of 5-HT release is the
somatodendritic serotonin receptor 1A (5-HT1A). This
autoreceptor is activated by 5-HT to inhibit firing of
serotonergic raphe neurons (18). The role of 5-HT in
human depression is supported by studies showing
decreased plasma tryptophan levels, reduced 5HIAA levels,
decreased 5-HT uptake and polymorphisms in 5-HT1A
receptor (19) and SERT (20–22).
The ability of 13-cis-RA to cause depression-related
behavior in adolescent mice and the ability of retinoids to
affect neuronal processes and organismal behavior led us to
hypothesize that serotonergic function may be altered in the
raphe nuclei in response to 13-cis-RA treatment. In this study,
as our model. These cells are serotonergic and express the 5-
HT1Areceptor and SERT (23, 24). We show that treatment of
RN46A-B14 cells with 13-cis-RA leads to increased intra-
cellular 5-HT content. Intracellular 5HIAA content also tends
to be increased. In addition, both the 5-HT1Areceptor and
SERT are increased following 13-cis-RA treatment.
Materials and Methods
Tissue Culture. RN46A-B14 cells (a gift from Dr.
Scott Whittemore, University of Louisville, Louisville, KY)
were used to investigate the effects of 13-cis-RA on
components of the serotonergic system. The RN46A parent
cell line was isolated from embryonic Day 13 rat medullary
raphe nuclei and immortalized with a SV40 large T antigen
(25). The RN46A-B14 cell line is a stably transfected
RN46A cell line that expresses full-length human brain–
derived neurotrophic factor (BDNF), required for seroto-
nergic differentiation (24). RN46A-B14 cells were main-
tained in Dulbecco’s modified Eagle’s medium (DMEM)/
F12 medium containing 10% fetal bovine serum (FBS) and
antibiotics (1000 units/ml penicillin and 1000 lg/ml
streptomycin) at 338C. Before differentiation, cells were
plated at a density of 4 3 106cells/plate in 100-mm dishes
and allowed to grow for 2–3 days until reaching
approximately 70% confluency. Cells were differentiated
as described (24) by incubation at 398C in DMEM/F12
medium containing 1% FBS, antibiotics (1000 units/ml
penicillin and 1000 lg/ml streptomycin), 5 lg/ml insulin, 20
nM progesterone, 100 lM putrescine, 1 lg/ml transferrin,
and 1% ovalbumin (w/v). Medium was changed every day
for the first 4 days, and on Day 4, 40 mM potassium chloride
(KCl) were added. Depolarization with KCl enhances the
serotoninergic phenotype of these cells (23, 25). Medium
was then changed every 48 hrs and contained 40 mM KCl
for the duration of the experiment. After 8 days at 398C,
differentiated cells were treated with 0, 2.5, or 10 lM 13-
cis-RA dissolved in an ethanol vehicle for 48 or 96 hrs. The
Accutane package insert reports that the steady-state plasma
concentration of 13-cis-RA ranges from a minimum (mean
6 SD) of 352.32 6 184.44 ng/ml (1.2 lM) to a maximum
of 731.98 6 361.86 ng/ml (2.5 lM). Thus, the 2.5 lM
concentration of 13-cis-RA was chosen to reflect the
average steady-state concentration of 13-cis-RA, and the
10 lM concentration of 13-cis-RA may be reached soon
after 13-cis-RA administration. All cells, including control,
were treated with an equal amount of vehicle. All retinoid
manipulations were performed under subdued light.
High-Performance Liquid Chromatography
(HPLC) Analysis of Intracellular 5-HT and 5HIAA
Content. Isocratic HPLC with electrochemical detection
was used to examine the effect of 13-cis-RA on 5-HT
production in RN46A-B14 cells. Cells were grown and
differentiated as described above. Although the medium
contained 66 lM tryptophan, to ensure adequate levels of 5-
HT for detection by HPLC analysis after 48 or 96 hrs of 13-
cis-RA treatment, cells were incubated with 10 lM
tryptophan and 10 lM chlorgyline for 30 mins and then
with 10 lM fluoxetine for an additional 30 mins as
described (23). Tryptophan is the precursor for 5-HT
synthesis, chlorgyline inhibits monoamine oxidase A, and
fluoxetine blocks SERT and, therefore, 5-HT entry into the
cell, thereby allowing for detection of 5-HT. Cells were left
1196O’REILLY ET AL
attached to the plate and washed twice with phosphate-
buffered saline (PBS) and lysed by freezing at ?808C. The
next day, 200 ll of e-pure water was added to the plate, the
plate was scraped, and the lysed cells were harvested,
transferred to an eppendorf tube, and centrifuged at 13,000 g
for 10 mins. The supernatant was stored at ?208C until
analysis. The protein content of each sample was deter-
mined using a portion of the supernatant and the BioRad
(Hercules, CA) DC protein assay kit.
5-HT and 5HIAA levels in the lysate were determined in
the College of Pharmacy Analytical Instrumentation Facility
Core (University of Texas at Austin, Austin, TX) by HPLC
electrochemical method modified from that of Bai et al. (26).
Briefly, the samples (60–100 ll) were injected into the
HPLC system, which consisted of a Shimadzu SCL-10A
system controller and a LC-10AD pump equipped with a
SIL-10A auto-sampler (Shimadzu, Columbia, MD), coupled
to a four-channel CoulArray electrochemical detector (ESA
Biosciences Inc., Chelmsford, MA). The isocratic mobile
phase consisted of 4 mM citrate, 8 mM ammonium acetate,
20 mg/l EDTA, 120 lM 1-octanesulfonic acid sodium salt
(SOS) at pH 3.5, and 5% methanol. 5-HT and 5HIAA were
separated by a 4.6 3 80 mm reverse-phase HR-80, 3-lm
particle-size, 120-A˚, column (ESA Biosciences) at a flow
rate of 1 ml/min and analyzed by electrochemical detection
using a CoulArray electrochemical detector (ESA Bioscien-
ces). The potential of Channels 1–4 of the CoulArray were
set at?50, 0, 300, and 400 mV, respectively (Fig. 1A). Peak
area (nC) of 5-HT or 5HIAA at the corresponding retention
time on the chromatogram resulting from 300 mV was
obtained using CoulArray for Windows, version 1.12
software (ESA Biosciences). The peak area was used to
quantify the 5-HT or 5HIAA in each sample injected, based
on the standard curve. The total 5-HT or 5HIAA concen-
tration was corrected for sample volume loaded and
normalized to protein content.
Northern Blot Analysis of 5-HT1A and SERT
mRNA. For probe generation, total RNA was isolated from
differentiated, vehicle control-treated RN46A-B14 cells or
adult rat hippocampus with RNA Stat-60 (Tel-Test,
Friendswood, TX). Two micrograms of RN46A-B14 or
rat hippocampal total RNA were reverse-transcribed with
oligo-dT primers and the Reverse Transcription System
(Promega, Madison, WI) per manufacturer’s instructions.
Amplification of the 5-HT1A and GAPDH cDNA were
performed using RN46A-B14 cDNA and the following
primers: 5-HT1Aforward primer, 59-AGC ATC TCC GAC
GTG ACC TTC AGC TAC CA-39; reverse primer, 59-GCT
CCC TTC TTT TCC ACC TTC CTG ACA GT-39, resulting
in a 635-bp product (27). Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) forward primer, 59-CGT CTT
CAC CAC CAT GGA GA-39; reverse primer, 59-CGG
CCA TCA CGC CAC AGT TT-39, resulting in a 260-bp
product (28). Amplification of SERT cDNA was performed
using hippocampal cDNA and the following primers. SERT
forward primer, 59-TGA CCA GCA GCA TGG AGA
CC-39; reverse primer, 59-CCA CGG CAT AGC CAA TGA
C-39, resulting in a 304-bp product (29). 5-HT1A, GAPDH,
and SERT cDNA were amplified using Taq polymerase
(New England Biolabs Inc., Ipswich, MA) per manufac-
turer’s instructions under the following polymerase chain
reaction (PCR) conditions: 958C for 5 mins followed by 50
cycles of 958C for 30 secs, 608C for 30 secs, and 728C for
30 secs, with a final extension period of 7 mins at 728C.
PCR reactions were then electrophoresed in a 1.8% agarose
gel and products of the appropriate size were purified using
the Qiagen Gel Extraction kit (Qiagen, Valencia, CA) per
the manufacturer’s instructions. All products were se-
quenced to confirm their identity.
Total RNA was isolated from cells differentiated and
treated with 0, 2.5, or 10 lM 13-cis-RA for 48 or 96 hrs as
described above using RNA Stat-60 (Tel-Test). RNA was
electrophoresed through 1.2% agarose/2.2 M formamide
gels, transferred to nylon filters, and cross-linked to the
filters with a UV-Stratalinker (Stratagene) as described (30).
The cDNA probes for 5-HT1A, SERT, and GAPDH were
labeled with [a-32P]dCTP using the Random Primed DNA
Labeling Kit (Roche Applied Science, Indianapolis, IN) per
the manufacturer’s instructions. Membranes were prehybri-
dized overnight at 428C in hybridization solution [50% (w/
v) deionized formamide/0.2% bovine serum albumin
(BSA)/0.2% polyvinyl pyrrolidone/2% Ficoll (molecular
weight 400,000 g/mole)/50 mM Tris?HCl, pH 7.5/0.1%
sodium pyrophosphate/1% SDS/10% dextran sulfate/100
lg/ml salmon sperm DNA] and then hybridized overnight at
428C in hybridization solution containing radiolabeled
probe. After hybridization, the membranes were washed
and processed as described previously (30). The membranes
were then exposed to film for 24 hrs. All membranes were
stripped and re-hybridized with GAPDH to control for
loading differences. Autoradiographs were quantitated using
a BioRad Gel Documentation System.
Western Blot Analysis of 5-HT1A and SERT
Protein. The effect of 13-cis-RA on 5-HT1Aand SERT
protein levels in serotonergic cells was examined using
semiquantitative Western blot analysis. RN46A-B14 cells
were differentiated as described above and treated with 13-
cis-RA for 48 or 96 hrs. For 5-HT1Aand SERT analyses,
cells were lysed in radioimmunoprecipitation (RIPA) buffer
(150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1%
sodium dodecyl sulfate [SDS], 50 mM Tris, 1 mg/ml
leupeptin, 1 mM dithiothreitol [DTT], 2 mM sodium
orthovanadate, 1 mg/ml phenylmethylsulfonyl fluoride
[PMSF], 1 mg/ml trypsin inhibitor, and 10 mM aprotinin)
and equal amounts of protein (50 lg for 5-HT1Aor 75 lg
for SERT) were electrophoresed on 12% (5-HT1A) or 10%
(SERT) sodium dodecyl sulfate–polyacrylamide gel electro-
phoresis (SDS-PAGE) gels. Protein was quantitated using
BioRad DC protein assay kit. After electrophoresis, protein
was transferred to a nitrocellulose membrane.
For analysis of 5-HT1A levels, the blots were
prehybridized with 5% milk in Tris-buffered saline with
13-CIS-RA AND SEROTONERGIC FUNCTION1197
Tween-20 (TBST; 10 mM Tris, pH 8, 150 mM NaCl, and
0.5% Tween-20) for 1 hr at room temperature before
overnight incubation at 48C with 5-HT1Apolyclonal anti-
body (catalogue sc-10801; Santa Cruz Biotechnology Inc.,
Santa Cruz, CA) at a 1:300 dilution. For analysis of SERT
levels, blots were prehybridized with 5% milk in TBST
before incubation with a 1:200 dilution of polyclonal anti-
SERT antibody (catalogue sc-1458; Santa Cruz Biotechnol-
ogy) in 1% BSA in TBST at 48C overnight. Secondary
antibodies were goat anti-rabbit (catalogue #0031460,
Pierce, Rockford, Il) or bovine anti-goat (catalogue #sc-
2350; Santa Cruz Biotechnology Inc.), for 5-HT1Aor SERT
respectively, and were incubated in 5% milk for one hour at
room temperature. Immunoreactivity was detected using the
Horseradish Peroxidase Super Signal West Pico Chemilu-
minescent Substrate kit (Pierce, Rockford, IL). The
membranes were then stripped and re-probed with poly-
clonal b-actin antibody (catalogue A2066; Sigma, St. Louis,
MO) at a 1:10,000 dilution to control for differences in
loading. Densitometry was performed using a BioRad Gel
RT-PCR Analysis of RAR/RXR Expression.
RN46A-B14 cells were grown and differentiated for 8 days
as described, and on the Day 8, total RNA was extracted
from RN46A-B14 cells as described above. DNA was
removed by DNase digestion and total RNA (1 lg) was
reverse-transcribed with oligo-dT primers and the Reverse
Transcription System (Promega) per manufacturer’s in-
structions. cDNA was then subjected to 40 PCR cycles at a
melting temperature (Tm) of 608C, with the primers listed
below. For adult rat dorsal raphe tissue, total RNA was
extracted using Trizol reagent (Invitrogen, Carlsbad, CA).
DNA was removed by means of a DNase digest and total
RNA (0.25 lg) was reverse-transcribed using RT/ Platinum
Taq Mix (Superscript One-Step RT-PCR with Platinum
Taq, Invitrogen) per manufacturer’s instructions, with the
gene-specific primers listed. Amplification of RARa,
RARb, RARc, RXRa, and RXRb/c cDNA were achieved
using the following primers: RARa forward: 59-CTG GAG
ATG GAC GAT GCT GAG ACT-39, reverse: 59-CAC
AGA TGA GGC AGA TGG CAC TGA-39, resulting in a
54-bp product (31); RARb forward: 59-CAA AGC CTG
CCT CAG TGG ATT CA-39, reverse: 59-AGT GGT AGC
CCG ATG ACT TGT CCT-39, resulting in a 178-bp
product (31); RARc forward: 59-GGA ACT CAT CAC
CAA GGT CAG CAA-39, reverse: 59-CGC TTC GCA
AAC TCC ACA ATC TT-39 resulting in a 175-bp product
(31); RXRa forward: 59-CTT TGA CAG GGT GCT AAC
AGA GC-39, reverse: 59-ACG CTT CTA GTG ACG CAT
ACA CC-39, resulting in a 172-bp product (32); RXRb/c
forward: 59-AGG CAG GTT TGC CAA GCT TCT G-39,
reverse: 59-GGA GTG TCT CCA ATG AGC TTG A-39,
resulting in a 102-bp product (33).
Statistical Analyses. Statistical analyses were per-
formed using Excel (XP 2002; Microsoft, Redmond, WA).
Two-tailed, Student’s t tests were performed to test for
Figure 1. Effect of 13-cis-RA treatment on intracellular 5-HT and
5HIAA levels in cultured serotonergic cells. Representative HPLC
tracing (A) for cell lysate and (B) 1 pmol standard. 5-HT and 5HIAA
retention times are indicated. (C and D) Intracellular 5-HT and 5HIAA
content. Cells were differentiated for 8 days before treatment with 0,
2.5, or 10 lM 13-cis-RA for 48 or 96 hrs. Before harvesting, cells were
incubated with 10 lM tryptophan and 10 lM chlorgyline for 30 mins,
then 10 lM fluoxetine was added for another 30 mins. Cells were
lysed and 5-HT and 5HIAA content were detected electrochemically
viaHPLC. Resultsshown in(C) 5-HTand (D)5HIAA aremean6 SEM
for four separate experiments. Statistical analysis was performed
using t tests comparing each 13-cis-RA concentration to control.
1198 O’REILLY ET AL
differences between vehicle control and 13-cis-RA treat-
ments. Data are expressed as mean 6 SEM, n ¼ 3, unless
otherwise indicated, and differences were considered
significant at P , 0.05.
Effect of 13-cis-RA Treatment on Intracellular 5-
HT and 5HIAA Levels. HPLC was used to determine the
effect of 13-cis-RA treatment on intracellular 5-HT levels in
RN46A-B14 cells. The levels of 5HIAA, the primary
metabolite of 5-HT, were also determined. 5-HT and
5HIAA peak retention times reflect their respective stand-
ards at 300 mV (Fig. 1A and B). We found that 48 hrs of 2.5
lM 13-cis-RA treatment did not increase intracellular 5-HT
(Fig. 1C) or 5HIAA (Fig. 1D) levels in RN46A-B14 cells.
However, treatment with 10 lM 13-cis-RA for 48 hrs
decreased (P ¼ 0.01) intracellular 5-HT levels to 77.8 6
6.2% of control (Fig. 1C) but had no effect on 5HIAA (Fig.
1D). Extending treatment for 96 hrs with 10 lM 13-cis-RA
significantly increased the intracellular 5-HT concentration
to 206.9 6 37.1% of vehicle control (P ¼ 0.03) (Fig. 1C).
Both 2.5 and 10 lM 13-cis-RA tended to increase
intracellular 5HIAA levels to 132.4 6 18.8% (P ¼ 0.14)
and 181.7 6 38.1% of vehicle control (P ¼ 0.07),
respectively, after 96 hrs of treatment (Fig. 1D). These data
indicate that prolonged treatment of RN46A-B14 cells with
13-cis-RA tends to increase intracellular 5-HT and 5HIAA
Effect of 13-cis-RA Treatment on 5-HT1AmRNA
and Protein Levels. Because retinoids are well known
for their ability to induce gene transcription (6), Northern
blot analysis was used to examine the effect of 13-cis-RA on
5-HT1A mRNA levels in RN46A-B14 cells. 13-cis-RA
treatment tended (P ¼ 0.06) to slightly decrease 5-HT1A
mRNA levels (91.2 6 3.8%) after 48 hrs of treatment with
2.5 lM 13-cis-RA (Fig. 2A). 10 lM 13-cis-RA treatment
had no effect on 5-HT1AmRNA levels after 48 hrs (93.9 6
5.5% of control), and there were no differences in 5-HT1A
mRNA levels due to either 2.5 or 10 lM 13-cis-RA
treatment after 96 hrs (104.9 6 5.6% and 104.9 6 5.8% of
vehicle control, respectively, Fig. 2A).
To determine the effect of 13-cis-RA on 5-HT1A
protein levels in RN46A-B14 cells, semiquantitative West-
ern blotting was performed. Treatment of RN46A-B14 cells
with 2.5 lM and 10 lM 13-cis-RA for 48 hrs significantly
increased 5-HT1Alevels to 168.5 6 20.0% and 148.7 6
2.2% of control, respectively (Fig. 2B). This increase was
maintained after 96 hrs of 13-cis-RA treatment. Thus,
treatment of serotonergic cells with 13-cis-RA increases 5-
HT1A protein levels as early as 48 hrs after drug
administration but does not affect 5-HT1AmRNA levels.
Effect of 13-cis-RA Treatment on SERT mRNA
and Protein Levels. Northern blot analysis was also used
to determine the effect of 13-cis-RA treatment on SERT
mRNA levels. Treatment of RN46A-B14 cells with 2.5 or
10 lM 13-cis-RA for 48 hrs did not affect SERT mRNA
levels (Fig. 3A). In contrast, 96 hrs of treatment with 10 lM
13-cis-RA tended (P ¼ 0.10) to increase SERT mRNA
levels to 144.8 6 20.7% of vehicle control. In addition to
the increase in mRNA levels, semiquantitative Western
blotting revealed that SERT protein levels were significantly
increased after treatment with 2.5 lM and 10 lM 13-cis-RA
for 48 hrs, reaching 142.5 6 11.1% and 119.2 6 3.6% of
vehicle control, respectively (Fig. 3B). SERT protein levels
tended to remain elevated after 96 hrs of treatment with 13-
cis-RA (Fig. 3B). Therefore, treatment of serotonergic cells
with 13-cis-RA leads to increased levels of SERT protein as
early as 48 hrs after treatment and later to an increase in
RAR and RXR Expression. RAR and RXR mediate
gene transcription in response to retinoic acid. Therefore,
RT-PCR was used to examine RAR and RXR expression in
RN46A-B14 cells and adult rat dorsal raphe tissue. RARa, b,
and c, as well as the RXRa and b/c isoforms, were expressed
in both RN46A-B14 cells and the raphe nuclei (Fig. 4),
indicating that retinoid-mediated gene transcription via
RAR/RXR/RARE can occur. In RN46A-B14 cells, the
RARa expression level may be lower than RARb and c.
However, this RT-PCR method is not quantitative.
We show here that prolonged treatment with 13-cis-RA
increases intracellular 5-HT and 5HIAA levels, although the
effect of 13-cis-RA on 5HIAA levels was not significant.
Additionally, 13-cis-RA treatment increased 5-HT1Arecep-
tor and SERT protein levels, although it had little effect on
their mRNA levels. Previously, we showed that 13-cis-RA
administration induced depression-related behavior in
adolescent male mice (15). Disturbances in the serotonergic
system are known to be involved in depression, including
altered 5-HT availability and changes in expression of the 5-
HT1A autoreceptor and SERT (22). An increase in
expression of the 5-HT1Aautoreceptor and SERT levels in
the raphe nuclei following 13-cis-RA treatment may lead to
decreased serotonergic availability at the synapse and thus
contribute to the increase in depression-related behavior
observed in vivo (15).
We examined whether 13-cis-RA alters intracellular 5-
HT levels or the levels of its metabolite, 5HIAA and saw
that 13-cis-RA increased 5-HT and tended to increase
5HIAA in vitro. The increase in intracellular 5HIAA
parallels the increase in intracellular 5-HT; thus, we suspect
the increase in intracellular 5HIAA is due to the increase in
intracellular 5-HT levels and occurred before chlorgyline
treatment. Ferguson et al. (34) examined the effects of 13-
cis-RA treatment on monoaminergic systems in adult rats.
Although they found no effect on 5-HT or 5HIAA content
in brain tissue homogenates of either hippocampus or
frontal cortex, 5HIAA levels in the striatum were increased
in male rats administered 13-cis-RA. These data indicate
13-CIS-RA AND SEROTONERGIC FUNCTION 1199
that there are likely to be brain region–specific effects of 13-
cis-RA on 5-HT/5HIAA levels. Ferguson et al. (34) did not
examine the raphe nuclei, and thus, it remains possible that
13-cis-RA will cause an increase in 5-HT and 5HIAA in the
raphe in vivo. Although we did not examine release,
recycling, or reuptake of 5-HT in this study, we speculate
that increased levels of intracellular 5-HT due to 13-cis-RA
treatment could occur because of increased reuptake. In
these experiments, we pretreated the cells with fluoxetine
before HPLC analysis, this SERT inhibitor was not added
until we had preloaded the cells with tryptophan for 5-HT
synthesis. Therefore, greater 5-HT reuptake may be due to
the ability of 13-cis-RA to increase SERT protein levels.
Alternatively, increased intracellular 5-HT levels could
result from increased 5-HT synthesis due to the effects of
13-cis-RA on the levels of synthetic enzymes such as
tryptophan hydroxylase or amino acid decarboxylase.
Although retinoids are known for their ability to alter
gene transcription when binding to RAR/RXR heterodimers
on RARE, other roles for retinoids are becoming evident.
ATRA has been shown to affect mRNA stability. Although
the mechanism is not completely understood, ATRA can
reduce tumor necrosis factor a (TNF-a) mRNA stability in a
RXR-mediated manner in hepatocytes (10), increase keratin
19 mRNA stability in cultured keratinocytes (11), and
increase protein stability in P19 cells (12). Adult raphe and
RN46A-B14 cells express RARa/b/c and RXRa/b/c (Fig.
4) indicating presence of the cellular mechanisms for
retinoid receptor-mediated gene transcription. We observed
an increase in 5-HT1Aprotein after 48 and 96 hrs treatment
with 13-cis-RA without an increase in mRNA. It is possible
that the increase in 5-HT1Aprotein level in response to 13-
cis-RA treatment is due to increased translation of 5-HT1A
mRNA or increased stability of the 5-HT1A protein. In
contrast, we observed an increase in SERT mRNA after 96
hrs of 13-cis-RA treatment. This may reflect an increase in
stability of the mRNA, but given the delay in increase of
mRNA with treatment, it is possible that the SERT gene is
not directly regulated by 13-cis-RA. Instead, perhaps an
intermediate gene, such as a transcription factor or a
derepressor element that regulates SERT expression, is
transcriptionally activated by 13-cis-RA. Because SERT
protein, but not mRNA levels, are changed after 48 hrs of
treatment, 13-cis-RA treatment, in RN46A-B14 cells, may
initially increase SERT protein stability or mRNA trans-
lation. Increased transcription of early response genes that
are transcription factors for SERT would then lead to the
increase seen in SERT mRNA at 96 hrs and thus be the
underlying cause of later elevated levels of SERT protein.
Functionally, the 5-HT1Aautoreceptor is involved in
regulation of serotonergic neuron firing (18). Activation of
5-HT1A autoreceptors residing on raphe nuclei by 5-HT
results in reduced firing of the raphe nuclei and therefore the
amount of serotonin in the synaptic cleft. Both increased
and decreased expression of the 5-HT1Areceptor has been
reported in depressed and suicidal patients (22). In addition,
polymorphisms in the human 5-HT1Apromoter region have
been linked to depression in some (19), but not all, patients
(35). Interestingly, 5-HT1A-null mice exhibit a decrease in
immobility in both the forced swim test and the tail
suspension test, consistent with an antidepressant-like effect
Figure 2. Effect of 13-cis-RA on 5-HT1AmRNA and 5-HT1Aprotein
levels. Cells were differentiated for 8 days and then cultured with 0,
2.5, or 10 lM 13-cis-RA for 48 or 96 hrs. (A) Total RNA was
harvested and subjected to Northern blot analysis for 5-HT1Aand
GAPDH mRNA (1269 and 1307 bp, respectively) as described in
Materials and Methods. GAPDH was used to correct for loading
differences. Northern blot analysis was repeated four separate times
with similar results; a representative Northern blot is shown. (B) Total
protein was harvested and subjected to Western blot analysis for 5-
HT1A and b-actin (30 and 42 kDa respectively) as described in
Materials and Methods. b-Actin was used to correct for loading
differences. Western blot analysis was performed three separate
times with similar results; a representative Western blot is shown.
Values reported are mean 6 SEM. Statistical analysis was
performed using t tests comparing each 13-cis-RA concentration to
control. * P , 0.05, ** P , 0.01; significantly different from control.
1200O’REILLY ET AL
in the tail suspension test (36). In these knockout mice, there
is an increase in basal firing of serotonergic neurons (37)
that is accompanied by enhanced extracellular 5-HT release
in vivo in hippocampus and frontal cortex (38) but not in
striatum (39). Ultimately, increases in somatodendritic
autoinhibitory 5-HT1Alevels may reduce serotonergic cell
firing and thereby decrease serotonin signaling.
Abnormalities in SERT expression may also contribute
to depression (22). SERT removes 5-HT from synaptic cleft
to regulate serotonin signaling. Two functional polymor-
phisms in the human SERT gene are associated with
increased vulnerability to depression and to affect the
response to antidepressants (40). These polymorphisms of
the SERT gene are associated with reduced SERT
expression, reduced 5-HT reuptake rate (20, 41, 42) and
increased susceptibility to depression (20, 21, 43). Also,
SERT-null mice exhibit decreased immobility in the tail
suspension test (44) and siRNA knockdown of SERT in
adult mice reduced time spent immobile in the forced swim
test (45). Interestingly, SERT knockout mice exhibit a gene
dose-dependent decrease in SERT protein levels, a decrease
in 5-HT uptake, and an increase in extracellular 5-HT levels
(46–48). Because the function of SERT is to remove 5-HT
from the synaptic cleft, both increases and decreases in
SERT may disrupt serotonin signaling, affecting down-
stream neuronal targets, which may eventually result in
Given that the roles of the presynaptic 5-HT1Areceptor
and SERT are to regulate serotonin signaling, increases of
Figure 3. Effect of 13-cis-RA treatment on SERT mRNA and protein
levels. Cells were differentiated for 8 days and then cultured with 0,
2.5, or 10 lM 13-cis-RA for 48 or 96 hrs. (A) Total RNA was
harvested for northern blot analysis of SERT and GAPDH mRNA
(3190 and 1307 bp, respectively) as described in Materials and
Methods. GAPDH was used to demonstrate equal loading. (B) Total
protein was harvested and subjected to western blot analysis for
SERT and b-actin (75 and 42 kDa respectively) as described in
Materials and Methods. b-Actin was used to demonstrate equal
loading. These experiments were performed three separate times
with similar results; representative Western and Northern blots are
shown. Results are mean 6 SEM for three separate experiments.
Statistical analysis was done using t tests comparing each 13-cis-RA
concentration to control. * P , 0.05, ** P , 0.01; significantly
different from control.
Figure 4. Expression of RAR and RXR in RN46A-B14 cells and rat
raphe tissue. (A) RN46A-B14 cells were differentiated for 8 days, and
total RNA was reverse transcribed. cDNA was then amplified with
primers specific for RAR and RXR. (B) Total RNA from rat raphe
tissue was reverse-transcribed in a one-step RT-PCR with the same
primers for RAR and RXR. All isoforms of RAR and RXR were
expressed in RN46A-B14 cells and rat raphe tissue. The RARa,
RARb, RARc, PCR amplicons were 54, 178, and 175 bp in length.
The RXRa and RXRb/c amplicons were 172 and 102 bp in length.
13-CIS-RA AND SEROTONERGIC FUNCTION1201
the 5-HT1Apresynaptic-receptor and SERT because of 13-
cis-RA treatment may inhibit firing and reduce serotonin
signaling from the raphe to other brain regions. Such
impairment in serotonergic neurotransmission could con-
tribute to the 13-cis-RA-induced increase in depression-
related behaviors observed in mice.
We thank Dr. Scott Whittemore, University of Louisville, Louisville,
Kentucky. who generously provided the RN46A-B14 cells. Neurotrans-
mitter HPLC was performed by Dr. Herng-Hsiang Lo in the University of
Texas at Austin CRED Analytical Instrumentation Facility Core.
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