ArticlePDF Available

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

Authors:

Abstract and Figures

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.
Content may be subject to copyright.
Mice Genetically Depleted of Brain Serotonin Do Not Display a
Depression-like Behavioral Phenotype
Mariana Angoa-Pé
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 eect 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 dierential between the onset of
5HT reuptake inhibition by SSRIs (minutes) and onset of their antidepressant ecacy (weeks to months), when considered with
their overall poor therapeutic eectiveness, 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 aective-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 signicant reductions in immobility time on the tail suspension test,
indicative of antidepressant drug eects. 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 decit hyperactivity disorder, sudden infant
death syndrome, and autism. Perhaps the strongest association
between impaired 5HT function and clinically signicant
psychopathology is for depression. Since the monoamine
theory of depression was posited about 50 years ago,
35
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 eects 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 6070% of patients without symptomatic
relief.
12,13
Some even question whether SSRIs have clinically
signicant therapeutic value beyond a placebo eect in treating
any form of depression.
14,15
Despite the long-held hypothesis
that 5HT neuronal dysfunction is an underlying cause of
depression,
1618
the relatively poor ecacy 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
eectively 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-specic 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, XXXXXX
mice.
2022
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 ring, 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.
19,20,2328
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 decits 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 dierent 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 uoxetine, 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 eect of days was
signicant (F3,66 = 4.73, p= 0.005, two-way repeated measures
ANOVA). Sucrose preference was 8085% for both groups.
Total uid 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
(2025% of total uid intake). The main eect of days (F3,66
= 4.13, p= 0.009) and genotype (F1,22 = 7.11, p= 0.014) were
signicant by two-way repeated measures ANOVA, whereas
their interaction was not. TPH2/mice actually drank
signicantly less quinine solution than wild-type controls on
day 2 (Bonferronis multiple comparison test, p< 0.05). Food
consumption was also measured in addition to uid intake, and
the results in Figure 1C indicate that the main eects of days
(F3,66 = 8.16, p= 0.0001) and the days ×genotype interaction
(F3,66 = 3.94, p= 0.011) were signicant, whereas the main
eect of genotype was not. Post hoc comparisons revealed that
food intake in the TPH2/mice was signicantly lower on
days 24(p< 0.050.0001, Bonferonnis 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 signicantly less quinine than WT controls while drinking signicantly more water. (C) KO mice have
slightly but not signicantly 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.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXXXXXB
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 eects of genotype (F1,22 = 5.05, p=
0.034) and trial (F1,22 = 32.41, p< 0.0001) were signicant by
two-way repeated measures ANOVA, but the genotype ×trial
interaction was not. TPH2/mice remained immobile for
signicantly less time than wild-type controls (i.e., less
depressed) on the rst trial (p< 0.05, Bonferroni multiple
comparison test) but this dierence 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 eects of weeks
(F6,126 = 25.71, p< 0.0001) and genotype (F1,21 = 9.85, p<
0.005) were signicant by two-way repeated measures ANOVA,
but their interaction was not. Post hoc analyses indicated that
both TPH2/(p< 0.0001, Bonferronis 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 signicantly less time immobile than WT controls on the rst 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. Eects 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 signicantly at weeks 36 and KO mice diered from WT only at the 3 week time point in the stress protocol. (B) Grooming time (in sec)
in the splash test diminished signicantly in KO mice but not in WT controls, and no dierence was seen between genotypes. (C) Body weights (in
g) of both WT and KO mice increased signicantly 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 signicant decreases in sucrose preference by comparison to their respective controls and
WT mice showed a signicantly 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.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXXXXXC
test) and TPH2+/+ mice (p< 0.050.0001, Bonferronis
multiple comparison test) degraded signicantly from their
starting coat status at weeks 36. Total grooming times are
presented in Figure 3B and show that the main eect of weeks
was signicant (F6,126 = 4.88, p= 0.0002, two-way repeated
measures ANOVA) while the main eect of genotype and the
weeks ×genotype interaction were not. Post hoc analyses
revealed that TPH2/mice grooming time was signicantly
decreased from starting values at weeks 46(p< 0.050.001,
Bonferronis multiple comparison test) whereas wild-type mice
did not vary over the weeks 06. Body weights of mice were
measured throughout the UCMS protocol, and the results are
presented in Figure 3C. The main eect of weeks was
signicant (F6,120 = 17.9, p< 0.0001), whereas the main eect
of genotype and the weeks ×genotype interaction were not.
Post hoc analyses revealed that the body weights of TPH2/
mice increased slightly at weeks 36 compared with starting
body weights (p< 0.0010.0001, Bonferronismultiple
comparison test) and wild-type controls diered signicantly
from their starting weights at weeks 16(p< 0.050.001,
Bonferronis 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 eects
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 signicant by two-way ANOVA. Wild-type mice
developed depression-like anhedonia with a signicant
reduction in sucrose preference from 80% to 30% (p<
0.0001, Bonferronis multiple comparison test). TPH2/mice
also showed a signicant reduction in sucrose preference from
80% to 45% after exposure to the UCMS protocol
(Bonferronis multiple comparison test, p< 0.0001). The
extent of the reduction in sucrose preference after the UCMS
procedure was signicantly greater in wild-type controls by
comparison to TPH2/mice (Bonferronis 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 eect of treatment was
signicant (F3,20 = 5.92, p= 0.005, one-way ANOVA), and both
genotypes showed a signicant increase in latency to escape on
the test day (p< 0.05 for both, Tukeys multiple comparison
test). The magnitude of the eect 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.
3236
Therefore, we tested TPH2/mice for their response to the
SSRIs uoxetine, 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 uoxetine, the main
eect of drug was signicant (F1,53 = 16.13, p= 0.0002, two-way
ANOVA) but the main eect of genotype and the drug ×
genotype interaction were not. Wild-type mice showed a
signicant reduction in immobility time after treatment with
uoxetine (p< 0.05, Bonferronis multiple comparison test).
When mice were treated with paroxetine (Figure 5B), the main
eects 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 signicant when analyzed by two-way ANOVA.
Post hoc comparisons showed that paroxetine signicantly
reduced immobility times on the TST for wild-type controls (p
< 0.0001, Bonferronis multiple comparison test) but did not
change the behavior of TPH2/mice. The eect of citalopram
on TST performance is presented in Figure 5C, and the gure
shows that the main eects 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 signicant by two-way
ANOVA. Wild-type mice showed signicant reductions in
immobility times after citalopram treatment (p< 0.0001,
Bonferronis multiple comparison test) whereas the slight
reduction seen in TPH2/mice did not reach signicance.
Although TPH2/mice did not respond as groups to SSRI
treatment with signicant reductions in TST immobility times
(Figure 5AC), we noted that some individual TPH2/mice
clearly displayed substantial reductions after drug treatment.
Depressed patients treated in clinical trials with SSRIs are
commonly classied as responders and nonresponders,
3739
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 classied 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 classied as non-
responders. Using this classication, it was observed that 33%
of WT controls and 19% of TPH2/mice were responders to
uoxetine. The results of this analytical approach are presented
in Figure 5DF. Treatment with uoxetine resulted in a
signicant main eect of drug (F2,51 = 27.38, p< 0.0001, two-
way ANOVA). The main eect of genotype and the drug ×
genotype interaction were not signicant. Post hoc compar-
isons revealed that TPH2+/+ mice that responded to uoxetine
had signicantly shorter immobility times than controls (p<
0.0001, Bonferronis multiple comparison test) and non-
responsive drug-treated mice (p< 0.001, Bonferronis multiple
Figure 4. Eects 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 signicant increases in latency to escape on the test day
compared with the NS condition. WT and KO mice did not dier in
the NS and S test conditions, respectively. Data are expressed as means
±SEM for 6 mice per group. The *indicates p< 0.05.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXXXXXD
comparison test). Fluoxetine nonresponders were not dierent
from untreated controls. Similarly, TPH2/responders
showed signicantly shorter immobility times by comparison
to both untreated controls (p< 0.0001, Bonferronis multiple
comparison test) and nonresponsive drug-treated mice (p<
0.001, Bonferronis multiple comparison test) and among
TPH2/mice, untreated controls were not dierent from
nonresponders. The pattern of response to paroxetine was
Figure 5. Eects 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) uoxetine (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 signicantly 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 dened
in Supporting Information, Tables 1 and 2. Both WT and KO mice responded signicantly to (D) uoxetine 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 dier from NR mice in the uoxetine 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. Eects 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) uoxetine (10 μM), (B) paroxetine (50 μM), and
(C) citalopram (50 μM). All SSRIs signicantly 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.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXXXXXE
similar to that of uoxetine 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 classied as citalopram responders whereas
only 31% of treated TPH2/mice were responders. The main
eects 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 signicant when analyzed by two-way
ANOVA. Post hoc comparisons revealed that TPH2+/+ mice
that responded to citalopram had signicantly shorter
immobility times than controls (p< 0.0001, Bonferronis
multiple comparison test) and nonresponsive drug-treated mice
(p< 0.05, Bonferronis multiple comparison test). Citalopram
nonresponders were not dierent from untreated controls.
Similarly, TPH2/responders showed signicantly shorter
immobility times by comparison to both untreated controls (p
< 0.0001, Bonferronis multiple comparison test) and non-
responsive drug-treated mice (p< 0.0001, Bonferronis multiple
comparison test) and among TPH2/mice, untreated controls
were not dierent from nonresponders. The number of
TPH2+/+ and TPH2/mice classied as responders and
nonresponders (and specied by sex) to the SSRIs along with
the immobility time cutovalues used to make these
classications 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
signicant 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 eects via inhibition of SERT, it was
important to conrm 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 signicantly higher
in TPH2/mice compared with wild-type controls. This result
is consistent with our previous nding of slightly increased
synaptosomal [3H]-5HT uptake in TPH2/mice.
23
The main
eect of drug on uptake was signicant for uoxetine (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 eect of genotype was not signicant
for any drug. Figure 6A shows that uoxetine leads to a
signicant reduction in [3H]-5HT uptake in synaptosomes
from both wild-type (p< 0.05, Tukeys multiple comparison
test) and TPH2/mice (p<0.001,Tukeysmultiple
comparison test). Paroxetine also signicantly reduced [3H]-
5HT uptake in wild-type (p<0.05,Tukeysmultiple
comparison test) and TPH2/mice (p< 0.05, Tukeys
multiple comparison test) as shown in Figure 6B. Finally, it can
be seen in Figure 6C that citalopram signicantly reduced the
uptake of [3H]-5HT in wild-type (p< 0.05, Tukeys multiple
comparison test) and TPH2/mice (p< 0.05, Tukeys
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 uoxetine, citalopram,
and paroxetine on the TST. Defects in 5HT neurotransmission
have long been implicated as causal factors in depression.
1618
Three dierent 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 eect
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 dierent tests of depressive-
like behavior can yield conicting 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
signicantly 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 ve 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 eects 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 dier from wild-type controls in the TST
and FST.
45,46
Savelieva and colleagues
26
reported that only
male TPH2/mice spent signicantly 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 dierent 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
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXXXXXF
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
signicant 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 signicant 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 dierent 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 ndings show that while some of the variation
in responsiveness to SSRIs can be attributed to the specic 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 dier 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 dierent 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 ospring 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
eects of SSRIs, which increase synaptic 5HT, but mice
genetically modied 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
signicantly decreased by uoxetine, paroxetine, and citalo-
pram, whereas those of TPH2/mice were not. These results
conrm 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 eects 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 nding, we classied
both TPH2/and wild-type controls as drug responders and
nonresponders much as depressed individuals are classied 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 nding emerged and conrmed that
some TPH2/mice indeed responded to SSRI treatment with
signicant reductions in immobility times on the TST.
Assuming that the SSRIs exert this eect 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 eects 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 signi-
cantly to SSRI treatment than TPH2+/+ mice (2036%
TPH2/responders compared to 3392% TPH2+/+ res-
ponders). Nevertheless, the reductions in TST immobility
times for responsive TPH2/mice were quite large and highly
statistically signicant. 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
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXXXXXG
interpreted with caution and should be expanded to include
additional behavioral tests, it is still quite interesting that these
mice show anti-depressant-likechanges 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, uoxetine, 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 eects of the SSRIs on selected
TPH2/mice are mediated by norepinephrine in the absence
of 5HT. In addition, these drugs can have broad inuence on
brain function via interactions with ion channels,
67
protein
kinases,
6870
phosphatases,
71
and phosphodiasterases.
72
At least
uoxetine 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 ecacy in the
treatment of depression.
7779
Together, the present results
suggest the possibility that SSRIs can exert antidepressant
eects 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 ecacy 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. Ospring 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 (46 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 reect groups of mice from at least 34
independent litters. Separate cohorts of adult mice (1012 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 eect 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 Scientic, Braintree,
MA) containing tap water for 2 days. Sipper tubes contained ball
bearings to minimize loss of uid 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 uid intake (i.e., 1 g = 1 mL of uid).
To assess uid 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 uid 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 quantied 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
despairwas 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 (11.5 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 dened
as a lack of movement or struggling and motionless hanging. The time
spent immobile by mice was recorded by 23 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 rst 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 rst 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 dened 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 23 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
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXXXXXH
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 23 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).
Briey, 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 uoxetine (20 mg/kg),
paroxetine (10 mg/kg), or citalopram (20 mg/kg). Drugs were
dissolved in 0.9% phosphate-buered 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 eectiveness 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 classied
as responders and nonresponders,
3739,96
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 classied 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 classied 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 uoxetine (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 Bonferonnis multiple comparison
test. Results from the TST (Figure 2A) were analyzed using a
Studentsttest. Results from the post-UCMS sucrose preference test
(Figure 3D), LH test (Figure 4), and all tests of the eects of SSRIs on
SERT uptake of 5HT (Figure 6) were analyzed using a one-way
ANOVA, and post hoc comparisons were made using Tukeys multiple
comparison test. Results on the eects of SSRIs on the TST (Figure 5)
were analyzed using a two-way ANOVA, and post hoc comparisons
were made using Bonferronis multiple comparison test. The pvalues
<0.05 were deemed statistically signicant. 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 classied as
responders or nonresponders (specied by sex) to the SSRIs
uoxetine, citalopram, and paroxetine, immobility time cuto
values for dening whether a subject was classied as a
responder or nonresponder to SSRIs, and information on the
specic 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 nal version of
the manuscript.
Funding
A Department of Veterans Aairs grant to D.M.K. (No.
RX000458) supported this research.
Notes
The authors declare no competing nancial 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, 557618.
(2) Lucki, I. (1998) The spectrum of behaviors influenced by
serotonin. Biol. Psychiatry 44, 151162.
(3) Prange, A. J. (1964) The pharmacology and biochemistry of
depression. Dis. Nerv. Syst. 25, 217221.
(4) Schildkraut, J. J. (1965) The catecholamine hypothesis of
affective disorders: A review of supporting evidence. Am. J. Psychiatry
122, 509522.
(5) Bunney, W. E., and Davis, J. M. (1965) Norepinephrine in
depressive reactions. A review. Arch. Gen. Psychiatry 13, 483494.
(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, XXXXXXI
(7) Wenthur, C. J., Bennett, M. R., and Lindsley, C. W. (2014)
Classics in chemical neuroscience: Fluoxetine (Prozac). ACS Chem.
Neurosci. 5,1423.
(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, 19051917.
(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, 10641070.
(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, 593602.
(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), 219.
(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 163,2840.
(13) Holtzheimer, P. E., and Mayberg, H. S. (2011) Stuck in a rut:
Rethinking depression and its treatment. Trends Neurosci. 34,19.
(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, 549570.
(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, 24442459.
(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, 24262443.
(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, 595605.
(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, 1033210337.
(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, 171181.
(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, 974984.
(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, 267281.
(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, 512517.
(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, 110112.
(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, 257264.
(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, 315322.
(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, 193200.
(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, 171213.
(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, 11251128.
(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, 495506.
(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, 879889.
(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, XXXXXXJ
(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, 252264.
(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, 771783.
(43) OLeary, 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, 357371.
(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, 162167.
(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, 260269.
(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, 14311443.
(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, 13331338.
(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,
1116.
(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, 694704.
(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, 758765.
(53) Trullas, R., Jackson, B., and Skolnick, P. (1989) Genetic
differences in a tail suspension test for evaluating antidepressant
activity. Psychopharmacology (Berlin, Ger.) 99, 287288.
(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, 81658172.
(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, 137150.
(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. 431,2125.
(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, 19861998.
(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, 196206.
(59) DAvanzato, 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,
401404.
(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, 10411051.
(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,
10011003.
(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, 26502658.
(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, 82708275.
(64) Hamon, M., and Blier, P. (2013) Monoamine neurocircuitry in
depression and strategies for new treatments. Prog. Neuropsychophar-
macol. Biol. Psychiatry 45,5463.
(65) Willner, P., Scheel-Kruger, J., and Belzung, C. (2013) The
neurobiology of depression and antidepressant action. Neurosci.
Biobehav. Rev. 37, 23312371.
(66) Cryan, J. F., OLeary, 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, 81868191.
(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, 14171424.
(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, 1154511550.
(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, 2476924780.
(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, 1564915658.
(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,
16.
(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, 934938.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn500096g |ACS Chem. Neurosci. XXXX, XXX, XXXXXXK
(74) Di Benedetto, M., DAddario, C., Candeletti, S., and Romualdi,
P. (2007) Alterations of CREB and DARPP-32 phosphorylation
following cocaine and monoaminergic uptake inhibitors. Brain Res.
1128,3339.
(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, 787802.
(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, 91049110.
(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, 293301.
(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, 458461.
(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,
20072017.
(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, 255259.
(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, 367370.
(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, 537541.
(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, 379391.
(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, 37853790.
(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, 319329.
(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, 358364.
(89) Monleon, S., DAquila, 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, 453457.
(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, 13571368.
(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. 175,4350.
(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, 130137.
(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, 153162.
(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,
165174.
(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, XXXXXXL
... Mice lacking TPH2 have been generated by several laboratories [56][57][58][59]. Characterization of these mice revealed a number of phenotypes associated with the loss of brain 5-HT, including early postnatal growth retardation, maternal neglect, increased aggression, sleep disturbances, social deficits, reduced anxiety-like behavior, and increased food intake [56,[60][61][62]. ...
... These results partially support our previous study, in which Tph2 −/− mice showed a reduced latency to immobility and increased immobility time in the FST, although, in this case, the swimming and climbing time were not assessed independently [60]. The other literature data regarding the behavior of Tph2 −/− mice in the FST are heterogeneous: either a reduced latency to immobility or a mild antidepressant phenotype were reported [57][58][59]. As previously discussed [60], the expression of depression-like behavior in Tph2 −/− mice in the FST may depend on specific protocols used to evaluate depressionlike behavior (e.g., FST session day), age of the animals, or genetic background on which the knockouts were created. ...
... Importantly, the increased ethanol consumption observed in mice depleted in brain 5-HT corresponds well to the characteristics of a subset of alcoholics with early-onset type II alcoholism (Cloninger type-2-like) that has also been associated with 5-HT hypofunction (for review [7]). Many common typologies, such as comorbid psychopathology (i.e., depression), antisocial phenotype, or no reactivity to SSRIs [7,58,62], have been found between Tph2 −/− mice and type II alcoholics, despite differences in the origin of impaired 5-HT transmission (Tph2 deletion; present study vs. homozygosity for the long (L) allele for 5-HTT gene; [7]). Future studies on Tph2 −/− mice will be instrumental in discovering novel non-serotoninergic medications for the treatment of type II alcoholics. ...
Article
Full-text available
Indirect evidence supports a link between disrupted serotonin (5-hydroxytryptamine; 5-HT) signaling in the brain and addictive behaviors. However, the effects of hyposerotonergia on ethanol drinking behavior are contradictory. In this study, mice deficient in tryptophan hydroxylase 2 (Tph2−/−), the rate-limiting enzyme of 5-HT synthesis in the brain, were used to assess the role of central 5-HT in alcohol drinking behavior. Life-long 5-HT depletion in these mice led to an increased ethanol consumption in comparison to wild-type animals in a two-bottle choice test. Water consumption was increased in naïve 5-HT-depleted mice. However, exposure of Tph2−/− animals to ethanol resulted in the normalization of water intake to the level of wild-type mice. Tph2 deficiency in mice did not interfere with ethanol-evoked antidepressant response in the forced swim test. Gene expression analysis in wild-type animals revealed no change in Tph2 expression in the brain of mice consuming ethanol compared to control mice drinking water. However, within the alcohol-drinking group, inter-individual differences in chronic ethanol intake correlated with Tph2 transcript levels. Taken together, central 5-HT is an important modulator of drinking behavior in mice but is not required for the antidepressant effects of ethanol.
... We observed altered neonatal USVs, anhedonia and increased activity during the FST in male mHFD offspring. These behaviours are associated with diminished brain 5-HT in mice [25][26][27][28][29] . Previous research has demonstrated that brain 5-HT levels during discrete windows of development are dependent on placental 5-HT synthesis 30 . ...
Article
Full-text available
High maternal weight is associated with detrimental outcomes in offspring, including increased susceptibility to neurological disorders such as anxiety, depression and communicative disorders. Despite widespread acknowledgement of sex biases in the development of these disorders, few studies have investigated potential sex-biased mechanisms underlying disorder susceptibility. Here, we show that a maternal high-fat diet causes endotoxin accumulation in fetal tissue, and subsequent perinatal inflammation contributes to sex-specific behavioural outcomes in offspring. In male offspring exposed to a maternal high-fat diet, increased macrophage Toll-like receptor 4 signalling results in excess microglial phagocytosis of serotonin (5-HT) neurons in the developing dorsal raphe nucleus, decreasing 5-HT bioavailability in the fetal and adult brains. Bulk sequencing from a large cohort of matched first-trimester human samples reveals sex-specific transcriptome-wide changes in placental and brain tissue in response to maternal triglyceride accumulation (a proxy for dietary fat content). Further, fetal brain 5-HT levels decrease as placental triglycerides increase in male mice and male human samples. These findings uncover a microglia-dependent mechanism through which maternal diet can impact offspring susceptibility for neuropsychiatric disorder development in a sex-specific manner.
... We observed altered neonatal USVs, anhedonia and increased activity during the FST in male mHFD offspring. These behaviours are associated with diminished brain 5-HT in mice [25][26][27][28][29] . Previous research has demonstrated that brain 5-HT levels during discrete windows of development are dependent on placental 5-HT synthesis 30 . ...
... The cylinders were filled with 22 • C water by two-thirds so that the animals could not lean on the cylinder bottom with their paws or tail. The test was carried out either in the original technique according to Porsolt [60] with one 6 min session, or in a modified configuration [61] with two sessions performed with 24 h interval: a 10 min pretest and a 5 min test. The experiment was recorded on a video camera; the resulting video materials were processed using the ANY-maze program (Stoelting Co., Dublin, Ireland) with the calculation of the animals' total immobility time. ...
Article
Full-text available
Neurotrophins are considered as an attractive target for the development of antidepressants with a novel mechanism of action. Previously, the dimeric dipeptide mimetics of individual loops of nerve growth factor, NGF (GK-6, loop 1; GK-2, loop 4) and brain-derived neurotrophic factor, BDNF (GSB-214, loop 1; GTS-201, loop 2; GSB106, loop 4) were designed and synthesized. All the mimetics of NGF and BDNF in vitro after a 5–180 min incubation in a HT-22 cell culture were able to phosphorylate the tropomyosin-related kinase A (TrkA) or B (TrkB) receptors, respectively, but had different post-receptor signaling patterns. In the present study, we conduct comparative research of the antidepressant-like activity of these mimetics at acute and subchronic administration in the forced swim test in mice. Only the dipeptide GSB-106 that in vitro activates mitogenactivated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and phospholipase C-gamma (PLCγ) post-receptor pathways exhibited antidepressant-like activity (0.1 and 1.0 mg/kg, ip) at acute administration. At the same time, the inhibition of any one of these signaling pathways completely prevented the antidepressant-like effects of GSB106 in the forced swim test. All the NGF mimetics were inactive after a single injection regardless of post-receptor in vitro signaling patterns. All the investigated dipeptides, except GTS201, not activating PI3K/AKT in vitro unlike the other compounds, were active at subchronic administration. The data obtained demonstrate that the low-molecular weight BDNF mimetic GSB106 that activates all three main post-receptor TrkB signaling pathways is the most promising for the development as an antidepressant.
... S2). Brain tissue from Tph2-null mice lacking expression of the rate-limiting serotonin synthetic enzyme in the central nervous system (CNS) (43) was homogenized in aCSF to provide a tissue environment devoid of endogenous serotonin (Fig. 4C). As shown in Fig. 4D, serotonin added exogenously was detected via aptamer-FET neuroprobes over a concentration range similar to that recorded in aCSF (i.e., fM to M), indicating that biofouling occurring during the measurement period of ~1 hour did not interfere with serotonin detection. ...
Article
Full-text available
While tools for monitoring in vivo electrophysiology have been extensively developed, neurochemical recording technologies remain limited. Nevertheless, chemical communication via neurotransmitters plays central roles in brain information processing. We developed implantable aptamer-field-effect transistor (FET) neuroprobes for monitoring neurotransmitters. Neuroprobes were fabricated using high-throughput microelectromechanical system (MEMS) technologies, where 150 probes with shanks of either 150-or 50-m widths and thicknesses were fabricated on 4-inch Si wafers. Nanoscale FETs with ultrathin (~3 to 4 nm) In 2 O 3 semiconductor films were prepared using sol-gel processing. The In 2 O 3 surfaces were coupled with synthetic oligonucleotide receptors (aptamers) to recognize and to detect the neurotransmitter serotonin. Aptamer-FET neuroprobes enabled femtomolar serotonin detection limits in brain tissue with minimal biofouling. Stimulated serotonin release was detected in vivo. This study opens opportunities for integrated neural activity recordings at high spatiotemporal resolution by combining these aptamer-FET sensors with other types of Si-based implantable probes to advance our understanding of brain function.
Article
Full-text available
Breastfeeding is an important component of optimal nutrition provided by the mother to support child development and health later in life. Lack of key macronutrients and micronutrients in breast milk leads to early metabolic changes with lifelong health consequences. The relationship between breastfeeding and postpartum depression is poorly understood. As a result of the analysis of breast milk of mice with a knockout of the TPH2 gene, a decrease in the intensities of protein fractions characteristic of albumin, a group of caseins, and whey acidic protein was found; a decrease in the intensity of peaks characteristic of saturated and unsaturated fatty acids, as well as protein structures of tryptophan and tyrosine. The data indicate a lack of nutrients of the protein-lipid profile in the breast milk of heterozygous female mice with impaired serotonin synthesis in the brain.
Article
Monitoring neurochemical signaling across time scales is critical to understanding how brains encode and store information. Flexible (vs stiff) devices have been shown to improve in vivo monitoring, particularly over longer times, by reducing tissue damage and immunological responses. Here, we report our initial steps toward developing flexible and implantable neuroprobes with aptamer-field-effect transistor (FET) biosensors for neurotransmitter monitoring. A high-throughput process was developed to fabricate thin, flexible polyimide probes using microelectromechanical-system (MEMS) technologies, where 150 flexible probes were fabricated on each 4 in. Si wafer. Probes were 150 μm wide and 7 μm thick with two FETs per tip. The bending stiffness was 1.2 × 10-11 N·m2. Semiconductor thin films (3 nm In2O3) were functionalized with DNA aptamers for target recognition, which produces aptamer conformational rearrangements detected via changes in FET conductance. Flexible aptamer-FET neuroprobes detected serotonin at femtomolar concentrations in high-ionic strength artificial cerebrospinal fluid. A straightforward implantation process was developed, where microfabricated Si carrier devices assisted with implantation such that flexible neuroprobes detected physiological relevant serotonin in a tissue-hydrogel brain mimic.
Article
The dimeric dipeptide mimetic hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301) was created on the basis of the structure of the exposed region of the neurotrophin-3 4th loop. The new compound, as well as the full-length neurotrophin, activated the TrkC and TrkB receptors. GTS-301 showed neuroprotective activity in experiments on HT-22 mouse hippocampal cells under conditions of oxidative stress and glutamate toxicity at concentrations of 10-12 and 10-8 M, respectively, and antidepressant-like activity in the forced swimming test on mice with 7-day intraperitoneal administration in doses of 10-40 mg/kg.
Article
The various stressors in chronic unpredictable stress (CUS) triggers depressive behavior, impairs learning, and decision-making abilities. The present study investigated the effects of Celastrus paniculatus seed oil (CPSO) alone and in combination with fluoxetine (FLU) in modified CUS (mCUS) induced depression in mice. In this study, adult albino mice were subjected to a modified version of CUS protocol having six different stressors and were applied daily consistently for 15 days. The post-treatment with CPSO (50 and 100 mg/kg) and FLU (10 mg/kg) alone and in combination from day 16th to 36th. Group I: normal control; group II: diseased control (mCUS subjected group); group III: CPSO (50 mg/kg); group IV: CPSO (100 mg/kg); group V: CPSO (50 mg/kg)+FLU (10 mg/kg); group VI: CPSO (100 mg/kg)+FLU (10 mg/kg); group VII: FLU (10 mg/kg); group VIII: FLU (20 mg/kg). During experimentation, various behavioral, biochemical, oxidative stress, inflammatory, and neurotransmitters level were checked. The CUS treated mice exhibited increased escaped latency, decreased number of open arm entries, increased immobility time, decreased percentage of sucrose consumption, and number of the boxes crossed as compared to the normal group. The post-treatment with the CPSO 50 + FLU 10, CPSO 100 + FLU 10, FLU 10 significantly (p < 0.05) attenuated behavioral, biochemical, inflammation, corticosteroid, and neurotransmitters level as compared to CPSO 50, CPSO 100, and FLU 20 alone. CPSO along with FLU appreciably achieved anti-depressant effect via lowering stress, inflammation, corticosteroid level, and restoration of neurotransmitters level in mCUS induced depression mice model.
Article
Full-text available
Spirituality has been gaining recognition as a potential treatment modality. Our paper aimed to provide a systematic overview of existing research examining the use of spirituality as a treatment method for depression. All articles published between 2000 and 2018 that scientifically evaluated therapeutic interventions with elements of spirituality were included in the review. Ten studies met the inclusion criteria. Their analysis showed that there were elements of spirituality-based treatments that were repeatedly mentioned, including gratitude, forgiveness, self-acceptance, and compassion. Most often, spirituality was used together with psychotherapy. The review also noted the emergence of digital interventions.
Article
Full-text available
A novel test procedure for antidepressants was designed in which a mouse is suspended by the tail from a lever, the movements of the animal being recorded. The total duration of the test (6 min) can be divided into periods of agitation and immobility. Several psychotropic drugs were studied: amphetamine, amitriptyline, atropine, desipramine, mianserin, nomifensine and viloxazine. Antidepressant drugs decrease the duration of immobility, as do psychostimulants and atropine. If coupled with measurement of locomotor activity in different conditions, the test can separate the locomotor stimulant doses from antidepressant doses. Diazepam increases the duration of immobility. The main advantages of this procedure are (1) the use of a simple, objective test situation, (2) the concordance of the results with the validated "behavioral despair" test from Porsolt and, (3) the sensitivity to a wide range of drug doses.
Article
Full-text available
Over recent decades, encouraging preclinical evidence using rodent models pointed to innovative pharmacological targets to treat major depressive disorder. However, subsequent clinical trials have failed to show convincing results. Two explanations for these rather disappointing results can be put forward, either animal models of psychiatric disorders have failed to predict the clinical effectiveness of treatments, or clinical trials have failed to detect the effects of these new drugs. A careful analysis of the literature reveals that both statements are true. Indeed, in some cases clinical efficacy has been predicted on the basis of inappropriate animal models, though the contrary is also true, as some clinical trials have not targeted the appropriate dose or clinical population. On the one hand, refinement of animal models requires using species that have better homological validity, designing models that rely on experimental manipulations inducing pathological features, and trying to model subtypes of depression. On the other hand, clinical research should consider carefully results from preclinical studies, in order to study these compounds at the correct dose, in the appropriate psychiatric nosological entity or symptomatology, in relevant subpopulations of patients characterized by specific biomarkers. To achieve these goals, translational research has to strengthen the dialogue between basic and clinical science.Neuropsychopharmacology Reviews accepted article preview online, 18 December 2013. doi:10.1038/npp.2013.342.
Article
Full-text available
Fluoxetine (ProzacTM), a classic in chemical neuroscience, was the first major breakthrough for the treatment of depression since the introduction of tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAOIs). Fluoxetine was the first US FDA approved selective serotonin reuptake inhibitor (SSRI), culminating after a 16 year journey to approval, offering superior efficacy and reduced side effects relative to TCAs and MAOIs. The exact mechanism by which the clinical efficacy of fluoxetine is manifested is still debated; however, the importance of fluoxetine and related SSRIs is unquestionable. Moreover, the trade name Prozac has permeated popular culture, and subsequently raised awareness of depression while also diminishing long-standing stigmas of patients suffering from major depression. In this review, we will highlight the history and significance of fluoxetine for both neuroscience and for the treatment of depression, as well as review the synthesis, medicinal chemistry, pharmacology, drug metabolism and adverse events of fluoxetine.
Article
Errors in Byline, Author Affiliations, and Acknowledgment. In the Original Article titled “Lifetime Prevalence and Age-of-Onset Distributions of DSM-IV Disorders in the National Comorbidity Survey Replication,” published in the June issue of the ARCHIVES (2005;62:593-602), an author’s name was inadvertently omitted from the byline and author affiliations footnote on page 592, and another author’s affiliation was listed incorrectly. The byline should have appeared as follows: “Ronald C. Kessler, PhD; Patricia Berglund, MBA; Olga Demler, MA, MS; Robert Jin, MA; Kathleen R. Merikangas, PhD; Ellen E. Walters, MS.” The author affiliations footnote should have appeared as follows: “Author Affiliations: Department of Health Care Policy, Harvard Medical School, Boston, Mass (Dr Kessler; Mss Demler and Walters; and Mr Jin); Institute for Social Research, University of Michigan, Ann Arbor (Ms Berglund); and Section on Developmental Genetic Epidemiology, National Institute of Mental Health, Rockville, Md (Dr Merikangas).” On page 601, the first sentence of the acknowledgment should have appeared as follows: “The authors appreciate the helpful comments of William Eaton, PhD, and Michael Von Korff, ScD.” Online versions of this article on the Archives of General Psychiatry Web site were corrected on June 10, 2005.
Article
Introduction LITTLE IS known about the possible etiological biochemical factors relating to depressive reactions. Clinical evidence suggests that many depressions respond to the following somatic treatment: electric convulsive therapy (ECT), the imipramine type of drugs, and the monoamine oxidase inhibitor group of drugs.2,3 Do these two classes of drugs have common factors in their mechanism of action and can this be related to the fact that one antihypertensive agent, namely, reserpine, produces severe depression in a small but consistent number of hypertensive patients?4-8Rosenblatt et al,9 in 1959, were among the first to specifically suggest that changes in brain norepinephrine (NEP) may be involved in depression. Based in part on the knowledge of the effect of reserpine and monoamine oxidase inhibitors on depressed mood and norepinephrine (NEP), they hypothesized that the depressive state might be associated with a relative decrease of norepinephrine
Article
Background Anxiety commonly co-occurs with bipolar disorders (BDs), but the significance of such “co-morbidity” remains to be clarified and its optimal treatment adequately defined. Methods We reviewed epidemiological, clinical, and treatment studies of the co-occurrence of BD and anxiety disorder through electronic searching of Pubmed/MEDLINE and EMBASE databases. ResultsNearly half of BD patients meet diagnostic criteria for an anxiety disorder at some time, and anxiety is associated with poor treatment responses, substance abuse, and disability. Reported rates of specific anxiety disorders with BD rank: panic ≥ phobias ≥ generalized anxiety ≥ posttraumatic stress ≥ obsessive-compulsive disorders. Their prevalence appears to be greater among women than men, but similar in types I and II BD. Anxiety may be more likely in depressive phases of BD, but relationships of anxiety phenomena to particular phases of BD, and their temporal distributions require clarification. Adequate treatment trials for anxiety syndromes in BD patients remain rare, and the impact on anxiety of treatments aimed at mood stabilization is not clear. Benzodiazepines are sometimes given empirically; antidepressants are employed cautiously to limit risks of mood switching and emotional destabilization; lamotrigine, valproate, and second-generation antipsychotics may be useful and relatively safe. Conclusions Anxiety symptoms and syndromes co-occur commonly in patients with BD, but “co-morbid” phenomena may be part of the BD phenotype rather than separate illnesses.