Mice Genetically Depleted of Brain Serotonin Do Not Display a Depression-like Behavioral Phenotype

Abstract

Reductions in function within the serotonin (5HT) neuronal system have long been proposed as etiological factors in depression. Serotonin selective reuptake inhibitors (SSRIs) are the most common treatment for depression and their therapeutic effect is generally attributed to their ability to increase the synaptic levels of 5HT. Tryptophan hydroxylase 2 (TPH2) is the initial and rate-limiting enzyme in the biosynthetic pathway of 5HT in the CNS and losses in its catalytic activity lead to reductions in 5HT production and release. The time differential between the onset of 5HT reuptake inhibition by SSRIs (minutes) and onset of their anti-depressant efficacy (weeks to months), when considered with their overall poor therapeutic effectiveness, has cast some doubt on the role of 5HT in depression. Mice lacking the gene for TPH2 are genetically depleted of brain 5HT and were tested for a depression-like behavioral phenotype using a battery of valid tests for affective-like disorders in animals. The behavior of TPH2-/- mice on the sucrose preference test, tail suspension test and forced swim test and their responses in the unpredictable chronic mild stress and learned helplessness paradigms was the same as wild-type controls. While TPH2-/- mice as a group were not responsive to SSRIs, a subset responded to treatment with SSRIs in the same manner as wild-type controls with significant reductions in immobility time on the tail suspension test, indicative of antidepressant drug effects. The behavioral phenotype of the TPH2-/- mouse questions the role of 5HT in depression. Furthermore, the TPH2-/- mouse may serve as a useful model in the search for new medications that have therapeutic targets for depression that are outside of the 5HT neuronal system.

Full text

Mice Genetically Depleted of Brain Serotonin Do Not Display a
Depression-like Behavioral Phenotype
Mariana Angoa-Pe
rez,
†,§
Michael J. Kane,
†,§
Denise I. Briggs,
†,§
Nieves Herrera-Mundo,
†,§
Catherine E. Sykes,
†,§
Dina M. Francescutti,
†,§
and Donald M. Kuhn*
,†,§
†
Research & Development Service, John D. Dingell VA Medical Center, Detroit, Michigan 48201, United States
§
Department of Psychiatry & Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan 48201,
United States
*
SSupporting Information
ABSTRACT: Reductions in function within the serotonin (5HT) neuronal system
have long been proposed as etiological factors in depression. Selective serotonin
reuptake inhibitors (SSRIs) are the most common treatment for depression, and their
therapeutic effect is generally attributed to their ability to increase the synaptic levels of
5HT. Tryptophan hydroxylase 2 (TPH2) is the initial and rate-limiting enzyme in the
biosynthetic pathway of 5HT in the CNS, and losses in its catalytic activity lead to
reductions in 5HT production and release. The time differential between the onset of
5HT reuptake inhibition by SSRIs (minutes) and onset of their antidepressant efficacy (weeks to months), when considered with
their overall poor therapeutic effectiveness, has cast some doubt on the role of 5HT in depression. Mice lacking the gene for
TPH2 are genetically depleted of brain 5HT and were tested for a depression-like behavioral phenotype using a battery of valid
tests for affective-like disorders in animals. The behavior of TPH2−/−mice on the sucrose preference test, tail suspension test,
and forced swim test and their responses in the unpredictable chronic mild stress and learned helplessness paradigms was the
same as wild-type controls. While TPH2−/−mice as a group were not responsive to SSRIs, a subset responded to treatment with
SSRIs in the same manner as wild-type controls with significant reductions in immobility time on the tail suspension test,
indicative of antidepressant drug effects. The behavioral phenotype of the TPH2−/−mouse questions the role of 5HT in
depression. Furthermore, the TPH2−/−mouse may serve as a useful model in the search for new medications that have
therapeutic targets for depression that are outside of the 5HT neuronal system.
KEYWORDS: Serotonin, TPH2, TPH2 knock out, depression-like behavior, SSRIs, SERT
The serotonin (5HT) neuronal system innervates nearly
the entire neuraxis from cell bodies located in the
mesencenphalon.
1
In its role as a neurotransmitter, 5HT
regulates a diverse array of physiological functions that include
feeding, aggression, and sleep.
2
Defects in 5HT neurochemical
function have been implicated in a large number of neuro-
psychiatric and developmental conditions that include schizo-
phrenia, attention deficit hyperactivity disorder, sudden infant
death syndrome, and autism. Perhaps the strongest association
between impaired 5HT function and clinically significant
psychopathology is for depression. Since the monoamine
theory of depression was posited about 50 years ago,
3−5
a great
deal of work has sharpened focus on the role played by reduced
5HT levels in the synapse in this condition.
6
The most widely
used pharmacotherapy for depression is the class of drugs
referred to as selective-serotonin-reuptake inhibitors (SSRIs
7,8
).
Fluoxetine and other SSRIs block the 5HT transporter and are
thought to exert their antidepressant effects by increasing the
synaptic levels of 5HT.
9
Depression is a very serious medical condition and accounts
for a disproportionate amount of disability and loss of
productivity among all of the major psychiatric diseases. The
lifetime prevalence of depression/mood disorders is approx-
imately 20%,
10
and depression is highly comorbid with anxiety
and other medical conditions.
11
Unfortunately, therapeutic
options for depression are somewhat limited and the best
treatments leave ∼60−70% of patients without symptomatic
relief.
12,13
Some even question whether SSRIs have clinically
significant therapeutic value beyond a placebo effect in treating
any form of depression.
14,15
Despite the long-held hypothesis
that 5HT neuronal dysfunction is an underlying cause of
depression,
16−18
the relatively poor efficacy of the SSRIs in
treating this condition and the high rates of remission
13
have
renewed interest in achieving a better understanding of the
neurobiological bases of this disorder and in developing more
effectively targeted drug therapies for it.
As part of a larger project to explore the involvement of 5HT
in neurodevelopment and psychiatric conditions, we developed
a mouse lacking the gene for the brain-specific form of
tryptophan hydroxylase (TPH), TPH2.
19
TPH2 is the initial
and rate-limiting enzyme in the biosynthesis of 5HT. Mice
lacking the gene for TPH2 are viable and fertile but show some
degree of developmental lag in comparison to wild-type
Received: March 24, 2014
Accepted: August 4, 2014
Research Article
pubs.acs.org/chemneuro
© XXXX American Chemical Society Adx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

mice.
20−22
TPH2−/−mice have no TPH2 protein, and brain
tissue from these mice cannot hydroxylate tryptophan to 5-
hydroxytryptophan.
19
Consequently, their central nervous
systems are devoid of 5HT. Other major elements of the
5HT neuronal system (i.e., receptors, neurons, axons, dendrites,
raphe unit firing, and the 5HT transporter) remain essentially
intact in these mice, and there appear to be few if any
compensatory alterations in other neurotransmitter sys-
tems.
19,20,23−28
In the course of characterizing the behavioral
phenotype of the TPH2−/−mouse, we and others have noted
numerous interesting and novel aspects of 5HT-behavioral
relations. TPH2−/−mice display intense impulsive and
compulsive behaviors and extreme aggressiveness,
23,29
as well
as autistic-like behavioral traits.
30
Surprisingly, these mice show
decreased anxiety-like behaviors in comparison to wild-type
control mice.
23,29,30
In light of the suspected roles played by deficits in 5HT
neurochemistry in depression, we carried out a battery of
extensively validated behavioral tests of depression-like
behaviors in TPH2−/−mice. We hypothesized that these
mice would show a profound behavioral phenotype indicative
of depression. However, TPH2−/−mice were not different from
wild-type controls on any of these tests and in some cases were
more resistant to the development of depression-like behaviors
than wild-type controls. What is more, a subset of TPH2−/−
mice responded to fluoxetine, paroxetine, and citalopram like
wild-type mice with reductions in immobility time on the tail
suspension test, indicative of antidepressant responses.
Together, these results suggest that the absence of brain 5HT
does not confer in mice a depression-like behavioral phenotype.
The TPH2−/−mouse questions the role of 5HT as an
etiological factor in depression and may serve as a valuable and
interesting starting point to search for new medications that
have therapeutic targets for depression that are outside of the
5HT neuronal system.
■RESULTS AND DISCUSSION
TPH2−/−mice were compared with wild-type controls for
sucrose preference to test for anhedonic-like behaviors. The
results in Figure 1A show that the main effect of days was
significant (F3,66 = 4.73, p= 0.005, two-way repeated measures
ANOVA). Sucrose preference was ∼80−85% for both groups.
Total fluid intake was identical for both groups of mice on each
day of this test (i.e., sucrose plus water; data not shown). Figure
1B presents results of the quinine preference test and indicates
that both genotypes expressed low preference for quinine
(∼20−25% of total fluid intake). The main effect of days (F3,66
= 4.13, p= 0.009) and genotype (F1,22 = 7.11, p= 0.014) were
significant by two-way repeated measures ANOVA, whereas
their interaction was not. TPH2−/−mice actually drank
significantly less quinine solution than wild-type controls on
day 2 (Bonferroni’s multiple comparison test, p< 0.05). Food
consumption was also measured in addition to fluid intake, and
the results in Figure 1C indicate that the main effects of days
(F3,66 = 8.16, p= 0.0001) and the days ×genotype interaction
(F3,66 = 3.94, p= 0.011) were significant, whereas the main
effect of genotype was not. Post hoc comparisons revealed that
food intake in the TPH2−/−mice was significantly lower on
days 2−4(p< 0.05−0.0001, Bonferonni’s multiple comparison
test) by comparison to their day 1 food intake. Food intake of
TPH2+/+ mice did not vary over the 4 day test period. The
results presented in Figure 1 indicate that the behavior of
TPH2−/−mice is normal and not indicative of anhedonia.
Figure 1. Sucrose and quinine preference and food intake in TPH2−/−and wild-type mice. Wild-type (WT) and TPH2−/−mice (KO) were tested
daily for drinking preference over the 4 day test for sucrose (A) or quinine (B) in a two-bottle choice paradigm. KO and WT mice show the same
preference for sucrose, but KO mice drink significantly less quinine than WT controls while drinking significantly more water. (C) KO mice have
slightly but not significantly greater food intake than WT controls. Data are presented as preference for liquid intake and in grams for food
consumption and are means ±SEM for 12 mice per independent group per test. The symbols are *p< 0.05 and ***p< 0.0001 compared with day 1
and #p< 0.05 compared with WT controls at day 2.
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The results in Figure 2A show that TPH2−/−mice were
immobile for the same amount of time on the tail suspension
test (TST) as wild-type controls. Performance of TPH2−/−and
wild-type mice on the TST was not complicated by tail
climbing, which can limit the use of the C57BL/6 strain on this
test.
31
Results in Figure 2B present data for the forced swim
test (FST). The main effects of genotype (F1,22 = 5.05, p=
0.034) and trial (F1,22 = 32.41, p< 0.0001) were significant by
two-way repeated measures ANOVA, but the genotype ×trial
interaction was not. TPH2−/−mice remained immobile for
significantly less time than wild-type controls (i.e., less
“depressed”) on the first trial (p< 0.05, Bonferroni multiple
comparison test) but this difference was no longer apparent on
the second trial when the behavior of TPH2−/−mice was the
same as wild-type controls. Taken together, results presented in
Figure 2 indicate that TPH2−/−mice do not exhibit depression-
like behaviors.
Results from the unpredictable chronic mild stress (UCMS)
model of depression are presented in Figure 3. Scores for coat
status are included in Figure 3A. The main effects of weeks
(F6,126 = 25.71, p< 0.0001) and genotype (F1,21 = 9.85, p<
0.005) were significant by two-way repeated measures ANOVA,
but their interaction was not. Post hoc analyses indicated that
both TPH2−/−(p< 0.0001, Bonferroni’s multiple comparison
Figure 2. Immobility times for TPH2−/−and wild-type control mice on the TST and FST. (A) TPH2−/−(KO) and wild-type controls (WT) have
the same immobility times in the TST. (B) KO mice spend significantly less time immobile than WT controls on the first trial of the FST but have
the same immobility times on the second trial. Immobility times are in sec for both tests and are means ±SEM of 13 WT and 14 KO for panel A and
12 WT and 12 KO mice for panel B. The *indicates p< 0.05.
Figure 3. Effects of UCMS on depression-like behavior of TPH2−/−and wild-type control mice. TPH2−/−(KO) and wild-type controls (WT) were
exposed to UCMS for 6 weeks and the emergence of depression-like behaviors was monitored weekly. (A) Coat status for both KO and WT mice
degraded significantly at weeks 3−6 and KO mice differed from WT only at the 3 week time point in the stress protocol. (B) Grooming time (in sec)
in the splash test diminished significantly in KO mice but not in WT controls, and no difference was seen between genotypes. (C) Body weights (in
g) of both WT and KO mice increased significantly over time. (D) Sucrose preference (mL sucrose divided by total water plus sucrose intake) after
completion of the UCMS. Both WT and KO mice showed significant decreases in sucrose preference by comparison to their respective controls and
WT mice showed a significantly greater reduction in sucrose preference compared with KO mice. Data in each panel are means ±SEM for 12 mice
per group. The symbols are *p< 0.05, **p< 0.001, and ***p< 0.0001 by comparison to the respective 0 time point for KO and WT mice and ##p<
0.001 by comparison to the WT control at week 3.
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test) and TPH2+/+ mice (p< 0.05−0.0001, Bonferroni’s
multiple comparison test) degraded significantly from their
starting coat status at weeks 3−6. Total grooming times are
presented in Figure 3B and show that the main effect of weeks
was significant (F6,126 = 4.88, p= 0.0002, two-way repeated
measures ANOVA) while the main effect of genotype and the
weeks ×genotype interaction were not. Post hoc analyses
revealed that TPH2−/−mice grooming time was significantly
decreased from starting values at weeks 4−6(p< 0.05−0.001,
Bonferroni’s multiple comparison test) whereas wild-type mice
did not vary over the weeks 0−6. Body weights of mice were
measured throughout the UCMS protocol, and the results are
presented in Figure 3C. The main effect of weeks was
significant (F6,120 = 17.9, p< 0.0001), whereas the main effect
of genotype and the weeks ×genotype interaction were not.
Post hoc analyses revealed that the body weights of TPH2−/−
mice increased slightly at weeks 3−6 compared with starting
body weights (p< 0.001−0.0001, Bonferroni’smultiple
comparison test) and wild-type controls differed significantly
from their starting weights at weeks 1−6(p< 0.05−0.001,
Bonferroni’s multiple comparison test). These results indicate
that TPH2−/−mice maintain normal appetitive behavior over
the extended UCMS protocol. The post-UCMS sucrose
preference test results in Figure 3D show that the main effects
of treatment (F1,113 = 281.7, p< 0.0001) and genotype (F1,113 =
8.65, p= 0.004) and their interaction (F1,113 = 10.78, p= 0.001)
were significant by two-way ANOVA. Wild-type mice
developed depression-like anhedonia with a significant
reduction in sucrose preference from 80% to 30% (p<
0.0001, Bonferroni’s multiple comparison test). TPH2−/−mice
also showed a significant reduction in sucrose preference from
80% to 45% after exposure to the UCMS protocol
(Bonferroni’s multiple comparison test, p< 0.0001). The
extent of the reduction in sucrose preference after the UCMS
procedure was significantly greater in wild-type controls by
comparison to TPH2−/−mice (Bonferroni’s multiple compar-
ison test, p< 0.001). Together, the data in Figure 3 show that
stress-induced depression-like behaviors are induced in
TPH2−/−mice to the same extent displayed by wild-type mice.
TPH2−/−mice and wild-type controls were exposed to the
learned helplessness (LH) paradigm, and the results are
presented in Figure 4. The main effect of treatment was
significant (F3,20 = 5.92, p= 0.005, one-way ANOVA), and both
genotypes showed a significant increase in latency to escape on
the test day (p< 0.05 for both, Tukey’s multiple comparison
test). The magnitude of the effect was the same for both
genotypes, indicating that TPH2−/−mice develop depression-
like behavior in the LH model like wild-type controls.
Many strains of wild-type mice show decreases in immobility
in the FST or the TST when treated acutely with SSRIs.
32−36
Therefore, we tested TPH2−/−mice for their response to the
SSRIs fluoxetine, paroxetine, and citalopram in the TST with
the expectation that they would be unresponsive to these drugs
(i.e., TPH2−/−mice should not respond to SSRIs because they
lack brain 5HT and inhibition of the SERT could not possibly
increase synaptic 5HT levels). The results presented in Figure
5A show that when mice were treated with fluoxetine, the main
effect of drug was significant (F1,53 = 16.13, p= 0.0002, two-way
ANOVA) but the main effect of genotype and the drug ×
genotype interaction were not. Wild-type mice showed a
significant reduction in immobility time after treatment with
fluoxetine (p< 0.05, Bonferroni’s multiple comparison test).
When mice were treated with paroxetine (Figure 5B), the main
effects of genotype (F1,43 = 52.4, p< 0.0001) and drug (F1,43 =
21.85, p< 0.0001), as well as their interaction (F1,43 = 13.12, p
= 0.0008) were significant when analyzed by two-way ANOVA.
Post hoc comparisons showed that paroxetine significantly
reduced immobility times on the TST for wild-type controls (p
< 0.0001, Bonferroni’s multiple comparison test) but did not
change the behavior of TPH2−/−mice. The effect of citalopram
on TST performance is presented in Figure 5C, and the figure
shows that the main effects of genotype (F1,50 = 17.3, p=
0.0001) and drug (F1,50 = 20.6, p< 0.0001) and their
interaction (F1,50 = 6.42, p = 0.014) were significant by two-way
ANOVA. Wild-type mice showed significant reductions in
immobility times after citalopram treatment (p< 0.0001,
Bonferroni’s multiple comparison test) whereas the slight
reduction seen in TPH2−/−mice did not reach significance.
Although TPH2−/−mice did not respond as groups to SSRI
treatment with significant reductions in TST immobility times
(Figure 5A−C), we noted that some individual TPH2−/−mice
clearly displayed substantial reductions after drug treatment.
Depressed patients treated in clinical trials with SSRIs are
commonly classified as responders and nonresponders,
37−39
generally based on a predetermined reduction in depression
scores (e.g., usually 50%). The concept of responsive and
nonresponsive subjects has also been recognized in animal
models of antidepressant resistance.
40
Therefore, we re-
examined the data from SSRI treatment of wild-type and
TPH2−/−mice presented above and classified mice in either
group as drug responders if their immobility times were 2
standard deviations lower than the mean of their respective
nontreated controls. Mice that had the same immobility (<2
standard deviations from the control mean) or higher times
compared with nontreated controls were classified as non-
responders. Using this classification, it was observed that 33%
of WT controls and 19% of TPH2−/−mice were responders to
fluoxetine. The results of this analytical approach are presented
in Figure 5D−F. Treatment with fluoxetine resulted in a
significant main effect of drug (F2,51 = 27.38, p< 0.0001, two-
way ANOVA). The main effect of genotype and the drug ×
genotype interaction were not significant. Post hoc compar-
isons revealed that TPH2+/+ mice that responded to fluoxetine
had significantly shorter immobility times than controls (p<
0.0001, Bonferroni’s multiple comparison test) and non-
responsive drug-treated mice (p< 0.001, Bonferroni’s multiple
Figure 4. Effects of LH on depression-like behavior of TPH2−/−and
wild-type control mice. TPH2−/−(KO) and wild-type controls (WT)
not exposed to foot shocks (NS) or exposed to inescapable foot
shocks (S) were tested for shock escape. Both WT and KO mice
showed significant increases in latency to escape on the test day
compared with the NS condition. WT and KO mice did not differ in
the NS and S test conditions, respectively. Data are expressed as means
±SEM for 6 mice per group. The *indicates p< 0.05.
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comparison test). Fluoxetine nonresponders were not different
from untreated controls. Similarly, TPH2−/−responders
showed significantly shorter immobility times by comparison
to both untreated controls (p< 0.0001, Bonferroni’s multiple
comparison test) and nonresponsive drug-treated mice (p<
0.001, Bonferroni’s multiple comparison test) and among
TPH2−/−mice, untreated controls were not different from
nonresponders. The pattern of response to paroxetine was
Figure 5. Effects of SSRIs on immobility times in the TST for TPH2−/−and wild-type control mice. Independent groups of TPH2−/−(KO) mice or
wild-type controls (WT) were injected acutely with (A) fluoxetine (20 mg/kg), (B) paroxetine (10 mg/kg), or (C) citalopram (20 mg/kg) or with
vehicle controls, and immobility times were tested 30 min after treatment. Only WT mice responded significantly to each SSRI with reductions in
immobility times. Data from drug-treated WT and KO mice was re-examined to classify mice as responders (R) or nonresponders (NR) as defined
in Supporting Information, Tables 1 and 2. Both WT and KO mice responded significantly to (D) fluoxetine and (F) citalopram by comparison to
controls and nonresponsive mice for each respective drug. Statistical tests for mice treated with (E) paroxetine could not be carried out because the
WT nonresponder group contained just one mouse. WT and KO controls did not differ from NR mice in the fluoxetine and citalopram groups.
Immobility times are in sec and are means ±SEM for 27 WT and 29 KO mice in panels A and D, 23 WT and 24 KO mice in panels B and E, and 27
WT and 27 KO mice in panels C and F. Symbols are *p< 0.05, **p< 0.001, and ***p< 0.0001.
Figure 6. Effects of SSRIs on SERT-mediated uptake of [3H]-5HT by hippocampal synaptosomes. Uptake of [3H]-5HT into synaptosomes from
TPH2−/−(KO) and wild-type control mice (WT) was measured in the absence or presence of (A) fluoxetine (10 μM), (B) paroxetine (50 μM), and
(C) citalopram (50 μM). All SSRIs significantly inhibited uptake to the same extent in KO and WT tissue. Uptake of [3H]-5HT is expressed as
nmol/(g·min) and is the mean ±SEM of four independent experiments. The symbols are *p< 0.05 and **p< 0.001 comparing drug to control for
each genotype.
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similar to that of fluoxetine and is presented in Figure 5E.
Approximately 92% of TPH2+/+ mice responded to paroxetine,
whereas only 38% of TPH2−/−mice were responders.
Statistical testing of paroxetine TPH2+/+ nonresponders to
other groups could not be done because this group contained
only one mouse. Results from treatment of mice with
citalopram are presented in Figure 5F. Approximately 81% of
TPH2+/+ mice were classified as citalopram responders whereas
only 31% of treated TPH2−/−mice were responders. The main
effects of genotype (F1,48 = 13.82, p= 0.0005) and drug (F2,48 =
55.48, p< 0.0001) and the genotype ×drug interaction (F2,48 =
3.22, p= 0.048) were significant when analyzed by two-way
ANOVA. Post hoc comparisons revealed that TPH2+/+ mice
that responded to citalopram had significantly shorter
immobility times than controls (p< 0.0001, Bonferroni’s
multiple comparison test) and nonresponsive drug-treated mice
(p< 0.05, Bonferroni’s multiple comparison test). Citalopram
nonresponders were not different from untreated controls.
Similarly, TPH2−/−responders showed significantly shorter
immobility times by comparison to both untreated controls (p
< 0.0001, Bonferroni’s multiple comparison test) and non-
responsive drug-treated mice (p< 0.0001, Bonferroni’s multiple
comparison test) and among TPH2−/−mice, untreated controls
were not different from nonresponders. The number of
TPH2+/+ and TPH2−/−mice classified as responders and
nonresponders (and specified by sex) to the SSRIs along with
the immobility time cutoffvalues used to make these
classifications are presented in Supporting Information Tables
1 and 2, respectively. Taken together, the results in Figure 5
show that while TPH2−/−mice did not respond to SSRIs as full
groups, some individual mice did show substantial and
significant reductions in immobility times on the TST.
In view of the results showing that a subset of TPH2−/−mice
respond to SSRIs with antidepressant-like reductions in
immobility time and considering that SSRIs are thought to
exert their antidepressant effects via inhibition of SERT, it was
important to confirm that SSRIs would bind to the SERT in
TPH2−/−mice. We tested this possibility by determining
whether SSRIs would block the synaptosomal uptake of [3H]-
5HT in TPH2−/−mice. It can be seen in all panels of Figure 6
that [3H]-5HT uptake was slightly but not significantly higher
in TPH2−/−mice compared with wild-type controls. This result
is consistent with our previous finding of slightly increased
synaptosomal [3H]-5HT uptake in TPH2−/−mice.
23
The main
effect of drug on uptake was significant for fluoxetine (F3,17 =
9.75, p= 0.0006), paroxetine (F3,18 = 7.59, p= 0.001), and
citalopram (F3,23 = 5.76, p= 0.0043) when analyzed by a one-
way ANOVA. The main effect of genotype was not significant
for any drug. Figure 6A shows that fluoxetine leads to a
significant reduction in [3H]-5HT uptake in synaptosomes
from both wild-type (p< 0.05, Tukey’s multiple comparison
test) and TPH2−/−mice (p<0.001,Tukey’smultiple
comparison test). Paroxetine also significantly reduced [3H]-
5HT uptake in wild-type (p<0.05,Tukey’smultiple
comparison test) and TPH2−/−mice (p< 0.05, Tukey’s
multiple comparison test) as shown in Figure 6B. Finally, it can
be seen in Figure 6C that citalopram significantly reduced the
uptake of [3H]-5HT in wild-type (p< 0.05, Tukey’s multiple
comparison test) and TPH2−/−mice (p< 0.05, Tukey’s
multiple comparison test). The results in Figure 6 show that
SERT function in TPH2−/−mice is the same as that in wild-
type controls and SSRIs block [3H-5HT] synaptosomal uptake
to the same extent in each group.
The data presented in this work document two rather
surprising behavioral characteristics of a mouse lacking brain
5HT. First, TPH2−/−mice do not display a depression-like
behavioral phenotype. Second, a subset of TPH2−/−mice show
an antidepressant response to the SSRIs fluoxetine, citalopram,
and paroxetine on the TST. Defects in 5HT neurotransmission
have long been implicated as causal factors in depression.
16−18
Three different tests widely used to assess mood disorders in
animals, which have high predictive validity for antidepressant
medications, the sucrose preference test, the TST, and the FST,
yielded results that were in good agreement and demonstrated
conclusively that the lack of 5HT in brain does not result in
depression-like behaviors. The UCMS and LH protocols, which
elicit depression-like behaviors in wild-type mice (e.g.,
diminished self-grooming and coat status, reduced sucrose
preference, increased shock escape times), have the same effect
on TPH2−/−mice. Thus, the genetic depletion of 5HT from
brain does not induce a depression-like phenotype, and it does
not prevent mice from developing a depression-like phenotype
upon exposure to chronic mild stress or inescapable footshock.
Because results using one or two different tests of depressive-
like behavior can yield conflicting outcomes (see below), we
consider it to be important to use a larger number of behavioral
tests for comparative purposes in the event that some tests
indicated that TPH2−/−mice displayed a depression-like
phenotype while others did not. Fortunately, the results of
the sucrose preference test, TST, FST, UCMS, and LH were in
excellent agreement, adding strength to the conclusion that
TPH2−/−mice do not express a depression-like phenotype. In
addition, we have previously shown that TPH2−/−mice display
significantly less novelty suppressed feeding than wild-type
controls.
23
This test has been used widely to probe the anxiety-
related component of depression in rodents,
41,42
and perform-
ance of TPH2−/−mice on this test was consistent with the
other tests used presently. We did not test TPH2−/−mice on
the repeated social defeat stress test of depression because their
extreme aggressiveness
23,29
prevents social defeat by other
mice.
It may appear that the present results stand in contrast to a
substantial body of research that has linked decreased 5HT
function with depression for the past five decades. However,
prior studies that have manipulated brain 5HT in animals via
pharmacological or genetic approaches actually reveal very mild
changes in behavior. For example, partial reductions in brain
5HT content with p-chlorophenylalanine, an inhibitor of
TPH2, while having minor effects itself on behavior on the
TST, do block reductions in immobility by SSRIs.
43,44
Mice
lacking the gene for PET-1 have ∼80% reductions in brain 5HT
neurons but do not differ from wild-type controls in the TST
and FST.
45,46
Savelieva and colleagues
26
reported that only
male TPH2−/−mice spent significantly less time immobile on
the FST than wild-type controls, indicating that these mice
were nondepressive. These same investigators also showed that
male TPH2−/−mice were not different from wild-type controls
on the TST and female TPH2−/−mice had no phenotype on
either the TST or FST.
26
Mosienko and colleagues reported
that TPH2−/−mice displayed increased immobility in the FST
but not on the TST.
29
The possibility exists that moderate reductions in brain 5HT,
versus the more drastic depletions seen in PET-1 and TPH2−/−
mice, would reveal a depression-like phenotype. Testing of this
possibility has been done using mice with partial reductions in
TPH2 expression. For instance, Beaulieu and colleagues
47
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generated a knockin mouse line that expressed a R439H
mutant of TPH2. This mutant is equivalent to a very rare
human TPH2 variant (R441H) seen in unipolar major
depression.
48
This loss of function mutant of TPH2 has 60−
80% reductions in brain 5HT as a result of lowered TPH2
enzymatic activity.
47,49
These TPH2 knockin mice indeed show
increased immobility time on the TST, indicative of depression-
like behavior.
47,50
However, this result is confusing because
mice heterozygous for the altered R439H TPH2 gene have
normal brain 5HT levels but, like homozygotes, show
significant increases in immobility time on the TST.
47
Zhang and colleagues
51
reported a functional single
nucleotide polymorphism in the mouse TPH2 gene. This
C1473G mutation replaces proline-447 with an arginine and
results in significant reductions in TPH2 catalytic activity and
5HT synthesis.
51,52
These investigators also made the very
interesting observation that BALB/c and DBA/2 mice are
homozygous for the 1473G allele and show much lower levels
of TPH2 activity and 5HT than C57BL/6 and 129X1/SvJ
strains, which are homozygous for the 1473C allele.
51
Studies
comparing mouse strains for depression-like behavior and
responsiveness to SSRIs are inconsistent in supporting a role
for TPH2 and reduced brain 5HT in depression. For instance, a
very large number of studies have shown that C57BL/6 mice
(1473C TPH2 allele) show more, less, or the same immobility
times on the TST or FST as BALB/c or DBA/2 mice, which
bear the 1473G TPH2 allele.
36
Mice with the same TPH2
1473G allele can even display the highest (BALB/c) or lowest
(DBA/2) immobility times among various mouse strains when
compared in the same study.
53
With regard to responsiveness to SSRIs on the TST or FST,
it also appears that the TPH2 genotype is not a determinant.
For example, it has been shown that mice with the 1473C allele
(C57BL/6 and 129Sv) are responsive to SSRIs in the FST
whereas strains bearing the 1473G allele (BALB/c and DBA/2)
are not responsive.
54
Mice with the same 1473C allele
(C57BL/6 and NMRI) are responsive or nonresponsive,
respectively, on the TST.
55
Mice with different alleles
(C57BL/6, C allele, and DBA/2, G allele) can be the most
responsive
34
or the least responsive (C57BL/6, C allele, and A/
J, G allele) to SSRIs on the TST among compared strains.
33
These complex findings show that while some of the variation
in responsiveness to SSRIs can be attributed to the specific drug
used or the behavioral test employed (TST versus FST), the
TPH2 allele is not a determining factor. Perhaps the most
conclusive demonstration that the TPH2 C1473G poly-
morphism is not related to depression-like behaviors has
been presented by Tenner and colleagues.
56
These investigators
bred the 1473G allele from DBA/2 mice onto a C57BL/6
background to generate congenic strains bearing the 1473C or
the 1473G alleles. These strains did not differ in either brain
5HT levels or immobility time on the FST.
56
Similarly, Berger
et al.
57
showed that mice bearing the TPH2 C1473G
polymorphism were not different from wild-type controls in
brain 5HT content, and they did not show a depression-like
phenotype on the TST, FST, sucrose preference test, or LH
paradigm. Taken together, our results agree with the majority of
mouse genetic/strain studies and do not support a role for
TPH2 or brain 5HT in the expression of a depression-like
behavioral phenotype in mice.
Three additional factors support the contention that mice
genetically depleted of 5HT do not show a depression-like
behavioral phenotype. First, it is well-known that anxiety
symptoms and syndromes are highly prevalent among patients
with depressive disorders,
10,58,59
which suggests that if
TPH2−/−mice were indeed depressed, they would also show
anxiety-like behaviors. It has been shown clearly that TPH2−/−
mice are the same as wild-type controls
23,30
or show decreases
in behaviors on tests that model anxiety.
29
Likewise, PET-1
knockout mice do not express anxiety-like behaviors.
46
Second,
mouse strains characterized by poor maternal care are highly
vulnerable to developing depression-like behaviors.
60
TPH2
20
and PET-1 knockout dams
61
show very poor maternal behavior
in the presence of their newborn pups, yet their offspring do
not develop depression-like behaviors despite drastic reductions
in brain 5HT. Third, it has been proposed that increased
hippocampal neurogenesis is a correlate of the antidepressant
effects of SSRIs, which increase synaptic 5HT, but mice
genetically modified to have low or no brain 5HT levels (i.e.,
PET-1, VMATf/f:SERT, and TPH2−/−mice) exhibit normal
levels of hippocampal neurogenesis.
62,63
Experiments designed to test the responsiveness of TPH2+/+
and TPH2−/−mice to SSRIs on the TST yielded interesting
results. The immobility times of TPH2+/+ mice were
significantly decreased by fluoxetine, paroxetine, and citalo-
pram, whereas those of TPH2−/−mice were not. These results
confirm numerous previous studies showing that wild-type
mice respond to SSRIs with antidepressant-like reductions in
immobility times. The failure of TPH2−/−mice to respond to
these drugs when group-analyzed is consistent with the
expectation that the antidepressant effects of SSRIs are
mediated by 5HT.
33,34,36
However, we noted that a subset
TPH2−/−mice responded to SSRIs with substantial reductions
in immobility times and some TPH2+/+ mice were non-
responders. Because of this unexpected finding, we classified
both TPH2−/−and wild-type controls as drug responders and
nonresponders much as depressed individuals are classified with
respect to their responsiveness to SSRIs. Mice of both
genotypes that showed reductions in immobility times after
drug treatment that was >2 standard deviations lower than the
means of the respective untreated controls were designated as
responders. Using this alternative analytical approach that takes
into account the individual responses of mice to drug
treatment, a unique finding emerged and confirmed that
some TPH2−/−mice indeed responded to SSRI treatment with
significant reductions in immobility times on the TST.
Assuming that the SSRIs exert this effect in wild-type controls
through blockade of the SERT to cause an increase in synaptic
5HT, it is very interesting that they could have antidepressant
behavioral effects in an animal totally lacking brain 5HT.
TPH2−/−mice have normal levels of a functional SERT (i.e.,
the SERT transports 5HT into synaptosomes and uptake can
be blocked by SSRI drugs as shown in Figure 6) but SSRI
binding to it cannot lead to increases in synaptic 5HT because
there is no 5HT available for transport into the presynaptic
process. Clearly, far fewer TPH2−/−mice responded signifi-
cantly to SSRI treatment than TPH2+/+ mice (20−36%
TPH2−/−responders compared to 33−92% TPH2+/+ res-
ponders). Nevertheless, the reductions in TST immobility
times for responsive TPH2−/−mice were quite large and highly
statistically significant. In many ways, this result is similar to the
situation in depressed humans that are nonresponsive to SSRI
treatment.
13
It has also been recognized that large proportions
of animals are nonresponsive to SSRIs in behavioral models of
depression-like behaviors.
40
Therefore, while the results on the
partial responsiveness of TPH2−/−mice to SSRIs must be
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interpreted with caution and should be expanded to include
additional behavioral tests, it is still quite interesting that these
mice show “anti-depressant-like”changes in behavior that are
not mediated by increases in synaptic 5HT caused by drug-
induced inhibition of the SERT.
The SERT is not the only target in brain with which the
SSRIs interact.
64,65
In particular, fluoxetine, sertraline, and
paroxetine fail to reduce TST immobility times in mice lacking
brain norepinephrine
66
suggesting that drug-induced increases
in synaptic norepinephrine are playing a role in the
antidepressant actions of selected SSRIs. In light of this, it is
certainly possible that the effects of the SSRIs on selected
TPH2−/−mice are mediated by norepinephrine in the absence
of 5HT. In addition, these drugs can have broad influence on
brain function via interactions with ion channels,
67
protein
kinases,
68−70
phosphatases,
71
and phosphodiasterases.
72
At least
fluoxetine reduces acid sphingomyelinase activity and lowers
brain ceramide levels while improving depressive-like behavior
in the UCMS model.
73
Long-term treatment with SSRIs can
also alter gene expression via interactions with transcription
factors,
74,75
and their ability to stimulate neurogenesis in the
adult brain
76
may play a role in therapeutic efficacy in the
treatment of depression.
77−79
Together, the present results
suggest the possibility that SSRIs can exert antidepressant
effects by acting at targets other than the SERT and do so in a
manner that is independent of 5HT. In light of the modest
therapeutic efficacy of current treatments for depression and
considering the high rates of treatment resistance and
remission,
13
the TPH2−/−mouse model is ideally suited to
allow new studies that search for new therapeutic sites of action
for the SSRIs. In the process, these studies are also likely to
yield new information on the neurobiological bases of
depression.
■METHODS
Subjects. TPH2−/−mice were generated by deleting exon 1 of the
TPH2 gene as described.
19
These mice have no brain TPH2 protein,
and they express no other compensatory enzymatic or chemical
mechanism to hydroxylate tryptophan, so their brains are devoid of
5HT and 5-hydroxyindoleacetic acid (5HIAA). 5HT neurons and
processes remain intact in TPH2−/−mice and show normal expression
of the SERT and 5HT receptors. The SERT is also capable of
transporting 5HT into synaptosomes to the same extent as that in
wild-type mice.
30
Wild-type and TPH2−/−mice used in this study were
derived from matings of heterozygous (TPH2+/−)malesand
heterozygous (TPH2+/−) females on a mixed C57BL/6-Sv129
background. Genome scanning analysis by The Jackson Laboratory
revealed that our strain background was 95.4% C57BL/6. Hetero-
zygous TPH2+/−mice have the same brain levels of 5HT, SERT, and
TPH2 as wild-type mice and were not tested presently. Offspring were
housed with their mothers until weaning at PND21 and thereafter
litters were housed together for an additional week. Litters were then
separated by sex, and males and females from the same litter were
housed as groups (4−6 mice per cage) in 27 cm ×48 cm ×20 cm
cages for at least 4 weeks prior to testing. For tests that required
individual housing of mice during the test (e.g., sucrose preference
test), subjects were housed singly overnight before experimentation.
Results of all tests reflect groups of mice from at least 3−4
independent litters. Separate cohorts of adult mice (10−12 weeks of
age) were acclimated to the behavioral testing rooms for 1 h prior to
all testing (10 am to 4 pm daily). Independent groups were used for
each behavioral test, and experimenters scoring behaviors were blind
to genotype. Equal numbers of male and female mice were used in all
tests. However, because we did not observe a main effect of sex on any
test, data from male and female mice was pooled for each genotype.
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health. The protocol was
approved by the Institutional Care and Use Committee of Wayne
State University (Permit Number A3310-01).
Sucrose and Quinine Preference and Food Intake. The
sucrose preference test was used as a measure of hedonia/anhedonia
and was carried out as previously described.
80,81
Singly housed mice
were habituated to the presence of two graduated drinking tubes (100
mL glass tubes with 1 mL graduations; Braintree Scientific, Braintree,
MA) containing tap water for 2 days. Sipper tubes contained ball
bearings to minimize loss of fluid to drippage. For the following 4 days,
mice were given a two-bottle choice of 3% sucrose in tap water versus
tap water. To eliminate potential side preferences, the position of the
bottles was switched daily. Consumption of water and sucrose and
total liquid intake was measured once daily. Fluid intake was
determined by weighing each bottle at the start of the test period
and the weight of the bottles after 24 h on each cage was subtracted
from the starting weight to yield fluid intake (i.e., 1 g = 1 mL of fluid).
To assess fluid loss to drippage during the test periods, 10 bottles were
placed on empty cages (1 per cage) throughout the cage rack and loss
was determined as described above. Loss of fluid from sipper bottles
was negligible (0.8% of total volume over 24 h). Preference for sucrose
is expressed as % of consumed sucrose divided by the total volume of
liquid consumed. Consumption of a solution of 0.0024% quinine was
measured in the same manner described above for sucrose. Diminished
preference for sucrose versus water is indicative of depression-like
behavior. For measures of food intake, mice were individually housed
and water was available ad libitum. Animals had access to standard
chow, and their food consumption was quantified by weighing food
pellets daily for 5 consecutive days.
Tail Suspension Test. The tail suspension test (TST) was carried
out as originally described by Steru et al.
82
This model of “behavioral
despair”was originally devised as a test for screening antidepressant
drugs but is now used widely in phenotyping depression-like behaviors
in rodents.
83,84
Mice are secured by the tail to a plastic band (∼4cm
wide) with medical adhesive tape (1−1.5 cm of the distal tail) and
suspended head-down 30 cm above the laboratory bench. Mice are
scored for immobility over a 6 min test period. Immobility is defined
as a lack of movement or struggling and motionless hanging. The time
spent immobile by mice was recorded by 2−3 investigators blinded to
the genotype of the subject undergoing testing. Increased immobility is
indicative of depression-like or resignation behavior.
Forced Swim Test. The forced swim test (FST) was carried out
according to the method of Porsolt.
83,85
This model, like the TST, was
originally designed as a test for screening antidepressant drugs but is
now used widely to assess depression-like behaviors.
83,86
Mice are
tested on two separate occasions. On the first test, mice are placed
individually into a 2 L glass beaker (OD = 13.1 cm, H= 19.3 cm)
containing 1.5 L of tap water (12 cm deep) at 25 °C for 15 min, and
the time of immobility is scored only in the first 5 min. The second
test is administered 24 h later, and mice are placed into the testing
chamber for 5 min, and the time of immobility was scored throughout.
Immobility is defined as minimal movement required for a mouse to
keep its head and nostrils above the water level. Increased immobility
is indicative of depression-like or resignation behavior.
Unpredictable Chronic Mild Stress (UCMS). The UCMS
procedure was based on those designed for rats
65,87,88
and mice.
89
In this test, mice were exposed to a series of mild stressors including
altered cage bedding, social stress, cage tilt, light/dark cycle disruption,
cage exchange, and predator sounds, presented in random order over a
period of 6 weeks basically as described previously.
36,90,91
The order of
presentation of all stressors in the UCMS is included in Supporting
Information Table 3. Body weight and coat status was assessed before
initiation of the UCMS protocol and weekly during the procedure.
Coat status from eight body parts (head, neck, dorsal coat, ventral
coat, tail, forepaws, and hind paws) was scored as 0 for well groomed
and 1 for unkempt by 2−3 observers blind to mouse genotype. The
total score per test was derived by summing the individual scores for
each body part.
78,87,92
Immediately after scoring coat status, mice were
exposed to the splash test,
93,94
which involves spraying a 10% sucrose
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solution onto the dorsal coat of the mouse in its home cage. This
mildly sticky solution induces self-grooming, the duration of which was
recorded for 5 min by 2−3 observers blinded to mouse genotype.
Finally, after completion of the 6 week UCMS procedure, mice were
assessed for sucrose preference in a single overnight session as
described above. Worsening coat status scores over time and decreased
self-grooming are indicative of depression-like behavior.
Learned Helplessness (LH). The LH paradigm was carried out
essentially as described by Fukui et al.
95
The apparatus consisted of a
shuttle cage (36 cm ×18 cm ×30 cm) where the compartments were
separated by a sliding door (Coulbourn Instruments, Whitehall, PA).
Briefly, group-housed animals were divided evenly and designated as
foot-shock (FS) or no-foot-shock (NFS). Training was given in two
sessions separated by 24 h. Learned helplessness was induced in FS
mice by administering 100 inescapable 2-s foot shocks (0.2 mA) with
an intertrial interval of 10 s. NFS mice were placed in the cage but did
not receive any shocks and were allowed to explore for an equivalent
time period. Approximately 24 h after the second training session,
escape testing was performed, and both groups received 30 trials of
escapable 0.2 mA foot shocks. Animals had a 5 min acclimatization
period with the door between the compartments closed prior to
initiation of the escape testing. The door opened with shock onset, and
the trial terminated when the mouse crossed through the gate into the
adjacent safe chamber or if they failed to escape within 20 s.
Treatment of Mice with SSRIs. In order to test the response of
TPH2−/−mice and their wild-type controls to SSRIs, mice of both
genotypes were injected intraperitoneally with fluoxetine (20 mg/kg),
paroxetine (10 mg/kg), or citalopram (20 mg/kg). Drugs were
dissolved in 0.9% phosphate-buffered saline and injected in volumes of
0.2 mL per 20 g of body weight. Mice were subsequently tested in the
TST (see above) 30 min after injection. Doses of SSRIs were selected
from previous studies showing their effectiveness in the TST after
acute treatment and without causing disruption in locomotor
activity.
33,66,86
We noted that subsets of mice from both genotypes
were responsive to SSRIs in the TST and some were not responsive.
Human subjects given SSRIs in clinical studies are frequently classified
as responders and nonresponders,
37−39,96
as are mice treated with
antidepressants in behavioral tests,
40,86
so we followed this convention
presently. Data from SSRI treatment of wild-type and TPH2−/−
groups of mice was re-examined, and we classified mice in either
group as drug responders if their immobility times were 2 standard
deviations lower than the mean of their respective nontreated controls.
Mice that had the same immobility (<2 standard deviations from the
control mean) or higher times as nontreated controls were classified as
nonresponders.
Functional Characterization of the SERT in TPH2−/−mice.
The functional status of the SERT in wild-type and TPH2−/−mice was
assessed by measuring [3H]-5HT uptake into hippocampal synapto-
somes as previously described.
23
The amount of [3H]-5HT
accumulated by synaptosomes in a 10 min reaction was determined
by liquid scintillation counting and is expressed as nmol 5HT/(g·min).
The ability of the SSRIs fluoxetine (10 μM), paroxetine (50 μM), and
citalopram (50 μM) to block uptake of 5HT by the SERT was
determined by adding drugs to synaptosomes 15 min prior to
initiation of the uptake reaction with the addition of substrate. The
SSRI concentrations used were selected from published reports
showing inhibition of synaptosomal [3H]-5HT uptake.
Data Analysis. Data from the sucrose preference test (Figure 1A),
quinine preference test (Figure 1B), food intake (Figure 1C), FST
(Figure 2B), UCMS coat status (Figure 3A), UCMS grooming time
(Figure 3B), and UCMS body weights (Figure 3C) were analyzed
using a two-way repeated measures ANOVA, and post hoc
comparisons were made using Bonferonni’s multiple comparison
test. Results from the TST (Figure 2A) were analyzed using a
Student’sttest. Results from the post-UCMS sucrose preference test
(Figure 3D), LH test (Figure 4), and all tests of the effects of SSRIs on
SERT uptake of 5HT (Figure 6) were analyzed using a one-way
ANOVA, and post hoc comparisons were made using Tukey’s multiple
comparison test. Results on the effects of SSRIs on the TST (Figure 5)
were analyzed using a two-way ANOVA, and post hoc comparisons
were made using Bonferroni’s multiple comparison test. The pvalues
<0.05 were deemed statistically significant. All statistical analyses were
carried out using GraphPad Prism version 6.01 for Windows,
GraphPad Software, San Diego, CA, www.graphpad.com.
■ASSOCIATED CONTENT
*
SSupporting Information
Numbers of TPH2+/+ and TPH2−/−mice classified as
responders or nonresponders (specified by sex) to the SSRIs
fluoxetine, citalopram, and paroxetine, immobility time cutoff
values for defining whether a subject was classified as a
responder or nonresponder to SSRIs, and information on the
specific stressors used in the UCMS. This material is available
free of charge via the Internet at http://pubs.acs.org.
■AUTHOR INFORMATION
Corresponding Author
*Mailing address: Research & Development Service (11R),
John D. Dingell VA Medical Center, 4646 John R, Detroit, MI
48201-1916. Phone: 313-476-4457. Fax: 313-576-1112. E-mail:
donald.kuhn@wayne.edu.
Author Contributions
M.A.P., M.J.K., D.I.B., N.H.M., and C.E.S. conducted the in
vivo behavioral and pharmacological experiments. D.M.F. and
M.A.P. conducted the in vitro synaptosomal 5HT uptake
experiments. M.A.P., M.J.K., and D.M.K. interpreted the data.
M.A.P., M.J.K., and D.M.K. conceived of the project and wrote
the paper. All authors edited and approved the final version of
the manuscript.
Funding
A Department of Veterans Affairs grant to D.M.K. (No.
RX000458) supported this research.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
We thank Dr. Cynthia Arfken for her advice on the statistical
analyses of our data.
■ABBREVIATIONS
FST, forced swim test; LH, learned helplessness; SERT,
serotonin transporter; 5HT, serotonin; SSRI, selective-
serotonin-reuptake inhibitor; TST, tail suspension test;
TPH2, tryptophan hydroxylase 2; TPH, tryptophan hydrox-
ylase; UCMS, unpredictable chronic mild stress
■REFERENCES
(1) Steinbusch, H. W. (1981) Distribution of serotonin-immunor-
eactivity in the central nervous system of the rat-cell bodies and
terminals. Neuroscience 6, 557−618.
(2) Lucki, I. (1998) The spectrum of behaviors influenced by
serotonin. Biol. Psychiatry 44, 151−162.
(3) Prange, A. J. (1964) The pharmacology and biochemistry of
depression. Dis. Nerv. Syst. 25, 217−221.
(4) Schildkraut, J. J. (1965) The catecholamine hypothesis of
affective disorders: A review of supporting evidence. Am. J. Psychiatry
122, 509−522.
(5) Bunney, W. E., and Davis, J. M. (1965) Norepinephrine in
depressive reactions. A review. Arch. Gen. Psychiatry 13, 483−494.
(6) Serretti, A., and Artioli, P. (2004) The pharmacogenomics of
selective serotonin reuptake inhibitors. Pharmacogenomics J. 4, 233−
244.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXI

(7) Wenthur, C. J., Bennett, M. R., and Lindsley, C. W. (2014)
Classics in chemical neuroscience: Fluoxetine (Prozac). ACS Chem.
Neurosci. 5,14−23.
(8) Rush, A. J., Trivedi, M. H., Wisniewski, S. R., Nierenberg, A. A.,
Stewart, J. W., Warden, D., Niederehe, G., Thase, M. E., Lavori, P. W.,
Lebowitz, B. D., McGrath, P. J., Rosenbaum, J. F., Sackeim, H. A.,
Kupfer, D. J., Luther, J., and Fava, M. (2006) Acute and longer-term
outcomes in depressed outpatients requiring one or several treatment
steps: A STAR*D report. Am. J. Psychiatry 163, 1905−1917.
(9) Gartside, S. E., Umbers, V., Hajos, M., and Sharp, T. (1995)
Interaction between a selective 5-HT1A receptor antagonist and an
SSRI in vivo: Effects on 5-HT cell firing and extracellular 5-HT. Br. J.
Pharmacol. 115, 1064−1070.
(10) Kessler, R. C., Berglund, P., Demler, O., Jin, R., Merikangas, K.
R., and Walters, E. E. (2005) Lifetime prevalence and age-of-onset
distributions of DSM-IV disorders in the National Comorbidity Survey
Replication. Arch. Gen. Psychiatry 62, 593−602.
(11) Ressler, K. J., and Nemeroff, C. B. (2000) Role of serotonergic
and noradrenergic systems in the pathophysiology of depression and
anxiety disorders. Depress. Anxiety 12 (Suppl 1), 2−19.
(12) Trivedi, M. H., Rush, A. J., Wisniewski, S. R., Nierenberg, A. A.,
Warden, D., Ritz, L., Norquist, G., Howland, R. H., Lebowitz, B.,
McGrath, P. J., Shores-Wilson, K., Biggs, M. M., Balasubramani, G. K.,
and Fava, M. (2006) Evaluation of outcomes with citalopram for
depression using measurement-based care in STAR*D: Implications
for clinical practice. Am. J. Psychiatry 163,28−40.
(13) Holtzheimer, P. E., and Mayberg, H. S. (2011) Stuck in a rut:
Rethinking depression and its treatment. Trends Neurosci. 34,1−9.
(14) Kirsch, I., Deacon, B. J., Huedo-Medina, T. B., Scoboria, A.,
Moore, T. J., and Johnson, B. T. (2008) Initial severity and
antidepressant benefits: A meta-analysis of data submitted to the
Food and Drug Administration. PLoS Med. 5, No. e45.
(15) Khan, A., Faucett, J., Lichtenberg, P., Kirsch, I., and Brown, W.
A. (2012) A systematic review of comparative efficacy of treatments
and controls for depression. PLoS One 7, No. e41778.
(16) Araragi, N., and Lesch, K. P. (2013) Serotonin (5-HT) in the
regulation of depression-related emotionality: insight from 5-HT
transporter and tryptophan hydroxylase-2 knockout mouse models.
Curr. Drug Targets 14, 549−570.
(17) Jacobsen, J. P., Medvedev, I. O., and Caron, M. G. (2012) The
5-HT deficiency theory of depression: perspectives from a naturalistic
5-HT deficiency model, the tryptophan hydroxylase 2Arg439His
knockin mouse. Philos. Trans. R. Soc., B 367, 2444−2459.
(18) Lesch, K. P., Araragi, N., Waider, J., van den Hove, D., and
Gutknecht, L. (2012) Targeting brain serotonin synthesis: Insights
into neurodevelopmental disorders with long-term outcomes related
to negative emotionality, aggression and antisocial behaviour. Philos.
Trans. R. Soc., B 367, 2426−2443.
(19) Thomas, D. M., Angoa Perez, M., Francescutti-Verbeem, D. M.,
Shah, M. M., and Kuhn, D. M. (2010) The role of endogenous
serotonin in methamphetamine-induced neurotoxicity to dopamine
nerve endings of the striatum. J. Neurochem. 115, 595−605.
(20) Alenina, N., Kikic, D., Todiras, M., Mosienko, V., Qadri, F.,
Plehm, R., Boye, P., Vilianovitch, L., Sohr, R., Tenner, K., Hortnagl, H.,
and Bader, M. (2009) Growth retardation and altered autonomic
control in mice lacking brain serotonin. Proc. Natl. Acad. Sci. U.S.A.
106, 10332−10337.
(21) Narboux-Neme, N., Angenard, G., Mosienko, V., Klempin, F.,
Pitychoutis, P. M., Deneris, E., Bader, M., Giros, B., Alenina, N., and
Gaspar, P. (2013) Postnatal growth defects in mice with constitutive
depletion of central serotonin. ACS Chem. Neurosci. 4, 171−181.
(22) Gutknecht, L., Jacob, C., Strobel, A., Kriegebaum, C., Muller, J.,
Zeng, Y., Markert, C., Escher, A., Wendland, J., Reif, A., Mossner, R.,
Gross, C., Brocke, B., and Lesch, K. P. (2006) Tryptophan
hydroxylase-2 gene variation influences personality traits and disorders
related to emotional dysregulation. Int. J. Neuropsychopharmacol.,1−
12.
(23) Angoa-Perez, M., Kane, M. J., Briggs, D. I., Sykes, C. E., Shah,
M. M., Francescutti, D. M., Rosenberg, D. R., Thomas, D. M., and
Kuhn, D. M. (2012) Genetic depletion of brain 5HT reveals a
common molecular pathway mediating compulsivity and impulsivity. J.
Neurochem. 121, 974−984.
(24) Araragi, N., Mlinar, B., Baccini, G., Gutknecht, L., Lesch, K. P.,
and Corradetti, R. (2013) Conservation of 5-HT1A receptor-mediated
autoinhibition of serotonin (5-HT) neurons in mice with altered 5-HT
homeostasis. Front. Pharmacol. 4, 97.
(25) Waider, J., Proft, F., Langlhofer, G., Asan, E., Lesch, K. P., and
Gutknecht, L. (2013) GABA concentration and GABAergic neuron
populations in limbic areas are differentially altered by brain serotonin
deficiency in Tph2 knockout mice. Histochem. Cell Biol. 139, 267−281.
(26) Savelieva, K. V., Zhao, S., Pogorelov, V. M., Rajan, I., Yang, Q.,
Cullinan, E., and Lanthorn, T. H. (2008) Genetic disruption of both
tryptophan hydroxylase genes dramatically reduces serotonin and
affects behavior in models sensitive to antidepressants. PLoS One 3,
No. e3301.
(27) Gutknecht, L., Araragi, N., Merker, S., Waider, J., Sommerlandt,
F. M., Mlinar, B., Baccini, G., Mayer, U., Proft, F., Hamon, M., Schmitt,
A. G., Corradetti, R., Lanfumey, L., and Lesch, K. P. (2012) Impacts of
brain serotonin deficiency following Tph2 inactivation on develop-
ment and raphe neuron serotonergic specification. PLoS One 7,
No. e43157.
(28) Kriegebaum, C., Song, N. N., Gutknecht, L., Huang, Y., Schmitt,
A., Reif, A., Ding, Y. Q., and Lesch, K. P. (2010) Brain-specific
conditional and time-specific inducible Tph2 knockout mice possess
normal serotonergic gene expression in the absence of serotonin
during adult life. Neurochem. Int. 57, 512−517.
(29) Mosienko, V., Bert, B., Beis, D., Matthes, S., Fink, H., Bader, M.,
and Alenina, N. (2012) Exaggerated aggression and decreased anxiety
in mice deficient in brain serotonin. Transl. Psychiatry 2, e122.
(30) Kane, M. J., Angoa-Perez, M., Briggs, D. I., Sykes, C. E.,
Francescutti, D. M., Rosenberg, D. R., and Kuhn, D. M. (2012) Mice
genetically depleted of brain serotonin display social impairments,
communication deficits and repetitive behaviors: possible relevance to
autism. PLoS One 7, No. e48975.
(31) Mayorga, A. J., and Lucki, I. (2001) Limitations on the use of
the C57BL/6 mouse in the tail suspension test. Psychopharmacology
(Berlin, Ger.) 155, 110−112.
(32) David, D. J., Renard, C. E., Jolliet, P., Hascoet, M., and Bourin,
M. (2003) Antidepressant-like effects in various mice strains in the
forced swimming test. Psychopharmacology (Berlin, Ger.) 166, 373−
382.
(33) Crowley, J. J., Blendy, J. A., and Lucki, I. (2005) Strain-
dependent antidepressant-like effects of citalopram in the mouse tail
suspension test. Psychopharmacology (Berlin, Ger.) 183, 257−264.
(34) Lucki, I., Dalvi, A., and Mayorga, A. J. (2001) Sensitivity to the
effects of pharmacologically selective antidepressants in different
strains of mice. Psychopharmacology (Berlin, Ger.) 155, 315−322.
(35) Ripoll, N., David, D. J., Dailly, E., Hascoet, M., and Bourin, M.
(2003) Antidepressant-like effects in various mice strains in the tail
suspension test. Behav. Brain Res. 143, 193−200.
(36) Jacobson, L. H., and Cryan, J. F. (2007) Feeling strained?
Influence of genetic background on depression-related behavior in
mice: A review. Behav. Genet. 37, 171−213.
(37) Illi, A., Setala-Soikkeli, E., Viikki, M., Poutanen, O., Huhtala, H.,
Mononen, N., Lehtimaki, T., Leinonen, E., and Kampman, O. (2009)
5-HTR1A, 5-HTR2A, 5-HTR6, TPH1 and TPH2 polymorphisms and
major depression. Neuroreport 20, 1125−1128.
(38) Tzvetkov, M. V., Brockmoller, J., Roots, I., and Kirchheiner, J.
(2008) Common genetic variations in human brain-specific
tryptophan hydroxylase-2 and response to antidepressant treatment.
Pharmacogenet. Genomics 18, 495−506.
(39) Peters, E. J., Slager, S. L., McGrath, P. J., Knowles, J. A., and
Hamilton, S. P. (2004) Investigation of serotonin-related genes in
antidepressant response. Mol. Psychiatry 9, 879−889.
(40) Samuels, B. A., Leonardo, E. D., Gadient, R., Williams, A., Zhou,
J., David, D. J., Gardier, A. M., Wong, E. H., and Hen, R. (2011)
Modeling treatment-resistant depression. Neuropharmacology 61, 408−
413.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXJ

(41) Pollak, D. D., Rey, C. E., and Monje, F. J. (2010) Rodent
models in depression research: Classical strategies and new directions.
Ann. Med. 42, 252−264.
(42) Dulawa, S. C., and Hen, R. (2005) Recent advances in animal
models of chronic antidepressant effects: The novelty-induced
hypophagia test. Neurosci. Biobehav. Rev. 29, 771−783.
(43) O’Leary, O. F., Bechtholt, A. J., Crowley, J. J., Hill, T. E., Page,
M. E., and Lucki, I. (2007) Depletion of serotonin and catecholamines
block the acute behavioral response to different classes of
antidepressant drugs in the mouse tail suspension test. Psychopharma-
cology (Berlin, Ger.) 192, 357−371.
(44) Page, M. E., Detke, M. J., Dalvi, A., Kirby, L. G., and Lucki, I.
(1999) Serotonergic mediation of the effects of fluoxetine, but not
desipramine, in the rat forced swimming test. Psychopharmacology
(Berlin, Ger.) 147, 162−167.
(45) Wellman, C. L., Camp, M., Jones, V. M., Macpherson, K. P.,
Ihne, J., Fitzgerald, P., Maroun, M., Drabant, E., Bogdan, R., Hariri, A.
R., and Holmes, A. (2013) Convergent effects of mouse Pet-1 deletion
and human PET-1 variation on amygdala fear and threat processing.
Exp. Neurol. 250C, 260−269.
(46) Schaefer, T. L., Vorhees, C. V., and Williams, M. T. (2009)
Mouse plasmacytoma-expressed transcript 1 knock out induced 5-HT
disruption results in a lack of cognitive deficits and an anxiety
phenotype complicated by hypoactivity and defensiveness. Neuro-
science 164, 1431−1443.
(47) Beaulieu, J. M., Zhang, X., Rodriguiz, R. M., Sotnikova, T. D.,
Cools, M. J., Wetsel, W. C., Gainetdinov, R. R., and Caron, M. G.
(2008) Role of GSK3 beta in behavioral abnormalities induced by
serotonin deficiency. Proc. Natl. Acad. Sci. U.S.A. 105, 1333−1338.
(48) Zhang, X., Gainetdinov, R. R., Beaulieu, J. M., Sotnikova, T. D.,
Burch, L. H., Williams, R. B., Schwartz, D. A., Krishnan, K. R., and
Caron, M. G. (2005) Loss-of-function mutation in tryptophan
hydroxylase-2 identified in unipolar major depression. Neuron 45,
11−16.
(49) Jacobsen, J. P., Siesser, W. B., Sachs, B. D., Peterson, S., Cools,
M. J., Setola, V., Folgering, J. H., Flik, G., and Caron, M. G. (2012)
Deficient serotonin neurotransmission and depression-like serotonin
biomarker alterations in tryptophan hydroxylase 2 (Tph2) loss-of-
function mice. Mol. Psychiatry 17, 694−704.
(50) Sachs, B. D., Jacobsen, J. P., Thomas, T. L., Siesser, W. B.,
Roberts, W. L., and Caron, M. G. (2013) The effects of congenital
brain serotonin deficiency on responses to chronic fluoxetine. Transl.
Psychiatry 3, e291.
(51) Zhang, X., Beaulieu, J. M., Sotnikova, T. D., Gainetdinov, R. R.,
and Caron, M. G. (2004) Tryptophan hydroxylase-2 controls brain
serotonin synthesis. Science 305, 217.
(52) Sakowski, S. A., Geddes, T. J., and Kuhn, D. M. (2006) Mouse
tryptophan hydroxylase isoform 2 and the role of proline 447 in
enzyme function. J. Neurochem. 96, 758−765.
(53) Trullas, R., Jackson, B., and Skolnick, P. (1989) Genetic
differences in a tail suspension test for evaluating antidepressant
activity. Psychopharmacology (Berlin, Ger.) 99, 287−288.
(54) Cervo, L., Canetta, A., Calcagno, E., Burbassi, S., Sacchetti, G.,
Caccia, S., Fracasso, C., Albani, D., Forloni, G., and Invernizzi, R. W.
(2005) Genotype-dependent activity of tryptophan hydroxylase-2
determines the response to citalopram in a mouse model of
depression. J. Neurosci. 25, 8165−8172.
(55) Jacobsen, J. P., Nielsen, E. O., Hummel, R., Redrobe, J. P.,
Mirza, N., and Weikop, P. (2008) Insensitivity of NMRI mice to
selective serotonin reuptake inhibitors in the tail suspension test can
be reversed by co-treatment with 5-hydroxytryptophan. Psychophar-
macology (Berlin, Ger.) 199, 137−150.
(56) Tenner, K., Qadri, F., Bert, B., Voigt, J. P., and Bader, M. (2008)
The mTPH2 C1473G single nucleotide polymorphism is not
responsible for behavioural differences between mouse strains.
Neurosci. Lett. 431,21−25.
(57) Berger, S. M., Weber, T., Perreau-Lenz, S., Vogt, M. A., Gartside,
S. E., Maser-Gluth, C., Lanfumey, L., Gass, P., Spanagel, R., and
Bartsch, D. (2012) A functional Tph2 C1473G polymorphism causes
an anxiety phenotype via compensatory changes in the serotonergic
system. Neuropsychopharmacology 37, 1986−1998.
(58) Vazquez, G. H., Baldessarini, R. J., and Tondo, L. (2014) Co-
occurrence of anxiety and bipolar disorders: Clinical and therapeutic
overview. Depress. Anxiety 31, 196−206.
(59) D’Avanzato, C., Martinez, J., Attiullah, N., Friedman, M., Toba,
C., Boerescu, D. A., and Zimmerman, M. (2013) Anxiety symptoms
among remitted depressed outpatients: Prevalence and association
with quality of life and psychosocial functioning. J. Affect. Disord. 151,
401−404.
(60) Belzung, C. (2014) Innovative drugs to treat depression: Did
animal models fail to be predictive or did clinical trials fail to detect
effects? Neuropsychopharmacology 39, 1041−1051.
(61) Lerch-Haner, J. K., Frierson, D., Crawford, L. K., Beck, S. G.,
and Deneris, E. S. (2008) Serotonergic transcriptional programming
determines maternal behavior and offspring survival. Nat. Neurosci. 11,
1001−1003.
(62) Diaz, S. L., Narboux-Neme, N., Trowbridge, S., Scotto-
Lomassese, S., Kleine Borgmann, F. B., Jessberger, S., Giros, B.,
Maroteaux, L., Deneris, E., and Gaspar, P. (2013) Paradoxical increase
in survival of newborn neurons in the dentate gyrus of mice with
constitutive depletion of serotonin. Eur. J. Neurosci. 38, 2650−2658.
(63) Klempin, F., Beis, D., Mosienko, V., Kempermann, G., Bader,
M., and Alenina, N. (2013) Serotonin is required for exercise-induced
adult hippocampal neurogenesis. J. Neurosci. 33, 8270−8275.
(64) Hamon, M., and Blier, P. (2013) Monoamine neurocircuitry in
depression and strategies for new treatments. Prog. Neuropsychophar-
macol. Biol. Psychiatry 45,54−63.
(65) Willner, P., Scheel-Kruger, J., and Belzung, C. (2013) The
neurobiology of depression and antidepressant action. Neurosci.
Biobehav. Rev. 37, 2331−2371.
(66) Cryan, J. F., O’Leary, O. F., Jin, S. H., Friedland, J. C., Ouyang,
M., Hirsch, B. R., Page, M. E., Dalvi, A., Thomas, S. A., and Lucki, I.
(2004) Norepinephrine-deficient mice lack responses to antidepres-
sant drugs, including selective serotonin reuptake inhibitors. Proc. Natl.
Acad. Sci. U.S.A. 101, 8186−8191.
(67) Tytgat, J., Maertens, C., and Daenens, P. (1997) Effect of
fluoxetine on a neuronal, voltage-dependent potassium channel
(Kv1.1). Br. J. Pharmacol. 122, 1417−1424.
(68) Prasad, H. C., Zhu, C. B., McCauley, J. L., Samuvel, D. J.,
Ramamoorthy, S., Shelton, R. C., Hewlett, W. A., Sutcliffe, J. S., and
Blakely, R. D. (2005) Human serotonin transporter variants display
altered sensitivity to protein kinase G and p38 mitogen-activated
protein kinase. Proc. Natl. Acad. Sci. U.S.A. 102, 11545−11550.
(69) Carneiro, A. M., and Blakely, R. D. (2006) Serotonin-, protein
kinase C-, and Hic-5-associated redistribution of the platelet serotonin
transporter. J. Biol. Chem. 281, 24769−24780.
(70) Steiner, J. A., Carneiro, A. M., Wright, J., Matthies, H. J., Prasad,
H. C., Nicki, C. K., Dostmann, W. R., Buchanan, C. C., Corbin, J. D.,
Francis, S. H., and Blakely, R. D. (2009) cGMP-dependent protein
kinase Ialpha associates with the antidepressant-sensitive serotonin
transporter and dictates rapid modulation of serotonin uptake. Mol.
Brain 2, 26.
(71) Zhu, C. B., Carneiro, A. M., Dostmann, W. R., Hewlett, W. A.,
and Blakely, R. D. (2005) p38 MAPK activation elevates serotonin
transport activity via a trafficking-independent, protein phosphatase
2A-dependent process. J. Biol. Chem. 280, 15649−15658.
(72) Zhu, C. B., Hewlett, W. A., Francis, S. H., Corbin, J. D., and
Blakely, R. D. (2004) Stimulation of serotonin transport by the cyclic
GMP phosphodiesterase-5 inhibitor sildenafil. Eur. J. Pharmacol. 504,
1−6.
(73) Gulbins, E., Palmada, M., Reichel, M., Luth, A., Bohmer, C.,
Amato, D., Muller, C. P., Tischbirek, C. H., Groemer, T. W.,
Tabatabai, G., Becker, K. A., Tripal, P., Staedtler, S., Ackermann, T. F.,
van Brederode, J., Alzheimer, C., Weller, M., Lang, U. E., Kleuser, B.,
Grassme, H., and Kornhuber, J. (2013) Acid sphingomyelinase-
ceramide system mediates effects of antidepressant drugs. Nat. Med.
19, 934−938.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXK

(74) Di Benedetto, M., D’Addario, C., Candeletti, S., and Romualdi,
P. (2007) Alterations of CREB and DARPP-32 phosphorylation
following cocaine and monoaminergic uptake inhibitors. Brain Res.
1128,33−39.
(75) Frechilla, D., Otano, A., and Del Rio, J. (1998) Effect of chronic
antidepressant treatment on transcription factor binding activity in rat
hippocampus and frontal cortex. Prog. Neuropsychopharmacol. Biol.
Psychiatry 22, 787−802.
(76) Malberg, J. E., Eisch, A. J., Nestler, E. J., and Duman, R. S.
(2000) Chronic antidepressant treatment increases neurogenesis in
adult rat hippocampus. J. Neurosci. 20, 9104−9110.
(77) Perera, T. D., Dwork, A. J., Keegan, K. A., Thirumangalakudi, L.,
Lipira, C. M., Joyce, N., Lange, C., Higley, J. D., Rosoklija, G., Hen, R.,
Sackeim, H. A., and Coplan, J. D. (2011) Necessity of hippocampal
neurogenesis for the therapeutic action of antidepressants in adult
nonhuman primates. PLoS One 6, No. e17600.
(78) Surget, A., Saxe, M., Leman, S., Ibarguen-Vargas, Y., Chalon, S.,
Griebel, G., Hen, R., and Belzung, C. (2008) Drug-dependent
requirement of hippocampal neurogenesis in a model of depression
and of antidepressant reversal. Biol. Psychiatry 64, 293−301.
(79) Snyder, J. S., Soumier, A., Brewer, M., Pickel, J., and Cameron,
H. A. (2011) Adult hippocampal neurogenesis buffers stress responses
and depressive behaviour. Nature 476, 458−461.
(80) Strekalova, T., Spanagel, R., Bartsch, D., Henn, F. A., and Gass,
P. (2004) Stress-induced anhedonia in mice is associated with deficits
in forced swimming and exploration. Neuropsychopharmacology 29,
2007−2017.
(81) Papp, M., Willner, P., and Muscat, R. (1991) An animal model
of anhedonia: attenuation of sucrose consumption and place
preference conditioning by chronic unpredictable mild stress.
Psychopharmacology (Berlin, Ger.) 104, 255−259.
(82) Steru, L., Chermat, R., Thierry, B., and Simon, P. (1985) The
tail suspension test: a new method for screening antidepressants in
mice. Psychopharmacology (Berlin, Ger.) 85, 367−370.
(83) Porsolt, R. D., Brossard, G., Hautbois, C., Roux, S. (2001)
Rodent models of depression: Forced swimming and tail suspension
behavioral despair tests in rats and mice, Current Protocols in
Neuroscience, Chapter 8, Unit 8.10A, J. Wiley, New York.
(84) Tye, K. M., Mirzabekov, J. J., Warden, M. R., Ferenczi, E. A.,
Tsai, H. C., Finkelstein, J., Kim, S. Y., Adhikari, A., Thompson, K. R.,
Andalman, A. S., Gunaydin, L. A., Witten, I. B., and Deisseroth, K.
(2013) Dopamine neurons modulate neural encoding and expression
of depression-related behaviour. Nature 493, 537−541.
(85) Porsolt, R. D., Anton, G., Blavet, N., and Jalfre, M. (1978)
Behavioural despair in rats: A new model sensitive to antidepressant
treatments. Eur. J. Pharmacol. 47, 379−391.
(86) Thompson, B. J., Jessen, T., Henry, L. K., Field, J. R., Gamble, K.
L., Gresch, P. J., Carneiro, A. M., Horton, R. E., Chisnell, P. J., Belova,
Y., McMahon, D. G., Daws, L. C., and Blakely, R. D. (2011)
Transgenic elimination of high-affinity antidepressant and cocaine
sensitivity in the presynaptic serotonin transporter. Proc. Natl. Acad.
Sci. U.S.A. 108, 3785−3790.
(87) Willner, P. (1997) Validity, reliability and utility of the chronic
mild stress model of depression: a 10-year review and evaluation.
Psychopharmacology (Berlin, Ger.) 134, 319−329.
(88) Willner, P., Towell, A., Sampson, D., Sophokleous, S., and
Muscat, R. (1987) Reduction of sucrose preference by chronic
unpredictable mild stress, and its restoration by a tricyclic
antidepressant. Psychopharmacology (Berlin, Ger.) 93, 358−364.
(89) Monleon, S., D’Aquila, P., Parra, A., Simon, V. M., Brain, P. F.,
and Willner, P. (1995) Attenuation of sucrose consumption in mice by
chronic mild stress and its restoration by imipramine. Psychopharma-
cology (Berlin, Ger.) 117, 453−457.
(90) Ibarguen-Vargas, Y., Surget, A., Touma, C., Palme, R., and
Belzung, C. (2008) Multifaceted strain-specific effects in a mouse
model of depression and of antidepressant reversal. Psychoneuroendoc-
rinology 33, 1357−1368.
(91) Mineur, Y. S., Belzung, C., and Crusio, W. E. (2006) Effects of
unpredictable chronic mild stress on anxiety and depression-like
behavior in mice. Behav. Brain Res. 175,43−50.
(92) Farooq, R. K., Isingrini, E., Tanti, A., Le Guisquet, A. M., Arlicot,
N., Minier, F., Leman, S., Chalon, S., Belzung, C., and Camus, V.
(2012) Is unpredictable chronic mild stress (UCMS) a reliable model
to study depression-induced neuroinflammation? Behav. Brain Res.
231, 130−137.
(93) Ducottet, C., and Belzung, C. (2005) Correlations between
behaviours in the elevated plus-maze and sensitivity to unpredictable
subchronic mild stress: evidence from inbred strains of mice. Behav.
Brain Res. 156, 153−162.
(94) Yalcin, I., Aksu, F., and Belzung, C. (2005) Effects of
desipramine and tramadol in a chronic mild stress model in mice
are altered by yohimbine but not by pindolol. Eur. J. Pharmacol. 514,
165−174.
(95) Fukui, M., Rodriguiz, R. M., Zhou, J., Jiang, S. X., Phillips, L. E.,
Caron, M. G., and Wetsel, W. C. (2007) Vmat2 heterozygous mutant
mice display a depressive-like phenotype. J. Neurosci. 27, 10520−
10529.
(96) Sackeim, H. A. (2001) The definition and meaning of
treatment-resistant depression. J. Clin.Psychiatry 62 (Suppl 16), 10−
17.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXL