The effects of congenital brain serotonin deficiency on responses to chronic fluoxetine

Article (PDF Available)inTranslational Psychiatry 3(8):e291 · August 2013with27 Reads
DOI: 10.1038/tp.2013.65 · Source: PubMed
Abstract
The importance of reversing brain serotonin (5-HT) deficiency and promoting hippocampal neurogenesis in the mechanisms of action for antidepressants remain highly controversial. Here we examined the behavioral, neurochemical and neurogenic effects of chronic fluoxetine (FLX) in a mouse model of congenital 5-HT deficiency, the tryptophan hydroxylase 2 (R439H) knock-in (Tph2KI) mouse. Our results demonstrate that congenital 5-HT deficiency prevents a subset of the signature molecular, cellular and behavioral effects of FLX, despite the fact that FLX restores the 5-HT levels of Tph2KI mice to essentially the levels observed in wild-type mice at baseline. These results suggest that inducing supra-physiological levels of 5-HT, not merely reversing 5-HT deficiency, is required for many of the antidepressant-like effects of FLX. We also demonstrate that co-administration of the 5-HT precursor, 5-hydroxytryptophan (5-HTP), along with FLX rescues the novelty suppressed feeding (NSF) anxiolytic-like effect of FLX in Tph2KI mice, despite still failing to induce neurogenesis. Thus, our results indicate that brain 5-HT deficiency reduces the efficacy of FLX and that supplementation with 5-HTP can restore some antidepressant-like responses in the context of 5-HT deficiency. Our findings also suggest that feeding latency reductions in the NSF induced by chronic 5-HT elevation are not mediated by drug-induced increments in neurogenesis in 5-HT-deficient animals. Overall, these findings shed new light on the impact of 5-HT deficiency on responses to FLX and may have important implications for treatment selection in depression and anxiety disorders.
OPEN
ORIGINAL ARTICLE
The effects of congenital brain serotonin deficiency on responses
to chronic fluoxetine
BD Sachs
1
, JPR Jacobsen
1
, TL Thomas
1
, WB Siesser
1
, WL Roberts
1
and MG Caron
1,2
The importance of reversing brain serotonin (5-HT) deficiency and promoting hippocampal neurogenesis in the mechanisms of
action for antidepressants remain highly controversial. Here we examined the behavioral, neurochemical and neurogenic effects of
chronic fluoxetine (FLX) in a mouse model of congenital 5-HT deficiency, the tryptophan hydroxylase 2 (R439H) knock-in (Tph2KI)
mouse. Our results demonstrate that congenital 5-HT deficiency prevents a subset of the signature molecular, cellular and
behavioral effects of FLX, despite the fact that FLX restores the 5-HT levels of Tph2KI mice to essentially the levels observed in wild-
type mice at baseline. These results suggest that inducing supra-physiological levels of 5-HT, not merely reversing 5-HT deficiency,
is required for many of the antidepressant-like effects of FLX. We also demonstrate that co-administration of the 5-HT precursor,
5-hydroxytryptophan (5-HTP), along with FLX rescues the novelty suppressed feeding (NSF) anxiolytic-like effect of FLX in Tph2KI
mice, despite still failing to induce neurogenesis. Thus, our results indicate that brain 5-HT deficiency reduces the efficacy of FLX
and that supplementation with 5-HTP can restore some antidepressant-like responses in the context of 5-HT deficiency. Our
findings also suggest that feeding latency reductions in the NSF induced by chronic 5-HT elevation are not mediated by drug-
induced increments in neurogenesis in 5-HT-deficient animals. Overall, these findings shed new light on the impact of 5-HT
deficiency on responses to FLX and may have important implications for treatment selection in depression and anxiety disorders.
Translational Psychiatry (2013) 3, e291; doi:10.1038/tp.2013.65; published online 13 August 2013
Keywords: antidepressant; anxiety; depression; neurogenesis; serotonin; tryptophan hydroxylase 2
INTRODUCTION
Major depression and anxiety disorders are highly prevalent
diseases that rank among the leading causes of disability
worldwide.
1–3
The negative impact of these disorders is
exacerbated by the poor remission rates obtained with standard
treatments.
4
Most antidepressants acutely increase the
extracellular levels of serotonin (5-HT), but mood improvements
typically do not emerge until after weeks of treatment.
5
Consequently, the clinical effects of antidepressants have been
hypothesized to result from long-term adaptive responses to
antidepressant administration, such as increased neurogenesis.
6–8
Antidepressant effects have also been hypothesized to result
from the correction of endogenous 5-HT deficiency,
9
but recent
studies have shown that mutant forms of the 5-HT synthesis gene,
tryptophan hydroxylase 2 (Tph2),
10,11
which could result in
impaired 5-HT synthesis,
12
are associated with poor
antidepressant treatment responses.
13–15
These observations
suggest that congenital brain 5-HT deficiency might reduce
antidepressant efficacy. Here we used a genetically engineered
mouse line, the Tph2 (R439H) knock-in (Tph2KI) mouse, which
exhibits B60–80% reductions in brain 5-HT
16
throughout
postnatal development,
17
to examine the effects of 5-HT
deficiency on responses to fluoxetine (FLX) in the novelty
suppressed feeding (NSF) and tail suspension tests (TSTs). The
NSF is a particularly interesting preclinical measure of
antidepressant-like effects in that the efficacy of antidepressants
in this test requires chronic administration and has been reported
to be neurogenesis dependent.
18,19
However, the neurogenesis
dependence of antidepressant-like effects in the NSF have been
challenged,
20
and the role of hippocampal neurogenesis in the
etiology and treatment of depression and anxiety disorders
remains highly controversial.
21
Our data indicate that 5-HT deficiency impairs NSF responses to
FLX and prevents the induction of neurogenesis by FLX. However,
we also show that FLX reduces immobility time in the TST in both
wild-type (WT) and Tph2KI mice, despite the fact that the
magnitude of the FLX-induced increase in 5-HT is markedly
reduced in Tph2KI compared with that of WT animals. In addition,
we demonstrate that supplementation with 5-hydroxytryptophan
(5-HTP), the 5-HT precursor, can restore the anxiolytic-like effects
of FLX in the NSF in Tph2KI mice, despite failing to restore FLX’s
pro-neurogenic effects in these animals. Thus, our results suggest
that 5-HT deficiency can impair a subset of antidepressant effects
and indicate that antidepressant-like effects in the NSF do not
require antidepressant-induced increases in hippocampal neuro-
genesis, at least under conditions of congenital 5-HT deficiency.
MATERIALS AND METHODS
Animals and drug treatments
The Tph2KI mouse line is on a mixed background (c57BL6/J—129S6/SvEv)
and has been described previously.
16
Homozygous Tph2 R439H KI mice
and WT littermate controls were derived from heterozygous breeding pairs
and were housed two to five per cage in a facility maintained at 23
±
2 1C
on a 12 h light–dark cycle. Eight- to 10-week-old age-matched littermates
were used for all experiments. Male mice were exclusively used for all
1
Department of Cell Biology, Duke University, Durham, NC, USA and
2
Department of Neurobiology, Duke University Medical Center, Durham, NC, USA. Correspondence:
Dr MG Caron, Department of Cell Biology, Duke University Medical Center, 487 CARL Building, Box 3287, Durham, NC 27710, USA.
E-mail: m.caron@cellbio.duke.edu
Received 12 June 2013; accepted 10 July 2013
Citation: Transl Psychiatry (2013) 3, e291; doi:10.1038/tp.2013.65
&
2013 Macmillan Publishers Limited All rights reserved 2158-3188/13
www.nature.com/tp
behavioral analyses and the subsequent FLX/neurogenesis studies. The
baseline neurogenesis experiments (Figure 2) included balanced numbers
of males and females, but the study was not sufficiently powered to reveal
any significant sex differences. FLX (Spectrum Chemical Corporation, New
Brunswick, NJ, USA) and desipramine (DES; Sigma, St Louis, MO, USA) were
administered to mice via their drinking water (155 mg l
1
) for a total of 4
weeks. Behavioral testing was conducted after 3 weeks of FLX adminis-
tration, and mice were killed and processed for immunohistichemistry
(IHC) analysis 1 week after behavioral testing. In our hands, this treatment
regimen results in a dose equivalent of FLX to B20 mg kg
1
per day for
both WT and Tph2KI mice.
22
An analogous treatment paradigm has been
shown to be effective for DES.
23
Supplementation with 5-HTP was performed by injecting WT and Tph2KI
mice intraperitoneally twice daily (at 0900 and 1700 h) with 5-HTP
(20 mg kg
1
) for 4 weeks, while administering FLX as described above.
Again, behavioral testing was performed after 3 weeks (2 h following the
first daily injection) and the mice were killed for IHC analysis 1 week later
(2 h after the final 5-HTP injection). This treatment paradigm partially
restores tissue levels of 5-HT in Tph2KI mice 2 h after administration.
22
Antidepressant-containing drinking water was administered via opaque
bottles and was replaced twice weekly. Chlordiazepoxide (7.5 mg kg
1
,
Sigma) was administered intraperitoneally 20 min before performing the
NSF. For proliferation studies, bromodeoxyuridine (BrdU; Sigma) solutions
were prepared fresh daily by dissolution in saline (10 mg ml
1
) and were
intraperitoneally injected (100 mg kg
1
) into mice 4, 18 and 24 h before
killing. For survival and double-labeling studies, BrdU was administered via
the drinking water (1 g l
1
) in opaque bottles that were replaced twice
weekly. All experiments were conducted in accordance with an animal
protocol that was approved by the Duke University Institutional Animal
Care and Use Committee.
Microdialysis
Surgery. Mice were anesthetized using isoflurane and placed in a Kopf
(Tujunga, CA, USA) stereotaxic frame equipped with a mouse adapter
(Stoelting Mouse and Neonatal Rat Adaptor, Wood Dale, IL, USA). A guide
cannula (catalog number 5-300004; Brainlink, Groningen, The Netherlands)
was implanted into the hippocampus (HIP; anterior-posterior: 3.3 mm,
medial-lateral: 3.0 mm, dorsal-ventral: 1.5 mm), according to the Franklin
and Paxinos mouse brain atlas. Each cannula was fixed in place with two
anchor screws (CMA, Chelmsford, MA, USA) and carboxylate dental cement
(CMA). Operated mice were single housed, treated with antibiotics (1.2 mg
sulfamethoxazole per ml and 0.24 mg trimethoprim per ml) in the drinking
water (with or without FLX) and allowed to recover 48–96 h after
implantation.
Dialysate collection. Sterile artificial cerebrospinal fluid (147 m
M NaCl,
2.7 m
M KCl, 0.85 mM MgCl2, 1.2 mM CaCl2; CMA) was delivered from a CMA
400 syringe pump at a flow rate of 0.45 ml min
1
. Sixteen to 24 h before
the start of sample collection, each mouse was gently restrained and a
microdialysis probe (catalog number 5-140040, 2 mm membrane; Brain-
link) was inserted into the guide cannula. Each mouse was then placed in a
circular cage with bedding, chow and water (with or without FLX) available
ad libitum. The probe tubing was stiffened with laboratory paper tape to
avoid biting. A two-channel swivel (catalog number 375/D/22QM; Instech,
Plymouth Meeting, PA, USA) allowed for unimpeded movement of the
mouse. Between 0900 and 1100 h, one baseline dialysate (30 min duration)
was collected on ice, shielded from light, immediately frozen on dry ice
and stored at 80 1C.
High-performance liquid chromatography–electrochemical detection analysis.
The high-performance liquid chromatography system consisted of a BASi
(West Lafayette, IN, USA) LC-4C detector coupled to a BASi LCEC radial flow
cell. The potential was set at þ 650 mV. Flow was provided by a Shimadzu
(Columbia, MD, USA) LC-20AD solvent delivery module. The pump was
preceded by an online degasser series 1100 from Agilent (Santa Clara, CA,
USA). The chromatograms were analyzed using PowerChrom software
(eDAQ, Colorado Springs, CO, USA). Ten microliters of dialysate was
separated on a 1 100-mm UniJet microbore 3 mm octadecylsilyl column
(BASi, West Lafayette, IN, USA) at a flow rate of 80 mlmin
1
. The mobile
phase consisted of 24 m
M Na
2
HPO
4
,3mM octanesulfonic acid, 27.4 mM citric
acid, 107 m
M EDTA and 17–18.5% (v/v) MeOH, pH adjusted to 4.8 with NaOH,
5-HT eluted at 11–13 min.
Behavioral analyses
The TST and NSF were performed as described previously.
16,24
Immediately
following the NSF, mice were returned to the home cage, where the
latency to feed and quantity of food consumed within 5 min were
measured.
Immunohistochemistry
Tissue was processed for IHC analysis as described previously.
24
Primary
antibodies used were rabbit anti-doublecortin (DCX; Abcam, Cambridge,
MA, USA; 1:200), rabbit anti-cleaved caspase-3 (1:500; Cell Signaling,
Danvers, MA, USA), mouse anti-NeuN (Millipore, Temecula, CA, USA; 1:200)
and rat anti-BrdU (Accurate Chemical Corporation, Westbury, NY, USA;
1:200). Alexafluor 488- or Alexafluor 568-conjugated secondary antibodies
(Life Technologies, Carlsbad, CA, USA) diluted 1:500 in 5% bovine serum
albumin in phosphate-buffered saline-t were used. Coverslipping was
performed using SlowFade Gold Antifade Reagent with 4’,6-diamidino-2-
phenylindole (Life Technologies). For assessments of granule cell layer
(GCL) and medial habenula size, images of 4’,6-diamidino-2-phenylindole-
stained sections (six sections per mouse for the GCL and four sections per
mouse for the medial habenula) were taken, and the GCL and medial
habenula of each hemisphere was outlined and measured using Axiovision
software (Zeiss, Oberkochen, Germany).
For confocal analysis, sections were stained according to the above
protocol, except that they were counterstained with TOTO-3, a nuclear
marker, for 15 min (1:500 dilution, Life Technologies) before coverslipping
with SlowFade Gold Antifade Reagent without 4’,6-diamidino-2-phenylin-
dole (Life Technologies).
Non-overlapping images of the entire GCL and subgranular zone were
taken on a fluorescence microscope (Zeiss, Oberkochen, Germany) by an
individual unaware of genotype and treatment condition. The numbers of
BrdU þ , BrdU þ /NeuN þ , DCX þ or activated caspase-3 þ cells were
counted by an observer unaware of genotype and treatment condition. For
proliferation studies, only BrdU þ cells within two cell widths of the
subgranular zone were counted. However, for survival and double-labeling
studies, BrdU þ cells were counted throughout the entire GCL, as they are
known to migrate.
25
For BrdU/NeuN double-labeling experiments, at least
600 BrdU þ cells were examined in each group.
Real-time PCR
Mice were killed by cervical dislocation, decapitated and heads were
rapidly cooled by submersion in liquid nitrogen for B6 s. Brains were
removed and a 1.5-mm diameter punch of dorsal HIP (primarily dentate
gyrus) was obtained from a 1-mm coronal section, snap-frozen in liquid
nitrogen and stored at 80 1C until further use. RNA was extracted with
Trizol in combination with RNeasy minikits according to the manufacturer’s
protocol (Qiagen, Valencia, CA, USA). RNA was reverse transcribed using
the iScript cDNA synthesis kit according to the manufacturer’s protocol
(Bio-Rad, Hercules, CA, USA), and real-time PCR was performed using a
LightCycler (Roche Applied Science, Indianapolis, IN, USA). The glycer-
aldehydes 3-phosphate dehydrogenase primers used were: 5’-CAT GTT
CCA GTA TGA CTC CAC TC-3
0
and 5’-GGC CTC ACC CCA TTT GAT GT-3’. The
brain-derived neurotrophic factor (BDNF) primers used were: 5’-CAA TGC
CGA ACT ACC CAA-3’ and 5’-AAC ATA AAT CCA CTA TCT TCC CC-3’ The
cAMP response element-binding (CREB) primers used were: 5’-AGC CGG
GTA CTA CCA TTC TAC-3’ and 5’-GCA GCT TGA ACA ACA ACT TGG-3’.
Statistical analysis
Data were analyzed using Student’s t-tests or two-way analysis of variances
with Tukey’s post-hoc tests, where appropriate. In some cases (for
example,, microdialysis experiments), data were transformed (for example,
log-transformed) before performing statistical analyses. Statistical analyses
were performed using JMP software (SAS, Cary, NC, USA).
RESULTS
FLX increases extracellular 5-HT levels in the HIP of 5-HT-deficient
mice
Consistent with our previous results,
16,17
microdialysis revealed
that Tph2KI mice have reduced extracellular 5-HT (5-HT
EXT
) in the
HIP compared with WT controls (main effect of genotype:
F
(1,25)
¼ 135.8074, Po0.0001, Figure 1a). Chronic treatment with
FLX increased 5-HT
EXT
in both genotypes (main effect of FLX:
Brain 5-HT deficiency and antidepressant responses
BD Sachs et al
2
Translational Psychiatry (2013), 1 9 & 2013 Macmillan Publishers Limited
F
(1,25)
¼ 88.8577, Po0.0001, Figure 1a). However, the magnitude of
the FLX-induced increase in 5-HT
EXT
in Tph2KI mice (B2.25-fold,
P ¼ 0.0326) was markedly less than that observed in WT animals
(B6.4-fold, genotype by drug interaction: F
(1,25)
¼ 28.3908,
Po0.0001, Figure 1a). Importantly, the levels of 5-HT
EXT
in Tph2KI
mice after chronic FLX treatment were not significantly different
from those in untreated WT mice (P ¼ 0.3963), but they were only
12% of the levels achieved in FLX-treated WT animals. These
results suggest that although chronic FLX treatment essentially
reverses hippocampal 5-HT deficiency in Tph2KI mice, congenital
5-HT deficiency can significantly blunt the neurochemical effects
of selective serotonin reuptake inhibitors (SSRIs).
Chronic FLX fails to induce an antidepressant-like effect in the NSF
in Tph2KI mice
In the NSF, chronic FLX significantly reduced feeding latency (main
effect of treatment: F
(1,68)
¼ 6.0055, P ¼ 0.0168, Figure 1b), but a
significant genotype by treatment interaction was also observed
(F
(1, 68)
¼ 10.4593, P ¼ 0.0019, Figure 1b). Indeed, FLX reduced
feeding latency in WT mice (P ¼ 0.0012) but not in Tph2KI animals.
Importantly, FLX-treated WT mice exhibited significantly shorter
feeding latencies than FLX-treated Tph2KI animals (P ¼ 0.0414),
suggesting that the lack of effect in Tph2KI mice was not due to a
floor effect. A significant main effect of FLX on home-cage feeding
latency was also observed (F
(1, 76)
¼ 4.1378, P ¼ 0.0454, Figure 1c),
but the genotype by treatment interaction was not significant
(P ¼ 0.3142), thus suggesting that differential effects of FLX on
appetitive drive in WT and Tph2KI animals were not responsible
for the observed differential responses to FLX. In contrast to
chronic FLX, acute treatment with chlordiazepoxide, a benzodia-
zepine, reduced feeding latency in both genotypes (main effect of
treatment: F
(1,32)
¼ 15.809, P ¼ 0.0004, Figure 1d). Similarly, chronic
treatment with DES, a tricyclic antidepressant that preferentially
inhibits norepinephrine reuptake, reduced feeding latency in both
genotypes (main effect of treatment: F
(1,35)
¼ 11.3246, P ¼ 0.0019,
Figure 1e), suggesting that brain 5-HT deficiency does not impair
anxiolytic-like responses to non-5-HT-specific drugs.
Throughout the NSF experiments, control Tph2KI mice exhib-
ited a tendency towards reduced feeding latencies compared with
that of WT animals (Figures 1 b–e). To determine the basis for this,
we examined home-cage food consumption and feeding latency,
and weight loss following a 24-h food-deprivation period. No
significant genotype differences were observed in home-cage
food consumption or weight loss (BDS, unpublished observations).
Similar to our previous reports,
16,26
Tph2KI mice exhibited
increased immobility in the TST (main effect of genotype: F
(1, 97)
¼
4.9953, P ¼ 0.0277, Figure 1f). In contrast to the NSF, chronic
treatment with FLX significantly reduced immobility time in the
TST (main effect of treatment: F
(1,97)
¼ 21.3464, Po0.0001,
Figure 1f) with no significant genotype by treatment interaction.
Brain 5-HT deficiency does not impair baseline neurogenesis
Because of the reported importance of hippocampal neurogenesis
in the NSF,
18,19
we hypothesized that the lack of effect of FLX
in Tph2KI mice in the NSF might result from a defect in the
neurogenic response to FLX. As we have shown previously,
24
there
are no significant baseline differences between WT and Tph2KI
mice in BrdU incorporation in the subgranular zone (Figures 2a–c),
but a detailed analysis of baseline neurogenesis in Tph2KI mice
has not been performed previously. Interestingly, IHC analysis
for DCX, a marker of immature neurons, revealed a significant
37% increase in the number of DCX þ neurons in Tph2KI mice
compared with that in WT controls (Student’s t-test: P ¼ 0.0025,
degrees of freedom ¼ 31, Figures 2d–f).
To determine whether the increased numbers of DCX þ
neurons resulted from increased survival of adult-generated
neural progenitor cells, Tph2KI and WT mice were administered
BrdU for 1 week and were killed either 1 or 21 days later.
Importantly, no significant differences in the number of BrdU þ
cells were observed in WT and Tph2KI mice killed on day 1
(Figure 2g). However, Tph2KI mice killed on day 21 had
significantly more BrdU þ cells than WT controls (Student’s t-test:
P ¼ 0.0351, degrees of freedom ¼ 21, Figure 2h). In addition, IHC
analysis for activated caspase-3, a marker of apoptosis, revealed
Figure 1. Neurochemical and behavioral responses to chronic fluoxetine (FLX) in wild-type (WT) and tryptophan hydroxylase 2 (R439H) knock-
in (Tph2KI) mice. (a) Levels of extracellular 5-HT (5-HT
EXT
) in the hippocampus (HIP) were determined by microdialysis. (b) Feeding latency in
the novelty suppressed feeding (NSF) test after chronic FLX treatment. (c) Home-cage feeding latency in WT and Tph2KI mice following
chronic FLX. (d) Feeding latenc y in the NSF after acute chlordiazepoxide administration. (e) Feeding latency in the NSF following chronic
desipramine (DES) treatment in WT and Tph2KI mice. (f) Immobility time in the tail suspension test (TST) following chronic FLX. *Significant
main effect of treatment by two-way analysis of variance (ANOVA; Po0.05). **Po0.05 compared with WT control by Tukey’s post-hoc test.
^
Po0.05 compared with WT FLX by Tukey ’s post-hoc test.
@
Po0.05 by Tukey’s post-hoc test compared with control Tph2KI mice. ‘X’ denotes a
significant genotype by treatment interaction by two-way ANOVA (Po0.05) and ‘#’ denotes a significant main effect of genotype (Po0.05 by
two-way ANOVA); n ¼ 7–8 per group for a, n ¼ 19–21 per group for b, n ¼ 10 per group for c, n ¼ 9 per group for d, n ¼ 8–11 per group for e
and n ¼ 22–27 per group for f.
Brain 5-HT deficiency and antidepressant responses
BD Sachs et al
3
& 2013 Macmillan Publishers Limited Translational Psychiatry (2013), 1 9
decreased numbers of apoptotic cells within the GCL of Tph2KI
animals when compared with that in WT controls (Student’s t-test:
P ¼ 0.0142, degrees of freedom ¼ 19, Figure 2i). We did not
observe any statistically significant effects of 3 weeks of FLX
treatment (beginning after the cessation of BrdU administration)
on the survival of BrdU þ cells in either genotype (Figure 2j),
although Tph2KI animals again exhibited an overall increase in
survival compared with that of WT controls (main effect of
genotype: F
(1,20)
¼ 4.446, P ¼ 0.0478, Figure 2j). To evaluate
whether this increased survival of adult-generated neurons might
lead to a larger GCL, we compared GCL size in WT and in Tph2KI
mice under baseline conditions and following chronic FLX
administration. Tph2KI mice were observed to have a significantly
larger GCL than WT controls (main effect of genotype:
F
(1,31)
¼ 5.8218, P ¼ 0.0219, Figure 2k). Although chronic FLX
administration led to a slight increase in GCL size in WT mice,
this effect did not reach significance. As a control, no significant
genotype or treatment differences were observed in the size of
the medial habenula (Figure 2l).
Brain 5-HT deficiency prevents the neurogenic effects of FLX
After observing no significant effects of FLX on cell survival, we next
examined the effects of FLX on cell proliferation. No significant main
effects of chronic FLX treatment or genotype were observed on
BrdU incorporation. However, a significant genotype by treatment
interaction was observed (F
(1,37)
¼ 5.5383, P ¼ 0.024, Figures 3a–e).
As expected,
6
chronic treatment with FLX before BrdU
administration increased the number of BrdU þ cells in WT mice
(P ¼ 0.0259, Figures 3a, c and e). However, this treatment had no
effect on BrdU incorporation in Tph2KI animals (Figures 3b, d and e).
Chronic FLX significantly increased DCX immunoreactivity (main
effect of treatment: F
(1,55)
¼ 8.4018, P ¼ 0.0054, Figures 3f, h and j),
but a significant genotype by treatment interaction was also
observed (F
(1,55)
¼ 21.3702, Po0.0001, Figure 3j). Indeed, the
increased DCX was only apparent in WT mice (Po0.0001) and
not in Tph2KI animals. Similar to what was observed above
(Figure 2d), control Tph2KI animals exhibited a 30% increase in the
number of DCX þ cells, but this effect did not reach statistical
significance using Tukey’s post-hoc analysis, only with the less
conservative Student’s t-test (P ¼ 0.0437). We did not observe a
significant increase in BrdU or DCX immunoreactivity in response
to chronic DES treatment in either genotype (BDS and TLT,
unpublished observations).
We next performed double-labeling experiments to compare
the percentage of BrdU þ cells that become NeuN þ neurons
between the groups. In both WT and Tph2KI animals, B80% of
the BrdU þ cells in the GCL were also immunopositive for NeuN 3
weeks after a 1-week exposure to BrdU, and FLX administration
did not significantly affect the proportion of BrdU þ /NeuN þ cells
in either genotype (Figure 4a). We again observed a significant
increase in the number of surviving BrdU þ cells in Tph2KI mice
compared with WT animals (main effect of genotype:
F
(1,20)
¼ 5.3493, P ¼ 0.0315, Figure 4b). Similarly, the total number
of BrdU þ /NeuN þ neurons was greater in Tph2KI than in WT
animals (main effect of genotype: F
(1,20)
¼ 5.5470, P ¼ 0.0288,
Figure 4c). Representative images are shown in Figures 4 d–i.
Chronic FLX treatment fails to increase hippocampal BDNF mRNA
levels in Tph2KI mice
As expected,
27
chronic FLX treatment led to significantly increased
mRNA levels of BDNF (main effect of treatment: F
(3,37)
¼ 4.4874,
P ¼ 0.0409, Figure 5a); however, a significant genotype by
treatment interaction was also observed (F
(3,37)
¼ 4.5347,
P ¼ 0.0399). Tukey’s post-hoc tests revealed that the effect of FLX
was only significant in WT mice (P ¼ 0.0216), not in Tph2KI
animals. Tukey’s post-hoc tests also revealed that Tph2KI animals
Figure 2. Effects of brain serotonin (5-HT) deficiency on neurogenesis in the subgranular zone (SGZ). (a) Quantification of bromodeoxyuridine
(BrdU) þ cell number in the SGZ. Representative BrdU images from wild-type (WT) (b) and tryptophan hydroxylase 2 (R439H) knock-in
(Tph2KI) ( c) animals are shown with BrdU in red. (d) Quantification of the number of doublecortin (DCX) þ immature neurons is shown.
Representative DCX images from WT (e) and Tph2KI (f) animals are shown with DCX staining in green. (g) Quantification of BrdU þ cell
number one day after BrdU administration. (h) Quantification of BrdU þ cell number 3 weeks after BrdU administration. (i) Quantification of
activated caspase-3 staining. (j) Quantification of BrdU þ cell number 3 weeks after BrdU administration in WT and Tph2KI mice treated with
fluoxetine (FLX) for 3 weeks. (k) Quantification of granule layer size in control and FLX-treated WT and Tph2KI mice. (l) Quantification of medial
habenula size in control and FLX-treated WT and Tph2KI mice. TOTO-3 þ nuclei are shown in blue. ‘$’ Indicates Po0.05 by Student’s t-test. ‘#’
Denotes a significant main effect of genotype by two-way analysis of variance, Po0.05; n ¼ 12 per group for ac; n ¼ 16–17 per group for df;
n ¼ 9 per group for g, n ¼ 11–12 per group for h, n ¼ 10–11 per group for i, n ¼ 6 per group for j, n ¼ 8–9 per group in k and l. The scale bar
indicates 20 mm. Arrows denote BrdU þ cells, and arrowheads indicate DCX þ
cells.
Brain 5-HT deficiency and antidepressant responses
BD Sachs et al
4
Translational Psychiatry (2013), 1 9 & 2013 Macmillan Publishers Limited
exhibit increased hippocampal BDNF mRNA at baseline
(P ¼ 0.0446). The effects of FLX on CREB mRNA expression in the
HIP were dependent upon genotype (significant genotype by
treatment interaction: F
(3,36)
¼ 4.7420, P ¼ 0.0361, Figure 5b).
However, Tukey’s post-hoc tests did not reveal any significant
differences between the groups (although the less conser-
vative Student’s t-test revealed a slight increase in CREB levels in
FLX-treated WT mice, P ¼ 0.0367, as expected
28
). We did not
observe any significant effects of DES on hippocampal levels of
CREB or BDNF (Figures 5c and d).
Co-administration of 5-HTP restores the anxiolytic ability of FLX in
Tph2KI mice in the NSF
Unlike FLX alone, chronic co-administration of 5-HTP þ FLX
reduced feeding latency in both Tph2KI and WT animals
(significant main effect of treatment: F
(3,34)
¼ 16.3484, P ¼ 0.0003,
Figure 5e). Co-administration of 5-HTP þ FLX also led to an increase
in the number of BrdU þ (P ¼ 0.0474, Figure 5f) and DCX þ cells
(P ¼ 0.0187, Figure 5g) in WT mice. However, 5-HTP þ FLX
treatment failed to increase the number of BrdU þ (genotype by
treatment interaction: F
(3,41)
¼ 4.1731, P ¼ 0.0475, Figure 5f) or
DCX þ cells (genotype by treatment interaction: F
(3,63)
¼ 9.8198,
P ¼ 0.0027, Figure 5g) in Tph2KI mice. Similar to what was
observed above (Figures 2d and 3j), Tph2KI mice exhibited a
55% increase in the number of DCX þ neurons, but this effect did
not achieve statistical significance. Chronic 5-HTP þ FLX adminis-
tration also failed to induce a significant increase in BDNF or CREB
expression in Tph2KI animals (BDS, unpublished observations).
DISCUSSION
Our results suggest that 5-HT deficiency could reduce the efficacy
of FLX by limiting FLX-induced increases in 5-HT
EXT
, thus blocking
downstream cellular and molecular responses. This would be
consistent with prior work that has implicated variants in Tph2 in
antidepressant sensitivity in humans
14,15
and with prior preclinical
work showing that acute pharmacologic inhibition of 5-HT
synthesis blocks the acute effects of SSRIs in the TST
29
and
forced swim test
30–32
in rodents. Although only FLX was examined
here, it is likely that other SSRIs would be impacted by 5-HT
deficiency as well. A previous report demonstrated that acute
5-HTP administration can restore antidepressant-like responses to
acute SSRI treatment in otherwise SSRI-insensitive NMRI mice,
suggesting that combined 5-HTP þ SSRI therapy could represent
an antidepressant augmentation strategy,
33
a hypothesis that is
further supported by our finding that NSF behavior can be
modified in Tph2KI mice by chronic combined 5-HTP þ FLX
treatment.
Although the specific mutation expressed by Tph2KI mice is
extremely rare, 5-HT deficiency could result from many different
mutations in 5-HT system genes.
34
As such, we hypothesize that
the current results will be relevant for a wide range of genetic
insults leading to 5-HT deficiency. Although we feel that studies
using Tph2KI mice may be highly informative for psychiatric
conditions, such as depression and anxiety, we do not claim that
these animals completely recapitulate any disorder. Rather, we
view these animals as a model of 5-HT deficiency, not of
depression or anxiety per se. Similarly, we have utilized the TST
and the NSF because of their strong predictive validity for
antidepressant action, not on the basis of their face validity or
relevance to depression- or anxiety-like behavior. Future studies
examining the effects of 5-HT deficiency on responses to chronic
stressors may be useful in determining the importance of 5-HT
deficiency in regulating susceptibility to stress, which could, in
turn, have implications for our understanding of the gene by
environment interactions that lead to aberrant emotional
behavior.
Figure 3. Effects of chronic fluoxetine (FLX) treatment on neurogenesis in wild-type (WT) and tryptophan hydroxylase 2 (R439H) knock-in
(Tph2KI) mice.Representative bromodeoxyuridine (BrdU)-stained micrographs of WT control (a), Tph2KI control (b), WT FLX (c) and Tph2KI FLX
hippocampus (HIP) (d) are shown with quantifications shown in panel e. Arrows denote BrdU þ cells. Representative doublecortin (DCX)-
stained micrographs of WT control (f ), Tph2KI control (g), WT FLX (h) and Tph2KI FLX (i). Quantification of immunohistochemistry (IHC) for
DCX is shown (j). Data were analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc tests to compare individual
differences. ‘X’ denotes interaction between treatment and genotype (Po0.05) by two-way ANOVA. *Significant main effect of genotype
(Po0.05). **Po0.05 compared with WT control by Tukey ’s post-hoc test; n ¼ 10–11 per group for ae and n ¼ 12–14 per group for fj. The scale
bar indicates 20 mm.
Brain 5-HT deficiency and antidepressant responses
BD Sachs et al
5
& 2013 Macmillan Publishers Limited Translational Psychiatry (2013), 1 9
The observed trend towards a reduction in feeding latency in
the NSF in Tph2KI animals compared with that in WT controls is
consistent with a role for 5-HT in anxiety-like behavior and is
similar to the phenotypes reported in other transgenic models
of 5 -HT deficiency
35,36
and an acute rat model of 5-HT
depletion.
37
We hypothesize that the variance in baseline
feeding latencies in WT and Tph2KI mice (compare F igure 1 with
Figure 5) may be associated with the varying levels of
physiologic al arous al associa ted with different drug adminis-
tration paradigms (that is, dietary vs injections). Indeed,
previous reports from several groups, including o ur own, have
shown that performance in the NSF test is sensitive to
stress.
19,24
The importance of adult hippocampal neurogenesis in depres-
sion- and anxiety-like behavior and in responses to antidepres-
sants has been widely debated.
21,38,39
The reported relationships
between neurogenesis and stress,
40–44
along with the fact
that completely inhibiting neurogenesis prevents some of the
behavioral effects of antidepressants, have suggested a role for
hippocampal neurogenesis in the development and treatment of
mood disorders.
18,19,44–47
However, numerous studies, including
the current study, have found neurogenesis to be of limited
importance in depression-related behavior and/or in anti-
depressant-like responses.
20,38,48–50
Our finding that chronic 5-HTP þ FLX (or D ES) administration,
which does not increase neurogenesis in Tph2KI animals, reduces
feedin g latency in Tph2KI mice demonstrates that antidepressant-
induced increases in neurogenesis are not required for this effect,
at least not in 5-HT-deficient animals. These current results are
distinct from previous studies that used X-ray irradiation to ablate
all dividing cells, which revealed that antidepressants are
ineffective when neur ogenesis ha s been completely inhib-
ited.
18,19
Interestingly, it has been shown that promoting
neurogenesis is not sufficient to in duce antidepressant-like
effects in the NSF,
51
and the effects of several class es of
experimental antidepressants, such as corticotropin -releasing
factor 1 and vasopressin 1b antagonists, reportedly do not
require adult hipp ocampal neurogenesis in animal models.
52
Taken together, these data suggest that increased neurogenesis
is neither required nor sufficient for feeding latency reductions in
the NSF.
Although 5-HT elevation has been repeatedly shown to increase
the proliferation of adult hippocampal neural progenitor cells, the
reported effects of 5-HT on the survival of neural progenitor cells
have been inconsistent. Several groups have reported that chronic
FLX administration increases the survival of newly born neurons
in vivo,
19,53,54
but other studies have suggested that chronic FLX
increases both apoptosis and cell turnover in the HIP.
55,56
Our
Figure 4. A quantification of double immunofluorescence for bromodeoxyuridine (BrdU) and NeuN in wild-type (W T) and tryptophan
hydroxylase 2 (R439H) knock-in (Tph2KI) mice. (a) The average percentage of BrdU þ cells that are also NeuN þ .(b) The average total number
of BrdU þ and (c) the average total number of BrdU þ /NeuN þ cells in control and fluoxetine (FLX)-treated WT and Tph2KI mice.
Representative micrographs from control WT (d), control Tph2KI (e), FLX-treated WT (f) and FLX-treated Tph2KI (g) are shown. Higher
magnification images of the indicated area in e reveal a BrdU þ and an adjacent BrdU þ /NeuN þ cell (h, i). TOTO-3 þ nuclei are shown in
blue, NeuN is shown in green and BrdU is shown in red. The scale bar indicates 20 mmfordg and 10 mm for h and i. *Significant main effect of
genotype Po0.05, n ¼ 6 per group.
Brain 5-HT deficiency and antidepressant responses
BD Sachs et al
6
Translational Psychiatry (2013), 1 9 & 2013 Macmillan Publishers Limited
results did not reveal a significant effect of FLX on cell survival but
did demonstrate an unexpected increase in cell survival in 5-HT-
deficient animals compared with that in WT controls. It is possible
that the improved survival of adult-generated neurons in Tph2KI
mice is related to their increased levels of hippocampal BDNF,
which has been shown to have an important role in the survival
(but not proliferation or maturation) of adult neural progenitor
cells.
57
It is likely that the increased size of the GCL observed in
Tph2KI mice will have important implications for hippocampal
function and hippocampal-dependent behaviors, but future
research will be required to evaluate this possibility.
Overall, our data indicate that chronic treatment with FLX can
reverse brain 5-HT deficiency in Tph2KI mice but that FLX fails to
induce several of its key molecular, cellular and behavioral effects
in 5-HT-deficient animals. Importantly, several non-5-HTergic
agents (that is, DES and chlordiazepoxide) appear to retain their
efficacy in 5-HT-deficient animals, and behavioral responses to FLX
can be restored in 5-HT-deficient animals by cotreatment with
5-HTP. These results suggest that 5-HT deficiency may contribute
to insensitivity to SSRIs by limiting the magnitude of SSRI-induced
5-HT increments. In addition, the observed decrease in feeding
latency induced by combined 5-HTP þ FLX treatment in the
absence of increased neurogenesis further refines our under-
standing of the importance of hippocampal neurogenesis in
mediating the effects of antidepressants.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This work was supported in part by grants from the National Institutes of Health
(MH79201 and MH60451) to MGC. Support from the Lennon Family Foundation to
MGC for the initial part of this work is also greatly appreciated. WBS was the recipient
of an NRSA postdoctoral fellowship (F32-MH-083404) and BDS was the recipient of a
Figure 5. mRNA analysis and effects of chronic 5-hydroxytryptophan (5-HTP). (a) The effects of chronic fluoxetine (FLX) treatment on
hippocampal brain-derived neurotrophic factor (BDNF) mRNA in wild-type (WT) and tryptophan hydroxylase 2 (R439H) knock-in (Tph2KI)
mice. (b) The effects of chronic FLX on hippocampal cAMP response element-binding (CREB) mRNA. (c) The effects of chronic desipramine
(DES) on hippocampal BDNF mRNA in WT and Tph2KI mice. (d) The effects of chronic DES on hippocampal CREB mRNA. (e) The feeding
latencies of WT and Tph2KI mice chronically treated with FLX þ 5-HTP are shown. Quantification of the number of bromodeoxyuridine
(BrdU) þ (f ) and doublecortin (DCX) þ (g) cells in FLX þ 5-HTP-treated WT and Tph2KI mice are shown. *Significant main effect of FLX by two
way analysis of variance (ANOVA; Po0.05). **Po0.05 by Tukey’s post-hoc test compared with WT control. ‘X’ indicates significant genotype by
treatment interaction by two-way ANOVA (Po0.05); n ¼ 10–11 mice per group for a; n ¼ 9–11 mice per group for bd; n ¼ 9–10 per group for
e, n ¼ 11 per group for f and n ¼ 11–15 per group for g.
Brain 5-HT deficiency and antidepressant responses
BD Sachs et al
7
& 2013 Macmillan Publishers Limited Translational Psychiatry (2013), 1 9
Minority Supplement award from the National Institutes of Health (MH79201-03S1)
and is currently the recipi ent of an NRSA postdoctoral fellowship (F32-MH093092).
JPRJ is the grateful recipient of an individual grant from The Lundbeck Foundation of
Denmark. We thank Meghan Rudder for technical assistance.
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    • "Furthermore, a similar phenotype was displayed by wild-type mice with serotonin synthesis blocked at adult age by chronic administration of p-chlorophenylalanine, an inhibitor of tryptophan hydroxylase 2 (Tph2), the rate-limiting enzyme for the synthesis of central serotonin (Diaz et al. 2013). These observations have been replicated in another model of hyposerotonergy, the Tph2 knock-in mice on a mix C57BL/ 6J-129S6/SvEv background, in which an increased survival of DG cells is described without signicant changes in cell proliferation (Sachs et al. 2013). Likewise, normal neural progenitor proliferation was also seen in Tph2 À/À mice on a pure C57BL/6N background (Klempin et al. 2013 ) conrming that serotonin is not required for physiological DG cell proliferation. "
    [Show abstract] [Hide abstract] ABSTRACT: The Brain-Derived Neurotrophic Factor, BDNF, was discovered more than 30 years ago and, like other members of the neurotrophin family, this neuropeptide is synthetized as a proneurotrophin, the proBDNF, which is further cleaved to yield mature BDNF. The myriad of actions of these two BDNF isoforms in the central nervous system is constantly increasing and requires the development of sophisticated tools and animal models to refine our understanding. This review is focused on BDNF isoforms, their participation in the process of neurogenesis taking place in the hippocampus of adult mammals, and the modulation of their expression by serotonergic agents. Interestingly, around this triumvirate of BDNF, serotonin and neurogenesis, a series of recent research has emerged with apparently counterintuitive results. This calls for an exhaustive analysis of the data published so far and encourages thorough work in the quest for new hypotheses in the field. This article is protected by copyright. All rights reserved.
    Article · May 2016
    • "This is reasonable since prevalence of depression after 3 years of onset of stroke can impede the process of rehabilitation and has been associated with poorer outcomes and increased length of stay in hospital [6]. In fact, it has been observed that the pathogenesis of depression derives from disturbance of neuroplasticity and reduced neurogenesis [18]; therefore timely prescription of antidepressants by psychiatrists in stroke management may hold significant promise in relieving the possibility of PSD through its neuroprotective effects on brain plasticity and neurogenesis [19, 20]. Surprisingly, this practice is not currently applicable at the tertiary hospital where this study was conducted and, perhaps, likewise, other hospitals. "
    [Show abstract] [Hide abstract] ABSTRACT: Purpose . To identify stroke survivors with symptoms of poststroke depression and the extent of psychiatry needs and care they have received while on physiotherapy rehabilitation. Participants . Fifty stroke survivors (22 females and 28 males) at the outpatient unit of Physiotherapy Department, University of Nigeria Teaching Hospital, Enugu, who gave their informed consent, were randomly selected. Their age range and mean age were 26–66 years and 54.76 ± 8.79 years, respectively. Method . A multiple case study of 50 stroke survivors for symptoms of poststroke depression was done with Beck’s Depression Inventory, mini mental status examination tool, and Modified Motor Assessment Scale. The tests were performed independently by the participants except otherwise stated and scored on a scale of 0–6. Data were analyzed using Z -test for proportional significance and chi-square test for determining relationship between variables, at p < 0.05 . Results . Twenty-one (42.0%) stroke survivors had symptoms of PSD, which was significantly dependent on duration of stroke ( χ 2 = 21.680, df = 6, and p = 0.001 ), yet none of the participants had a psychiatry review. Conclusions . Symptoms of PSD may be common in cold compared to new cases of stroke and may need psychiatry care while on physiotherapy rehabilitation.
    Full-text · Article · Jan 2016
    • "The current study did not directly explore the mechanisms through which FLX administration leads to increased proliferation in the habenula and hypothalamus. However, our current results indicate that chronic FLX can increase BDNF mRNA expression in the habenula and hypothalamus, similar to what has been reported previously in the hippocampus (Nibuya et al., 1995; Sachs, Jacobsen, et al., 2013). Prior research has shown that infusions of BDNF can promote cell proliferation and neurogenesis in the hypothalamus and the habenula (Pencea et al., 2001). "
    [Show abstract] [Hide abstract] ABSTRACT: Chronic treatment with antidepressants has been shown to enhance neurogenesis in the adult mammalian brain. Although this effect was initially reported to be restricted to the hippocampus, recent work has suggested that fluoxetine, a selective serotonin reuptake inhibitor, also promotes neurogenesis in the cortex. However, whether antidepressants target neural progenitor cells in other brain regions has not been examined. Here, we used BrdU labeling and immunohistochemistry with a transgenic mouse line in which nestin+ neural progenitor cells can be inducibly labeled with the fluorescent protein, Tomato, following tamoxifen administration. We investigated the effects of chronic fluoxetine on cell proliferation and nestin+ progenitor cells in periventricular areas in the medial hypothalamus and medial habenula, two brain areas involved in stress and anxiety responses. Our data provide the first in vivo evidence that fluoxetine promotes cell proliferation and neurogenesis and increases the mRNA levels of BDNF in the hypothalamus and habenula. By identifying novel cellular targets of fluoxetine, our results may provide new insight into the mechanisms underlying antidepressant responses. © The Author 2015. Published by Oxford University Press on behalf of CINP.
    Full-text · Article · Oct 2014
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