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Serotonin-1A autoreceptors are necessary and sufficient for the
normal formation of circuits underlying innate anxiety
Jesse W Richardson-Jones1, Caryne P Craige2, Thanh H Nguyen3, Hank F Kung4, Alain M
Gardier3, Alex Dranovsky1,6, Denis J David3, Bruno P Guiard3, Sheryl G Beck2, René Hen*,
1,5,6, and E David Leonardo*,1,6
1 Department of Psychiatry, Columbia University, New York, NY, 10032 USA
2 Department of Anesthesiology, Children’s Hospital of Philadelphia and University of
Pennsylvania, Philadelphia, PA, 19312 USA
3 Faculté Pharmacie, Université Paris Sud, EA 3544, Chatenay-Malabry, F-92296 France
4 Department of Radiology, University of Pennsylvania, Philadelphia, PA, 19104USA
5 Department of Neuroscience, Columbia University, New York, NY, 10032 USA
6 The New York State Psychiatric Institute, New York, NY 10032 USA
Abstract
Identifying factors contributing to the etiology of anxiety and depression is critical for the
development of more efficacious therapies. Serotonin (5-HT) is intimately linked to both
disorders. The inhibitory serotonin-1A (5-HT1A) receptor exists in two separate populations with
distinct effects on serotonergic signaling: 1) an autoreceptor that limits 5-HT release throughout
the brain, and 2) a heteroreceptor that mediates inhibitory responses to released 5-HT. Traditional
pharmacologic and transgenic strategies have not addressed the distinct roles of these two receptor
populations. Here we use a recently developed genetic mouse system to independently manipulate
5-HT1A auto and heteroreceptor receptor populations. We show that 5-HT1A autoreceptors act to
affect anxiety-like behavior. In contrast, 5-HT1A heteroreceptors affect responses to forced swim
stress, without effects on anxiety-like behavior. Together with our previously reported work, these
results establish distinct roles for the two receptor populations, providing evidence that signaling
through endogenous 5-HT1A autoreceptors is necessary and sufficient for the establishment of
normal anxiety-like behavior.
Introduction
Anxiety and depression are highly co-morbid disorders with partially overlapping genetic
predisposition, environmental risk, symptom dimensions, and treatment profiles ( Ressler
and Nemeroff, 2000; Nemeroff, 2002). This overlap suggests that anxiety and depression
likely share some circuitry and neurochemistry, but can be influenced by distinct factors.
Serotonin (5-HT) participates in the etiology and treatment of both anxiety and depression
(Rush et al., 2006; Davidson, 2009). The most common treatments for major depressive
*To whom correspondence should be addressed: el367@columbia.edu, rh95@columbia.edu. Corresponding Author: E. David
Leonardo M.D., Ph.D., Department of Psychiatry, Columbia University/New York State Psychiatric Institute, 1051 Riverside Drive,
Box 87, New York, New York 10032, Tel 212-543-5266, el367@columbia.edu.
Conflict of Interest: R.H. receives compensation as a consultant for Braincells, Inc., in relation to the generation of novel
antidepressants. E.D.L. receives compensation as a consultant from PGx Health.
NIH Public Access
Author Manuscript
J Neurosci. Author manuscript; available in PMC 2011 October 20.
Published in final edited form as:
J Neurosci
. 2011 April 20; 31(16): 6008–6018. doi:10.1523/JNEUROSCI.5836-10.2011.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
disorder and many anxiety disorders are the selective serotonin reuptake inhibitors (SSRIs),
which are thought to exert their therapeutic effects by increasing extracellular 5-HT levels
(Gartside et al., 1995). However, in contrast to the beneficial effects of SSRIs in adults,
evidence from rodent models suggest that SSRI treatment during early development
increases anxiety or depression later in life (Caspi et al., 2003; Lira et al., 2003; Ansorge et
al., 2008; Olivier et al., 2008; Oberlander et al., 2009). Thus, 5-HT may affect immature and
mature mood-related circuitry differently.
Serotonin is released throughout the forebrain by axons emanating from cell bodies located
in the midbrain raphe (Barnes and Sharp, 1999). The largely neuromodulatory effects of 5-
HT are mediated through fourteen receptor subtypes, whereas 5-HT levels are limited by
two inhibitory autoreceptors expressed on 5-HT raphe neurons (Barnes and Sharp, 1999).
The 5-HT1A receptor, a major inhibitory receptor subtype, is expressed: 1) on 5-HT raphe
neurons as an autoreceptor, limiting released 5-HT at nerve terminals, and 2) in diverse
target regions as a heteroreceptor mediating cellular responses to released 5-HT. In
particular, 5-HT1A heteroreceptors are highly expressed in forebrain regions that regulate
mood and anxiety, such as the prefrontal cortex, hippocampus, and amygdala (Hamon et al.,
1990; Beck et al., 1992; Riad et al., 2000). Thus, the 5-HT1A receptor can influence anxiety
or depression by impacting either 5-HT levels (as an autoreceptor) or the limbic response to
released 5-HT (as a heteroreceptor).
Diverse evidence has demonstrated that 5-HT1A receptors contribute to the establishment of
mood disorders. A functional polymorphism in the promoter region of the human Htr1a
gene (coding for the human 5-HT1A receptor) that regulates receptor levels is linked to
depression, response to antidepressants, and amygdala reactivity (Lemonde et al., 2003; Le
Francois et al., 2008; Fakra et al., 2009). Mice lacking all 5-HT1A receptors throughout life
display increased anxiety-like behavior (Heisler et al., 1998; Parks et al., 1998; Ramboz et
al., 1998), an effect that is phenocopied by pharmacological blockade during the first few
postnatal weeks (Lo Iacono and Gross, 2008). Transgenic gain-of-function studies have
likewise suggested that the 5-HT1A receptor can stably impact anxiety-like behavior during
the first weeks of life (Gross et al., 2002; Bert et al., 2006). Given this evidence, 5-HT1A
receptors may influence anxiety and depression differentially not only between
subpopulations, but also across developmental stages in an animal.
Separating both the temporal (developmental versus adult) and spatial (autoreceptor versus
heteroreceptor) roles of the endogenous 5-HT1A receptor in anxiety and depression has
proven difficult with existing genetic or pharmacological techniques. To independently
assess the functional role of endogenous 5-HT1A autoreceptors and heteroreceptors, we
developed a genetic mouse system with temporal and spatial specificity and tested the adult
phenotype of these animals in a number of classic, mood-related paradigms.
Materials and Methods
Transgenic Mice
5-HT1A Autoreceptor KO mice—Mice with suppressible 5-HT1A receptors (Htr1atetO)
were made as described (Richardson-Jones et al 2010). Htr1atetO/tetO mice demonstrate a
pattern of 5-HT1A receptors that is indistinguishable from the wild-type pattern. Mice with
inducible suppression of 5-HT1A autoreceptors were homozygous for the tetO insertion
(Htr1atetO/tetO), possessed one copy of the raphe-specific tTS suppressor transgene, Pet1-
tTS, and were maintained in the absence of doxycycline (dox) throughout life (Richardson-
Jones et al., 2010).
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5-HT1A Heteroreceptor KO mice—Heteroreceptor-specific suppression was achieved
by placing tTS under transcriptional control of elements of the α-CamKII promoter
(Mayford et al., 1996). Briefly, an EcoRI-BamHI fragment containing the tTS coding
sequence was freed from the pAct tTS plasmid (Mallo et al., 2003). The fragment was
blunted and cloned into the EcoRV site of the pNN265 plasmid, flanking it with a generic 5′
intron and 3′ intron and SV40 polyA sequence (Mayford et al., 1996). The tTS sequence
flanked by introns was freed by digest with Not1 and cloned into the pMM403 plasmid,
placing it under the control of α-CamKII promoter fragments. Lastly, the promoter
fragments and tTS coding sequence (CamK-tTS) were freed by Sfi/Srf digest and subjected
to pronuclear injection into B6CBA/F2 Hybrid zygotes. Founders were screened by PCR for
tTS (as previously described) and southern blot analysis.
Seven founder lines were generated, bred to the Htr1atetO/tetO background, and screened for
suppression of 5-HT1A heteroreceptors. The three general patterns of suppression in these
lines corresponded to the previous reported activity of these promoter fragments: 1)
suppression of all receptors in the brain, 2) preferential suppression of forebrain receptors
while sparing receptors in the raphe, and 3) preferential suppression of dorsal hippocampal
receptors while sparing most other receptor populations (data not shown) (Gross et al., 2002;
Pittenger et al., 2002). One line was identified that displayed suppression of 5-HT1A
heteroreceptors without affecting autoreceptors, and this line was used for subsequent
experiments. Mice with inducible suppression of 5-HT1A heteroreceptors were homozygous
for the tetO insertion (Htr1atetO/tetO), possessed one copy of the forebrain-specific tTS
suppressor transgene, CamK-tTS, and were maintained in the absence of dox throughout
life.
For all experiments, male Htr1atetO/tetO/Pet-tTS+ and Htr1atetO/tetO/CamK-tTS+ mice were
bred to Htr1atetO/tetO females. Thus, the tTS transgene was transmitted through the male
germline, ensuring that all pups were raised by mothers of the same genotype, regardless of
dox status. All experiments were conducted on male offspring. Animals were maintained on
a mixed 129S6/Sv; C57B6; CBA background. To control for the effect of transgene
insertion and genetic background, controls for all experimental groups were generated by
feeding Htr1atetO/tetO/Pet-tTS+ or Htr1atetO/tetO/CamK-tTS+ animals chow containing
40mg/kg dox (Product # F5545, Bioserv, Frenchtown, NJ) throughout life. Dox exposure in
utero in these animals was accomplished by feeding mothers with the dox chow throughout
the course of breeding. Experimental animals were fed standard laboratory chow (Prolab
Isopro RMH 3000, PMI Nutrition International, Brentwood, MO) either throughout life
(Auto-KO and Hetero-KO) or beginning at postnatal day 50 (Adult Hetero-KO). To control
for the possible effects of dox on behavior, littermate controls lacking the tTS transgene, in
which dox had no effect on 5-HT1A receptor expression, were also tested in baseline
behavioral experiments. For all tests presented, dox exposure alone did not result in any
behavioral changes. (Data not shown).
Animal Husbandry
Animals were housed in groups of between 3 and 5 per cage and had access to food and
water, ad libitum. Animals were maintained on a 12:12 light/dark schedule, and all testing
was conducted during the light period. Animal protocols were approved by the Institutional
Animal Care and Use Committee and were conducted in accordance to the NIH Guide for
the Care and Use of Laboratory Animals Care was taken to minimize number of animals
used in all experiments and their suffering.
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Receptor autoradiography
Mice of the ages indicated were sacrificed by cervical dislocation and decapitation.
Extracted brains were frozen immediately on crushed dry ice (−75°C) and maintained at
−80°C until sectioning. Brains were cryosectioned at a thickness of 18μm, and sections were
thaw-mounted on Superfrost slides (Fisher, Fairlawn, NJ, USA). Sections were maintained
at −80°C until processing. Mounted sections were processed for 4-(2′-Methoxyphenyl)-1-
[2′-(n-2″-pyridinyl)-p-[125I]iodobenzamido]ethylpiperazine) (125I-MPPI) autoradiography
and receptor levels were quantified using a previously described method (Richardson-Jones
et al., 2010).
Electrophysiology
Whole cell recordings—Whole cell recordings of dorsal raphe and hippocampal CA1
pyramidal neurons were made as previously described (Beck et al., 2004; Tsetsenis et al.,
2007; Crawford et al., 2010). In brief, mice were sacrificed using decapitation. While
submerged in a cold aCSF solution where NaCl is replaced with sucrose (248 mM), the
coronal slices were cut at the level of the dorsal hippocampus and midbrain with a Leica
VT1000s vibratome (Leica Microsystems, Bannockburn, IL) to generate 200μm thick brain
slices. Slices were maintained in aCSF bubbled with 95%O2/5%CO2 at 36°C for 1 hour and
then at room temperature until used. Individual slices were then placed in a recording
chamber and continuously perfused with 32–34°C aCSF solution bubbled with
95%O2/5%CO2 with a solution flow rate of 1.5–2ml/min. The composition of the aCSF was
(in mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2.0 MgSO4, 2.5 CaCl2, 10 dextrose, and 26
NaHCO3. Neurons were visualized using Nikon E600 (Optical Apparatus, Ardmore, PA)
upright microscope fitted with a 40x water-immersion objective, DIC, and infrared filter.
Whole-cell recording pipettes fabricated on a Sutter Instruments pipette puller (P-97, Sutter
Instrument, Novato, CA) had a resistance of 6–10MΩ when filled with an intracellular
solution of 130mM Kgluconate, 5mM NaCl, 10mM Na-phosphocreatinine, 1mM MgCl2,
0.02mM EGTA, 10mM HEPES, 2mM MgATP, 0.5mM Na2GTP, and 0.1% biocytin (pH
7.3). Recordings were collected online with a Multiclamp 700B amplifier, Digidata 1320 A/
D converter, and Clampex 9.0 software (Molecular Devices, Union City, CA). In current
clamp recording, resting membrane potential (RMP), action potential (AP) threshold, AP
duration, after-hyperpolarization (AHP) amplitude, and the time it takes for the AHP to
depolarize to one-half its peak amplitude (AHP t1/2) were measured directly from traces as
previously described (Crawford et al., 2010). In voltage clamp, with a holding potential of
−60 mV, the magnitude of the outward current elicited by bath application of 100 nM 5-
carboxyamidotryptamine (5-CT) was measured. 5-CT was chosen for these experiments
instead of 8-OH-DPAT as it is a high affinity full agonist, is less lipophillic and washes out
more easily from the slices. Following the experiment, slices were fixed for 2–3 hours with
4% paraformaldehyde and the midbrain slices were processed for immunohistochemical
detection of tryptophan hydroxylase (TPH). Identity of all recorded cells was confirmed by
visualization of the biocytin-filled cells. Immunohistochemical identification of each neuron
recorded in patch clamp configuration was completed as previously described (Beck et al.,
2004; Lemos et al., 2006; Kirby et al., 2008). In brief, a standard immunohistochemistry
protocol was used on 200μm-thick slices using mouse anti-TPH (1:200, Sigma) along with
secondary donkey anti-mouse Alexa Fluor 488 (1:200, Invitrogen). For visualization of the
biocytin filled cell and streptavidin-conjugated Alex Fluor 638 (1:100, Invitrogen), and
NeuN (1:500, Sigma) along with secondary donkey anti-mouse Alexa Flour 488 (1:200,
Invitrogen). TPH labeling of biocytin filled cells was imaged on a Leica DMR fluorescent
microscope (Leica Microsystems) using OpenLab 3.0.9 software (Improvision) and
confirmed on a Leica DMIRE2 confocal microscope (Leica Microsystems, Bannockburn,
IL) using Leica confocal software (version 2.5, Leica Microsystems).
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Intracerebral in vivo microdialysis
Extracellular 5-HT levels were measured by in vivo microdialysis as previously described
(Guiard et al., 2008). Briefly, two concentric dialysis probes were implanted in the vHPC
and PFC (outer diameter × active length: 0.3 × 1.6 and 0.3 mm × 2 mm, respectively) of
anesthetized mice (chloral hydrate, 400mg/kg, i.p.). Stereotaxic coordinates (in mm) were,
PCF: A = 1.6, L = 1.3, V = 1.6; vHPC: A = −2.8, L = 3.0, V = 3.0 (Franklin and Paxinos,
1997). Animals were allowed to recover for a period of 24 hours. Following recovery,
probes were continuously perfused with aCSF, and dialysates were collected every 15
minutes for analysis by HPLC-amperometry (Guiard et al., 2008). Baseline 5-HT levels
were calculated as the average of the first four samples, ±SEM. Freely moving mice were
treated (t=0) with either a challenge dose of fluoxetine (18 mg/kg; i.p.) or its vehicle, and
dialysate samples were collected for a 0–120 min post-treatment period. The limit of
sensitivity for 5-HT was 0.5 fmol/sample (signal-to-noise ratio 2). Following sample
collection, brains were removed and sectioned to ensure proper probe placement.
Behavioral and Physiological Testing
All animals used for behavioral testing were age matched within two weeks. Animals were
initially tested at 11–13 weeks of age, at least four weeks after the cessation of dox in adult
heteroreceptor-KO animals. Baseline anxiety tests were completed before other behavioral
tests.
8-OH DPAT Induced Hypothermia—Body temperature was assessed intra-rectally,
using a lubricated probe inserted approximately 2 cm and a Thermalert TH-5 thermal
monitor (Physitemp, Clifton, NJ). Mice were singly housed in clean cages for 10 minutes,
and three baseline body temperature measurements were taken. Ten minutes after the third
baseline measurement, animals received 8-OH DPAT i.p. at the doses indicated and body
temperature was monitored every ten minutes for a total of 50 minutes. Temperatures are
represented as a change from the final baseline measurement.
Stress-Induced Hyperthermia—The stress induced hyperthermia paradigm measures a
physiologic response to a stressful stimuli (Adriaan Bouwknecht et al., 2007). Briefly,
animals in their home cages were moved to a testing room and allowed to acclimate for 1
hour. One animal per cage was removed and a baseline body temperature was measured
intrarectally. Each animal was then placed in a novel, clean cage for ten minutes, after which
a second body temperature was recorded.
Open Field Test—Exploration in response in response to a novel open field was measured
as described (Richardson-Jones et al., 2010). Dependent measures were total path length
(cm), number and percent distance in the center (distance traveled in the center divided by
the total distance traveled).
Light/Dark Choice Test—Exploration of the light/dark chamber was measured as
described (Richardson-Jones et al., 2010). Dependent measures were total distance and
percent time spent in the light compartment.
Modified Forced Swim Test—Behavioral response to forced swimming was assayed as
described previously (David et al., 2007). Briefly, mice were placed into clear plastic
buckets 20 cm in diameter and 23 cm deep filled 2/3 of the way with 26°C water and
videotaped from the side. Mice had a two minute pre-test period prior to initiating scoring,
after which two consecutive two minute periods were scored. All animals were exposed to
the swim test on two consecutive days. Scoring was done using an automated Viewpoint
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Videotrack software package (Montreal, Canada). Dependent variables were immobility,
swimming and climbing.
Statistical Analysis
In general, the effect of treatment or dose was analyzed using an analysis of variance
(ANOVA), using repeated measures where appropriate. Significant ANOVAs were followed
up with Fisher PLSD test for behavioral and physiological measures, and with Student-
Neuman-Keuls t-test for electrophysiological characterization.
Results
Conditional suppression of the 5-HT1A auto- and heteroreceptors
Previous attempts to target auto- versus heteroreceptor populations independently in mice
relied on transgenic gain-of-function strategies using heterologous promoters that resulted in
over- and mis-expression of the receptor (Gross et al., 2002; Kusserow et al., 2004; Bert et
al., 2006; Audero et al., 2008). Such approaches fall short of establishing causal links
between endogenous receptor populations and either circuit function or behavior. To
independently manipulate either 5-HT1A hetero- or autoreceptors while maintaining
endogenous expression of the other receptor population, we used the tTS/tetO system to
suppress 5-HT1A receptor transcription from the Htr1a gene locus. We previously reported
the generation of mice that allow for specific suppression of 5-HT1A autoreceptors, using
two engineered mouse lines: 1) a raphe-specific tTS transgenic line, and 2) a knock-in
mouse line with tetO sites inserted between the promoter and coding regions of the Htr1A
locus (Richardson-Jones et al., 2010). To suppress 5-HT1A autoreceptors in the raphe
throughout life (Auto-KO), the previously described Htr1atetO/tetO/Pet-tTS+ mice were
maintained in the absence of dox throughout life (Figure 1c, d). Controls for these mice, in
which 5-HT1A autoreceptors were not suppressed, were generated by maintaining
Htr1atetO/tetO/Pet-tTS+ mice in the presence of dox throughout life (Figure 1a, d).
Quantitative receptor autoradiography revealed that less than 20% of control 5-HT1A
receptor binding remained in the dorsal raphe of adult Auto-KO mice (one-tailed t test in the
dorsal raphe, t13 = −15.503, p <0.0001), whereas no changes were observed in binding of 5-
HT1A in areas of high heteroreceptor expression, such as the amygdala, hippocampus, and
entorhinal cortex (Repeated measures ANOVA with brain region as a within-subject factor
and group as a between-subject factor, main effect of group F1,13 = 1.398, p = 0.2583;
region by group interaction, F1,13 = 0.795, p = 0.46232) (Fig 2). Attempts with this line to
fully suppress the autoreceptor beginning in adulthood, had resulted in a modest 30%
reduction in raphe 5-HT1A expression. Physiological and behavioral characterization of
these animals has been reported elsewhere (Richardson-Jones et al., 2010). The reason for
more effective suppression of 5-HT1A transcription in animals maintained in the absence of
dox throughout life remains unclear, although it appears that early developmental
suppression at the 5-HT1A locus may be irreversible (data not shown).
To generate mice with suppression of 5-HT1A heteroreceptors without affecting
autoreceptors, we first created a transgenic mouse line with tTS expression directed by
promoter elements from the α-CamKII gene (Mayford et al., 1996). One mouse line was
identified that demonstrated suppression of only 5-HT1A forebrain heteroreceptors without
affecting 5-HT1A raphe autoreceptors (Hetero-KO) (ANOVA in dorsal raphe, F2,9 = 0.429,
p < 0.6635)(Figure 1b, e; Figure 2b, d). In Hetero-KO mice, receptor expression was
undetectable in the hippocampus, amygdala, or prefrontal cortex, whereas low levels of
receptor remained in the olive, entorhinal cortex, and the medial and lateral septum
(ANOVA for the effect of group in hippocampus, F2,9 = 20210, p < 0.0001) (Fig 2b, d).
Consistent with the expected delayed developmental activation of the α-CamKII promoter
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elements, we confirmed that suppression of receptors was complete by postnatal day 14 (Fig
2e). Control mice, in which 5-HT1A heteroreceptors were not suppressed, were generated by
maintaining Htr1atetO/tetO/CamKII-tTS+ mice in the presence of dox throughout life (Figure
1e). Adult suppression of 5-HT1A heteroreceptors (Adult-Hetero-KO) was achieved by
maintaining Htr1atetO/tetO/CamKII-tTS+ mice in the presence of dox until postnatal day 50,
then withdrawing dox for at least 4 weeks. Adult suppression of 5-HT1A receptors in these
animals was as effective as early developmental suppression (Figure 1e and Figure 2b, d).
Altered agonist response in mice with conditional suppression of 5-HT1A auto- and
heteroreceptors
To directly confirm the functional impact of receptor suppression revealed by
autoradiography, we performed whole cell recordings of neurons in acute slice in response
to the 5-HT1,7 agonist, 5-carboxyamidotryptamine (5-CT). Cellular responses were recorded
in: 1) the dorsal raphe of Auto-KO mice and their Controls, and 2) area CA1 of the
hippocampus in Hetero-KO mice and their Controls. Following recording, cells were filled
with biocytin. Pyramidal neurons in the hippocampus were confirmed by morphological
features, and raphe neurons were confirmed as serotonergic by immunohistochemistry for
tryptophan hydroxylase (data not shown).
In agreement with the decreased 5-HT1A autoreceptor levels indicated by autoradiography,
serotonergic neurons in the dorsal raphe of Auto-KO mice had a significantly decreased
current response to 5-CT (two-tailed t-test, t35 = 8.432, p < 0.0001) (Figure 3A-C). Indeed,
we were unable to detect any response to 5-CT (defined as current <5 pA) in 5-HT dorsal
raphe neurons of Auto-KO mice, consistent with a lack of functional 5-HT1A raphe
autoreceptors in these animals. The small amount of remaining receptor binding in the
dorsal raphe may represent either desensitized 5-HT1A autoreceptors or receptors on non-5-
HT raphe neurons (Kirby et al., 2003, Beck et al, 2004).
In pyramidal neurons in area CA1 of Hetero-KO mice, we observed a large decrease in the
average current response to 5-CT (Figure 3D-F) (one-tailed t test, t39 = −3.254, p = 0.0012),
consistent with the greatly decreased levels of heteroreceptor revealed by autoradiography.
To assess the in vivo functional status of 5-HT1A autoreceptors in both groups of mice, we
examined their hypothermic response to challenge with the 5-HT1A agonist, 8-OH-DPAT or
its saline vehicle. The controls for the Auto-KO, Hetero-KO, and Adult Hetero-KO mice
displayed a hypothermic response to 0.5mg/kg 8-OH DPAT consistent with previously
observed values (Figure 4a ii, b ii, and c ii) (repeated-measures ANOVA, main effect of
dose for: Hetero-KO Controls, F1,12 = 46.979, p < 0.0001; Adult Hetero-KO Controls, F1,9 =
33.055, p = 0.0003; Auto-KO Controls, F1,6 = 109.630, p < 0.0001). Consistent with a lack
of functional autoreceptor, 5-HT1A Auto-KO mice displayed no detectable body temperature
decrease in response to 8-OH DPAT, with responses indistinguishable from the response to
saline injection (Figure 4ci) (repeated-measures ANOVA for effect of treatment F1,6 = 1.38,
p = 0.284). Conversely, 5-HT1A Hetero-KO mice displayed a full hypothermic response to
8-OH-DPAT that was significantly different from the response to saline and
indistinguishable from the hypothermic response in their Controls (Figure 4ai, ii) (repeated-
measures ANOVA, effect of dose in Hetero-KO mice, F1,12 = 33.10, p < 0.0001; repeated
measures ANOVA, effect of group in 8-OH DPAT treated mice, F1,12 = 2.486, p = 0.141).
Likewise, 5-HT1A Adult Hetero-KO mice displayed a response to 8-OH DPAT that was
significantly different from the response to saline and indistinguishable from the response in
their matched controls (repeated measures ANOVA, effect of dose in Adult Hetero-KO
mice, F1,9 = 36.216, p = 0.0002; repeated measures ANOVA effect of group in 8-OH DPAT
treated mice, F1,10 = 0.071, p = 0.795) (Figure 4b i, ii). These findings are consistent with
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previous literature implicating 5-HT1A auto-, but not heteroreceptors in the 8-OH-DPAT
induced hypothermic response in mice (Martin et al., 1992).
Taken together, these results provide both a direct in vitro measurement and an in vivo
confirmation of the functional selectivity of genetic suppression of 5-HT1A auto- and
heteroreceptors, respectively, in the Auto-KO, Hetero-KO, and Adult Hetero-KO mice.
Increased serotonergic tone in mice lacking 5-HT1A autoreceptors throughout
life—The extracellular level of serotonin (5-HT)ext is generally thought to be under the
inhibitory control of both 5-HT1A and 5-HT1B autoreceptors. Here we sought to determine
how specific suppression of 5-HT1A autoreceptors throughout life was reflected at the
neurochemical level in adult animals. We observed higher basal cortical 5-HText in whole-
life Auto-KO mice compared to their wild-type littermates (F1,13 = 7.79, p = 0.015).
However, differences between the two strains of mice in the ventral hippocampus (vHPC)
were not significant (F1,13 = 1.74, p = 0.21) (Table 1).
To directly measure the effect of pharmacological inactivation of the 5-HT transporter in
mice lacking 5-HT1A autoreceptors, we challenged both Auto-KO mice and their controls
with the SSRI fluoxetine.
In the frontal cortex, a two-way ANOVA (treatment by genotype) on area under the curve
(AUC) values revealed no significant effect of genotype (F1,28 = 0.041, p = 0.841), but a
significant effect of treatment (F1,28 = 36.984, p < 0.0001). No genotype by treatment
interaction was detected (F1,28 = 0.02, p = 0.9672).
In the vHPC, a two-way ANOVA (treatment by genotype) on AUC values revealed both a
significant effect of genotype (F1,30 = 6.82, p = 0.0139), and a significant effect of treatment
(F1,30 = 58.21, p < 0.0001), as well as a genotype by treatment interaction (F1,30 = 6.62, p =
0.0153).
Thus, as expected, both control and Auto-KO mice displayed increased 5-HText in response
to acute treatment with the fluoxetine in the vHPC (Figure 5a, c) and frontal cortex (Figure
5b, d). Moreover, Auto-KO mice displayed a proportionately larger increase in 5-HText in
response to fluoxetine than their controls in the vHPC, but not in the frontal cortex (Figure
5c, d)(post-hoc for group, p = 0.0198). Together, these results demonstrate that suppression
of 5-HT1A autoreceptors throughout life results in regionally distinct increases in
serotonergic tone.
Increased anxiety-like behavior in mice lacking 5-HT1A autoreceptors—Mice
lacking all 5-HT1A receptors throughout life display reliably increased anxiety-like behavior
(Heisler et al., 1998; Parks et al., 1998; Ramboz et al., 1998). To independently assess the
role of endogenous 5-HT1A autoreceptors and heteroreceptors in anxiety-like behavior, we
tested Hetero-KO, Adult Hetero-KO, and Auto-KO mice in conflict/anxiety paradigms: the
open field, the light/dark choice test, and the elevated plus maze.
We were unable to detect any significant impact on anxiety-like behavior by suppression of
5-HT1A heteroreceptors beginning in early postnatal development. Specifically, we detected
no differences between 5-HT1A Hetero-KO mice and their controls in exploration of the
center of the open field (ANOVA, F1,32 = 0.025, p = 0.875), although a trend was seen for
decreased exploration of the total open field (repeated-measures ANOVA, F1,32 = 3.31, p =
0.078) (Figure 6a). Likewise, no differences were detected between 5-HT1A Hetero-KO
mice and their controls in total exploration (repeated-measures ANOVA, main effect of
group, F1,41 = 0.064, p = 0.802), entries into the light (ANOVA, main effect of group, F1,41
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= 0.258, p = 0.614), or time in the light compartment (ANOVA, main effect of group, F1,41
= 0.077, p = 0.7823) in the light/dark choice test (Figure 6b). Finally, no effects on anxiety-
like behavior were detected in the elevated plus maze (data not shown).
In Adult-Hetero-KO mice, we did not detect changes in exploration of the center of the open
field (ANOVA, F1,47 = 0.686, p = 0.412), although, similar to the results in Hetero-KO
mice, we observed a trend for a decrease in total exploration of the open field (Figure 6c)
(repeated-measures ANOVA F1,47 = 2.74, p = 0.105). No differences were detected between
Adult-Hetero-KO mice and their Controls in total exploration (ANOVA, F1,30 = 0.122, p =
0.730), exploration of the light (ANOVA, F1,30 = 2.11, p = 0.157), or entries into the light in
the L/D choice test (ANOVA, F1,30 = 2.314, p = 0.139) (Figure 6d). Together, these results
demonstrate that suppression of 5-HT1A heteroreceptors, beginning either in early postnatal
development or adulthood, is not sufficient to impact classic anxiety-like behavior.
However, we cannot rule out the contribution of 5-HT1A heteroreceptors to exploratory
behavior, as trends for decreased exploration in the open field were seen in both groups of
mice that lacked 5-HT1A heteroreceptors.
Having observed that suppression of 5-HT1A heteroreceptors alone either throughout life or
beginning in adulthood was not sufficient to impact anxiety-like behavior, we next tested
whether 5-HT1A autoreceptors alone might play a role in the establishment of anxiety-like
behavior. Compared to their controls, Auto-KO mice displayed decreased exploration of the
center of the open field compared to the total (ANOVA, F1,50 = 8.351, p = 0.0057) (Figure
6eii), and a decrease in total exploration (Figure 5ei) (repeated-measures ANOVA, F1,50 =
4.786, p = 0.033). Likewise, in the light/dark choice test, Auto-KO mice displayed decreased
total exploration (ANOVA, F1,37 = 19.74, p < 0.0001) and fewer entries into the light
compartment (ANOVA, F1,37 = 9.19, p = 0.004), but did not demonstrate a decrease in
percent distance in the light (ANOVA, F1,37 = 0.017, p = 0.896) (Figure 6f i-iii). No effects
on anxiety-like behavior were seen in the elevated plus maze (data not shown). The effects
seen in the open field and light-dark test did not reflect an effect of dox treatment, as
littermate control animals lacking the tTS transgene but fed the same chows throughout life
displayed no differences in any of the measures (data not shown). The phenotype observed
in Auto-KO mice is consistent with the increased anxiety-like behavior in conflict-based
tasks and decreased exploration of novel environments, and is similar to what has been
observed in constitutive 5-HT1A KO mice on at least three genetic backgrounds (Heisler et
al., 1998; Parks et al., 1998; Ramboz et al., 1998). These data suggest that endogenous 5-
HT1A autoreceptors are necessary and sufficient for the establishment of anxiety-like
behavior. Furthermore, these data suggest that normal signaling though 5-HT1A
heteroreceptors (with autoreceptors intact) is dispensable for the establishment of anxiety-
like behavior.
Mice lacking 5-HT1A heteroreceptors throughout life demonstrate increased
behavioral despair in the forced swim test (FST)—In addition to its role in anxiety,
diverse evidence has linked 5-HT1A receptors to depression and the response to stress. 5-
HT1A KO mice display altered behavior in the FST and a blunted response to
antidepressants as well as increased anxiety-like behavior (Ramboz et al., 1998; Santarelli et
al., 2003). However, it is unclear whether the anxiety phenotype, and other non-anxiety
related phenotypes like the one seen in the FST are impacted by the same populations of 5-
HT1A receptors, or whether the temporal and spatial role of 5-HT1A receptors for these two
behaviors are distinct.
To test directly the role of 5-HT1A hetero- and autoreceptors in mediating mood-related
behavior that does not capture dimensions of anxiety, we first tested Auto-KO mice in the
forced swim test. In this test, which is often used as a pharmacological screen for
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antidepressant activity, active swimming and climbing are scored as mobility, which
generally decreases over time. We observed that Auto-KO mice displayed decrease mobility
on the second day of testing that was indistinguishable from their controls (Fig 7c)
(repeated-measures ANOVA, main effect of group, F1,48 = 0.871, p = 0.3554; main effect of
time, F3,48 = 23.771, p <0.0001). This finding contrasted with our previously reported effect
of adult suppression of 5-HT1A autoreceptors (with decreased autoreceptor levels decreasing
behavioral despair)(Richardson-Jones et al., 2010), and suggests that compensation occurs
when 5-HT1A autoreceptors are suppressed throughout life.
As suppressing autoreceptors throughout life did not alter the response to forced swim
stress, we next tested the response of Hetero-KO and Adult-Hetero-KO mice. Although
Hetero-KO mice were indistinguishable from their controls on the first day of testing, they
displayed markedly less mobility, or more behavioral despair, on the second day of testing
(Fig 7a) (repeated measures ANOVA, group by time interaction, F3,34 = 2.97, p = 0.0345;
post hoc for Day 2 minutes 3–4 and 5–6 p = 0.0371 and p = 0.0018, respectively).
Conversely, adult suppression of 5-HT1A heteroreceptors was not sufficient to impact
behavioral despair on either day of the test (Figure 7b) (repeated-measures ANOVA, main
effect of group between Adult Hetero-KO mice and Controls, F1,44 = 2.132, p = 0.1513;
group by time interaction, F3,44 = 0.570, p = 0.6357). This demonstrates that 5-HT1A
heteroreceptors act developmentally to establish the circuitry underlying this behavior, and
establishes a dissociation, between the roles of 5-HT1A auto- and heteroreceptors in
modulating mood-related anxious and non-anxious behavior, respectively.
DISCUSSION
Independent suppression of endogenous 5-HT1A auto- and heteroreceptors
The 5-HT1A receptor exists as two distinct populations in the brain (auto- and
heteroreceptors) that, individually, could impact the circuitry underlying mood and anxiety
in both development and/or adulthood. We accomplished independent manipulation of 5-
HT1A auto- and heteroreceptors populations using the tTS/tetO system (Mallo et al., 2003;
Richardson-Jones et al., 2010). This system provides a number of advances over classic KO
and previous transgenic technology. First, it allows for spatial and temporal specificity,
neither of which are possible using constitutive KO mice. Given the hypothesized difference
in the roles of the 5-HT system in development and adulthood, temporal specificity was a
primary goal (Ansorge et al., 2007; Ansorge et al., 2008). Second, this genetic strategy
allows for examination of the role of endogenous receptors; unlike previous transgenic over-
expression strategies, the tTS/tetO strategy relies on suppressible gene expression from the
endogenous promoter elements. Moreover, we have recently demonstrated that this system
is generalizable to other genes (Tanaka et al., 2010).
Establishment of 5-HT tone
In agreement with the observation that the firing activity of serotonergic neurons and
consequently the release of 5-HT is under tonic inhibitory control by somatodendritic 5-
HT1A autoreceptors(Haddjeri et al., 2004), constitutive deletion of the 5-HT1A receptor
increases basal extracellular 5-HT level in some brain structures (He et al., 2001; Parsons et
al., 2001). This is consistent with data obtained here in Auto-KO mice (Table 1).
Nevertheless, the effect observed here is region-dependent, as basal 5-HText was increased
in the frontal cortex, but not in the vHPC of Auto-KO mice. Several hypotheses may explain
such differences. Regional differences in 5-HT re-uptake sites in mouse brain could account
for these neurochemical observations. Autoradiographic studies have reported a lower
density of SERT in the frontal cortex compared to the hippocampus in constitutive 5-HT1A
KO mice(Ase et al., 2001). Interestingly, SERT downregulation is also observed in double
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5-HT1A/1B KO mice (Guilloux et al., 2010: submitted). Thus, the increase in 5-HText
observed in the frontal cortex, (but not in the vHPC) of Auto-KO mice, may be attributed to
a specific reduction of SERT expression in the frontal cortex. A second possibility to be
explored with regard to the present results is the marked difference in the intrinsic
organization of 5-HT innervations. The frontal cortex is primarily innervated by the dorsal
raphe nucleus (DRN), while the hippocampus is mostly innervated by the median raphe
nucleus (MRN) (Azmitia and Segal, 1978; Imai et al., 1986; McQuade and Sharp, 1997). It
is thus possible that the enhancement of basal extracellular 5-HT levels specifically in the
frontal cortex in Auto-KO mice resulted from a greater effect of autoreceptor deletion on the
release of 5-HT from DRN as compared to the MRN.
The magnitude of 5-HT release in the presence of transporter blockade also displayed a
region-dependent difference. As expected, upon challenge with fluoxetine, 5-HText was
increased in both control and Auto-KO mice (Figure 5). However, the magnitude of the
response to SSRI in Auto-KO mice was greater in the vHPC than in the frontal cortex. These
neurochemical effects markedly contrast with levels observed at baseline. Owing to the
greater density of 5-HT uptake sites in the hippocampus in mice (compared to the frontal
cortex (Ase et al., 2001)), SSRIs are expected to cause a greater increase in 5-HText levels in
the hippocampus. Indeed, this was observed in the present study. In contrast with this
assumption, fluoxetine (Malagie et al., 1995) and paroxetine (Romero and Artigas, 1997)
have been shown to increase 5-HText to a comparable extent in the frontal cortex and
hippocampus of WT mice. These data can be interpreted in light of the distinct roles that 5-
HT1A heteroreceptors in the frontal cortex are thought to play in the negative feedback
inhibition of the raphe. Specifically, frontal cortex forebrain heteroreceptors are thought to
participate in feedback via polysynaptic connections to the raphe, whereas hippocampal
heteroreceptors are not (Ceci et al., 1994; Assie and Koek, 1996; Casanovas et al., 1999;
Hajos et al., 1999). Our results in the Auto-KO mouse suggest that autoreceptor feedback in
response to fluoxetine is stronger in raphe projections to hippocampus than frontal cortex,
since heteroreceptor based negative feedback plays a substantial role solely in frontal cortex.
Alternatively, the heterogeneous elevation of 5-HText induced by fluoxetine in Auto-KO
mice could be due to changes in the sensitivity of terminal 5-HT1B autoreceptors. Such
changes have been detected in constitutive 5-HT1A KO mice (Boutrel et al., 2002).
Establishment of anxiety-like behavior
Mice lacking all 5-HT1A receptors (both autoreceptors and heteroreceptors) throughout life
display increased anxiety-like behavior (Ramboz et al., 1998). Subsequent gain-of-function
experiments, in which 5-HT1A receptors were ectopically overexpressed in forebrain areas
such as the cortex and striatum (in the absence of autoreceptors), reversed the increased
anxiety behavior in 5-HT1A KO mice (Gross et al., 2002). These findings led to the theory
that endogenous 5-HT1A heteroreceptors in the forebrain control the normal establishment
of anxiety-like behavior (Gross and Hen, 2004; Akimova et al., 2009; Goodfellow et al.,
2009; Zhang et al., 2010). A separate body of literature has implicated serotonergic tone
during postnatal development in determining anxiety-like behavior, with whole-life or early
postnatal blockade of the 5-HT transporter increasing anxiety in rodents (Lira et al., 2003;
Ansorge et al., 2004; Jennings et al., 2006). As one of the major modulators of serotonergic
tone, the 5-HT1A autoreceptors may likewise participate in the developmental establishment
of anxiety. Thus, both 5-HT1A autoreceptors and 5-HT1A heteroreceptors are well-
positioned to affect the circuitry underlying anxiety-like behavior.
Here we demonstrate, using a loss-of-function approach, that suppression of endogenous 5-
HT1A autoreceptors throughout life is sufficient to increase anxiety in the adult. Conversely,
loss of endogenous heteroreceptors beginning either in the early postnatal period or in
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adulthood is not sufficient to impact anxiety-like behavior. Together these results suggest
that the anxious-like phenotype of the 5-HT1A KO mouse likely results from increased
serotonergic neuron excitability during development. Thus, our results suggest a new
framework for interpreting the role of the 5-HT1A receptor in the developmental
programming of anxiety behavior that de-emphasizes the role of the heteroreceptors in favor
of autoreceptor-mediated serotonergic tone.
The results presented here by no means discount the role of serotonergic signaling in the
developing forebrain in establishing anxiety circuitry. Indeed, forebrain neurons undergo a
shift in their response to 5-HT during this time that corresponds to differential coupling of
excitatory and inhibitory receptor subtypes (Beique et al., 2004; Goodfellow et al., 2009).
Thus the serotonergic system can impact anxiety circuitry both at the level of
neurotransmitter release and through the relative balance of inhibitory and excitatory
response in the developing forebrain. However, using our loss-of-function approach, we
conclude that under normal conditions endogenous 5-HT1A forebrain heteroreceptors are not
the primary mediators of 5-HT’s effect on developing anxiety circuitry. Whether these
receptors might play a more prominent role under adverse conditions (i.e. with higher 5-HT
as result of stress) is beyond the scope of these experiments but remains an intriguing
hypothesis (Goodfellow et al., 2009).
Finally, our results highlight the importance of distinguishing between endogenous functions
of a receptor (often most clearly revealed through loss-of-function manipulations) and the
ability of a receptor to ectopically impact circuits (often most clearly revealed through gain-
of-function manipulations)(Gross et al., 2002; Kusserow et al., 2004). Recent evidence
suggests that the 5-HT1A receptor has great utility in pharmacogenomic experiments
designed to probe circuit function, but the phenotypes conferred by such manipulations
should not be confused with the endogenous role of the receptor (Tsetsenis et al., 2007;
Audero et al., 2008).
Behavioral effects of heteroreceptor suppression
In contrast to the widely recognized antidepressant effect of increasing 5-HT in the adult,
several pieces of evidence suggest that increased 5-HT tone in early development can
increase adult depression as well as anxiety. Although antidepressant drugs that increase
extracellular 5-HT levels reliably decrease immobility in adult rodents in the forced swim
test, mice lacking 5-HT transporter activity either throughout life or only during a postnatal
developmental period show increased immobility in the this test (Lira et al., 2003; Wellman
et al., 2007). Moreover, mice lacking enzymes necessary for the synthesis of 5-HT
throughout life (which display lower levels of 5-HText) show decreased immobility in the
forced swim test in adulthood (Savelieva et al., 2008). Thus, the circuitry underlying
responsiveness in the forced swim test response can be influenced by extracellular 5-HT
levels during development. Indeed, it appears that manipulations of extracellular 5-HT levels
during postnatal development result in behaviors that are directly opposite to those caused
by pharmacological manipulation of serotonin levels with SSRIs in adult animals.
Here we demonstrate that mice lacking 5-HT1A autoreceptors throughout life that display
increased anxiety-like behavior, nevertheless display no differences from their controls in
forced swim behavior in adulthood. In contrast, we demonstrate that mice lacking 5-HT1A
heteroreceptors beginning in development display decreased mobility, or increased
behavioral despair, in adulthood (Fig. 8). This phenotype was not observed when
heteroreceptor suppression was initiated in adulthood, suggesting that serotonergic signaling
in the forebrain during development stably impacts the circuitry underlying the behavioral
response to forced swim stress without affect conflict based anxiety measures.
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Together, our findings demonstrate a functional dissociation across both spatial (raphe
versus forebrain) and temporal (development versus adulthood) domains within a single
receptor subtype, and may be relevant to the partially overlapping set of genes and circuitry
underlying anxiety and depression-related behavior.
Acknowledgments
We thank Matheus Araujo for technical assistance. This work was supported by NIH grants K08 MH076083 and
R01 MH091427 to E.D.L., a NARSAD Young Investigator award to E.D.L., R01 MH075047 to S.G.B, R01
MH068542 to R.H., a NARSAD Distinguished Investigator Award to R.H. and AstraZeneca grant CU08-8439 to
R.H.
References
Adriaan Bouwknecht J, Olivier B, Paylor RE. The stress-induced hyperthermia paradigm as a
physiological animal model for anxiety: a review of pharmacological and genetic studies in the
mouse. Neurosci Biobehav Rev. 2007; 31:41–59. [PubMed: 16618509]
Akimova E, Lanzenberger R, Kasper S. The serotonin-1A receptor in anxiety disorders. Biol
Psychiatry. 2009; 66:627–635. [PubMed: 19423077]
American Psychiatric Association., American Psychiatric Association. Task Force on DSM-IV.
Diagnostic and statistical manual of mental disorders: DSM-IV-TR. 4. Washington, DC: American
Psychiatric Association; 2000.
Ansorge MS, Hen R, Gingrich JA. Neurodevelopmental origins of depressive disorders. Curr Opin
Pharmacol. 2007; 7:8–17. [PubMed: 17188022]
Ansorge MS, Morelli E, Gingrich JA. Inhibition of serotonin but not norepinephrine transport during
development produces delayed, persistent perturbations of emotional behaviors in mice. J Neurosci.
2008; 28:199–207. [PubMed: 18171937]
Ansorge MS, Zhou M, Lira A, Hen R, Gingrich JA. Early-life blockade of the 5-HT transporter alters
emotional behavior in adult mice. Science. 2004; 306:879–881. [PubMed: 15514160]
Ase AR, Reader TA, Hen R, Riad M, Descarries L. Regional changes in density of serotonin
transporter in the brain of 5-HT1A and 5-HT1B knockout mice, and of serotonin innervation in the
5-HT1B knockout. J Neurochem. 2001; 78:619–630. [PubMed: 11483665]
Assie MB, Koek W. Possible in vivo 5-HT reuptake blocking properties of 8-OH-DPAT assessed by
measuring hippocampal extracellular 5-HT using microdialysis in rats. Br J Pharmacol. 1996;
119:845–850. [PubMed: 8922730]
Audero E, Coppi E, Mlinar B, Rossetti T, Caprioli A, Banchaabouchi MA, Corradetti R, Gross C.
Sporadic autonomic dysregulation and death associated with excessive serotonin autoinhibition.
Science. 2008; 321:130–133. [PubMed: 18599790]
Azmitia EC, Segal M. An autoradiographic analysis of the differential ascending projections of the
dorsal and median raphe nuclei in the rat. J Comp Neurol. 1978; 179:641–667. [PubMed: 565370]
Barnes NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology.
1999; 38:1083–1152. [PubMed: 10462127]
Beck SG, Choi KC, List TJ. Comparison of 5-hydroxytryptamine 1A-mediated hyperpolarization in
CA1 and CA3 hippocampal pyramidal cells. J Pharmacol Exp Ther. 1992; 263:350–359.
[PubMed: 1403796]
Beck SG, Pan YZ, Akanwa AC, Kirby LG. Median and dorsal raphe neurons are not
electrophysiologically identical. J Neurophysiol. 2004; 91:994–1005. [PubMed: 14573555]
Beique JC, Campbell B, Perring P, Hamblin MW, Walker P, Mladenovic L, Andrade R. Serotonergic
regulation of membrane potential in developing rat prefrontal cortex: coordinated expression of 5-
hydroxytryptamine (5-HT)1A, 5-HT2A, and 5-HT7 receptors. J Neurosci. 2004; 24:4807–4817.
[PubMed: 15152041]
Bert B, Fink H, Hortnagl H, Veh RW, Davies B, Theuring F, Kusserow H. Mice over-expressing the
5-HT(1A) receptor in cortex and dentate gyrus display exaggerated locomotor and hypothermic
response to 8-OH-DPAT. Behav Brain Res. 2006; 167:328–341. [PubMed: 16256213]
Richardson-Jones et al. Page 13
J Neurosci. Author manuscript; available in PMC 2011 October 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Boutrel B, Monaca C, Hen R, Hamon M, Adrien J. Involvement of 5-HT1A receptors in homeostatic
and stress-induced adaptive regulations of paradoxical sleep: studies in 5-HT1A knock-out mice. J
Neurosci. 2002; 22:4686–4692. [PubMed: 12040075]
Casanovas JM, Hervas I, Artigas F. Postsynaptic 5-HT1A receptors control 5-HT release in the rat
medial prefrontal cortex. Neuroreport. 1999; 10:1441–1445. [PubMed: 10380960]
Caspi A, Sugden K, Moffitt TE, Taylor A, Craig IW, Harrington H, McClay J, Mill J, Martin J,
Braithwaite A, Poulton R. Influence of life stress on depression: moderation by a polymorphism in
the 5-HTT gene. Science. 2003; 301:386–389. [PubMed: 12869766]
Ceci A, Baschirotto A, Borsini F. The inhibitory effect of 8-OH-DPAT on the firing activity of dorsal
raphe serotoninergic neurons in rats is attenuated by lesion of the frontal cortex.
Neuropharmacology. 1994; 33:709–713. [PubMed: 7936107]
Crawford LK, Craige CP, Beck SG. Increased intrinsic excitability of lateral wing serotonin neurons of
the dorsal raphe: a mechanism for selective activation in stress circuits. J Neurophysiol. 2010;
103:2652–2663. [PubMed: 20237311]
David DJ, Klemenhagen KC, Holick KA, Saxe MD, Mendez I, Santarelli L, Craig DA, Zhong H,
Swanson CJ, Hegde LG, Ping XI, Dong D, Marzabadi MR, Gerald CP, Hen R. Efficacy of the
MCHR1 antagonist N-[3-(1-{[4-(3,4-difluorophenoxy)phenyl]methyl}(4-piperidyl))-4-methylphen
yl]-2-methylpropanamide (SNAP 94847) in mouse models of anxiety and depression following
acute and chronic administration is independent of hippocampal neurogenesis. J Pharmacol Exp
Ther. 2007; 321:237–248. [PubMed: 17237257]
Davidson JR. First-line pharmacotherapy approaches for generalized anxiety disorder. J Clin
Psychiatry. 2009; 70(Suppl 2):25–31. [PubMed: 19371504]
Fakra E, Hyde LW, Gorka A, Fisher PM, Munoz KE, Kimak M, Halder I, Ferrell RE, Manuck SB,
Hariri AR. Effects of HTR1A C(−1019)G on amygdala reactivity and trait anxiety. Arch Gen
Psychiatry. 2009; 66:33–40. [PubMed: 19124686]
Franklin, KBJ.; Paxinos, G. The mouse brain in stereotaxic coordinates. San Diego: Academic Press;
1997.
Gartside SE, Umbers V, Hajos M, Sharp T. 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. 1995; 115:1064–1070. [PubMed: 7582504]
Goodfellow NM, Benekareddy M, Vaidya VA, Lambe EK. Layer II/III of the prefrontal cortex:
Inhibition by the serotonin 5-HT1A receptor in development and stress. J Neurosci. 2009;
29:10094–10103. [PubMed: 19675243]
Gross C, Hen R. The developmental origins of anxiety. Nat Rev Neurosci. 2004; 5:545–552. [PubMed:
15208696]
Gross C, Zhuang X, Stark K, Ramboz S, Oosting R, Kirby L, Santarelli L, Beck S, Hen R.
Serotonin1A receptor acts during development to establish normal anxiety-like behaviour in the
adult. Nature. 2002; 416:396–400. [PubMed: 11919622]
Guiard BP, David DJ, Deltheil T, Chenu F, Le Maitre E, Renoir T, Leroux-Nicollet I, Sokoloff P,
Lanfumey L, Hamon M, Andrews AM, Hen R, Gardier AM. Brain-derived neurotrophic factor-
deficient mice exhibit a hippocampal hyperserotonergic phenotype. Int J Neuropsychopharmacol.
2008; 11:79–92. [PubMed: 17559709]
Haddjeri N, Lavoie N, Blier P. Electrophysiological evidence for the tonic activation of 5-HT(1A)
autoreceptors in the rat dorsal raphe nucleus. Neuropsychopharmacology. 2004; 29:1800–1806.
[PubMed: 15127086]
Hajos M, Hajos-Korcsok E, Sharp T. Role of the medial prefrontal cortex in 5-HT1A receptor-induced
inhibition of 5-HT neuronal activity in the rat. Br J Pharmacol. 1999; 126:1741–1750. [PubMed:
10372816]
Hamon M, Gozlan H, el Mestikawy S, Emerit MB, Bolanos F, Schechter L. The central 5-HT1A
receptors: pharmacological, biochemical, functional, and regulatory properties. Ann N Y Acad Sci.
1990; 600:114–129. discussion 129–131. [PubMed: 2252305]
He M, Sibille E, Benjamin D, Toth M, Shippenberg T. Differential effects of 5-HT1A receptor
deletion upon basal and fluoxetine-evoked 5-HT concentrations as revealed by in vivo
microdialysis. Brain Res. 2001; 902:11–17. [PubMed: 11376590]
Richardson-Jones et al. Page 14
J Neurosci. Author manuscript; available in PMC 2011 October 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Heisler LK, Chu HM, Brennan TJ, Danao JA, Bajwa P, Parsons LH, Tecott LH. Elevated anxiety and
antidepressant-like responses in serotonin 5-HT1A receptor mutant mice. Proc Natl Acad Sci U S
A. 1998; 95:15049–15054. [PubMed: 9844013]
Imai H, Steindler DA, Kitai ST. The organization of divergent axonal projections from the midbrain
raphe nuclei in the rat. J Comp Neurol. 1986; 243:363–380. [PubMed: 2419370]
Jennings KA, Loder MK, Sheward WJ, Pei Q, Deacon RM, Benson MA, Olverman HJ, Hastie ND,
Harmar AJ, Shen S, Sharp T. Increased expression of the 5-HT transporter confers a low-anxiety
phenotype linked to decreased 5-HT transmission. J Neurosci. 2006; 26:8955–8964. [PubMed:
16943551]
Kirby LG, Pernar L, Valentino RJ, Beck SG. Distinguishing characteristics of serotonin and non-
serotonin-containing cells in the dorsal raphe nucleus: electrophysiological and
immunohistochemical studies. Neuroscience. 2003; 116:669–683. [PubMed: 12573710]
Kirby LG, Freeman-Daniels E, Lemos JC, Nunan JD, Lamy C, Akanwa A, Beck SG. Corticotropin-
releasing factor increases GABA synaptic activity and induces inward current in 5-
hydroxytryptamine dorsal raphe neurons. J Neurosci. 2008; 28:12927–12937. [PubMed:
19036986]
Kusserow H, Davies B, Hortnagl H, Voigt I, Stroh T, Bert B, Deng DR, Fink H, Veh RW, Theuring F.
Reduced anxiety-related behaviour in transgenic mice overexpressing serotonin 1A receptors.
Brain Res Mol Brain Res. 2004; 129:104–116. [PubMed: 15469887]
Le Francois B, Czesak M, Steubl D, Albert PR. Transcriptional regulation at a HTR1A polymorphism
associated with mental illness. Neuropharmacology. 2008
Lemonde S, Turecki G, Bakish D, Du L, Hrdina PD, Bown CD, Sequeira A, Kushwaha N, Morris SJ,
Basak A, Ou XM, Albert PR. Impaired repression at a 5-hydroxytryptamine 1A receptor gene
polymorphism associated with major depression and suicide. J Neurosci. 2003; 23:8788–8799.
[PubMed: 14507979]
Lemos JC, Pan YZ, Ma X, Lamy C, Akanwa AC, Beck SG. Selective 5-HT receptor inhibition of
glutamatergic and GABAergic synaptic activity in the rat dorsal and median raphe. Eur J Neurosci.
2006; 24:3415–3430. [PubMed: 17229091]
Lira A, Zhou M, Castanon N, Ansorge MS, Gordon JA, Francis JH, Bradley-Moore M, Lira J,
Underwood MD, Arango V, Kung HF, Hofer MA, Hen R, Gingrich JA. Altered depression-related
behaviors and functional changes in the dorsal raphe nucleus of serotonin transporter-deficient
mice. Biol Psychiatry. 2003; 54:960–971. [PubMed: 14625138]
Lo Iacono L, Gross C. Alpha-Ca2+/calmodulin-dependent protein kinase II contributes to the
developmental programming of anxiety in serotonin receptor 1A knock-out mice. J Neurosci.
2008; 28:6250–6257. [PubMed: 18550767]
Malagie I, Trillat AC, Jacquot C, Gardier AM. Effects of acute fluoxetine on extracellular serotonin
levels in the raphe: an in vivo microdialysis study. Eur J Pharmacol. 1995; 286:213–217.
[PubMed: 8605960]
Mallo M, Kanzler B, Ohnemus S. Reversible gene inactivation in the mouse. Genomics. 2003;
81:356–360. [PubMed: 12676559]
Martin KF, Phillips I, Hearson M, Prow MR, Heal DJ. Characterization of 8-OH-DPAT-induced
hypothermia in mice as a 5-HT1A autoreceptor response and its evaluation as a model to
selectively identify antidepressants. Br J Pharmacol. 1992; 107:15–21. [PubMed: 1422568]
Mayford M, Bach ME, Huang YY, Wang L, Hawkins RD, Kandel ER. Control of memory formation
through regulated expression of a CaMKII transgene. Science. 1996; 274:1678–1683. [PubMed:
8939850]
McQuade R, Sharp T. Functional mapping of dorsal and median raphe 5-hydroxytryptamine pathways
in forebrain of the rat using microdialysis. J Neurochem. 1997; 69:791–796. [PubMed: 9231740]
Nemeroff CB. Comorbidity of mood and anxiety disorders: the rule, not the exception? Am J
Psychiatry. 2002; 159:3–4. [PubMed: 11772680]
Oberlander TF, Gingrich JA, Ansorge MS. Sustained neurobehavioral effects of exposure to SSRI
antidepressants during development: molecular to clinical evidence. Clin Pharmacol Ther. 2009;
86:672–677. [PubMed: 19890255]
Richardson-Jones et al. Page 15
J Neurosci. Author manuscript; available in PMC 2011 October 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Olivier JD, Van Der Hart MG, Van Swelm RP, Dederen PJ, Homberg JR, Cremers T, Deen PM,
Cuppen E, Cools AR, Ellenbroek BA. A study in male and female 5-HT transporter knockout rats:
an animal model for anxiety and depression disorders. Neuroscience. 2008; 152:573–584.
[PubMed: 18295409]
Parks CL, Robinson PS, Sibille E, Shenk T, Toth M. Increased anxiety of mice lacking the
serotonin1A receptor. Proc Natl Acad Sci U S A. 1998; 95:10734–10739. [PubMed: 9724773]
Parsons LH, Kerr TM, Tecott LH. 5-HT(1A) receptor mutant mice exhibit enhanced tonic, stress-
induced and fluoxetine-induced serotonergic neurotransmission. J Neurochem. 2001; 77:607–617.
[PubMed: 11299323]
Pittenger C, Huang YY, Paletzki RF, Bourtchouladze R, Scanlin H, Vronskaya S, Kandel ER.
Reversible inhibition of CREB/ATF transcription factors in region CA1 of the dorsal hippocampus
disrupts hippocampus-dependent spatial memory. Neuron. 2002; 34:447–462. [PubMed:
11988175]
Ramboz S, Oosting R, Amara DA, Kung HF, Blier P, Mendelsohn M, Mann JJ, Brunner D, Hen R.
Serotonin receptor 1A knockout: an animal model of anxiety-related disorder. Proc Natl Acad Sci
U S A. 1998; 95:14476–14481. [PubMed: 9826725]
Ressler KJ, Nemeroff CB. Role of serotonergic and noradrenergic systems in the pathophysiology of
depression and anxiety disorders. Depress Anxiety. 2000; 12(Suppl 1):2–19. [PubMed: 11098410]
Riad M, Garcia S, Watkins KC, Jodoin N, Doucet E, Langlois X, el Mestikawy S, Hamon M,
Descarries L. Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-
HT1B serotonin receptors in adult rat brain. J Comp Neurol. 2000; 417:181–194. [PubMed:
10660896]
Richardson-Jones JW, Craige CP, Guiard BP, Stephen A, Metzger KL, Kung HF, Gardier AM,
Dranovsky A, David DJ, Beck SG, Hen R, Leonardo ED. 5-HT1A autoreceptor levels determine
vulnerability to stress and response to antidepressants. Neuron. 2010; 65:40–52. [PubMed:
20152112]
Romero L, Artigas F. Preferential potentiation of the effects of serotonin uptake inhibitors by 5-HT1A
receptor antagonists in the dorsal raphe pathway: role of somatodendritic autoreceptors. J
Neurochem. 1997; 68:2593–2603. [PubMed: 9166757]
Rush AJ, Trivedi MH, Wisniewski SR, Nierenberg AA, Stewart JW, Warden D, Niederehe G, Thase
ME, Lavori PW, Lebowitz BD, McGrath PJ, Rosenbaum JF, Sackeim HA, Kupfer DJ, Luther J,
Fava M. Acute and longer-term outcomes in depressed outpatients requiring one or several
treatment steps: a STAR*D report. Am J Psychiatry. 2006; 163:1905–1917. [PubMed: 17074942]
Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R,
Arancio O, Belzung C, Hen R. Requirement of hippocampal neurogenesis for the behavioral
effects of antidepressants. Science. 2003; 301:805–809. [PubMed: 12907793]
Savelieva KV, Zhao S, Pogorelov VM, Rajan I, Yang Q, Cullinan E, Lanthorn TH. Genetic disruption
of both tryptophan hydroxylase genes dramatically reduces serotonin and affects behavior in
models sensitive to antidepressants. PLoS One. 2008; 3:e3301. [PubMed: 18923670]
Tanaka KF, Ahmari SE, Leonardo ED, Richardson-Jones JW, Budreck EC, Scheiffele P, Sugio S,
Inamura N, Ikenaka K, Hen R. Flexible Accelerated STOP Tetracycline Operator-Knockin
(FAST): A Versatile and Efficient New Gene Modulating System. Biol Psychiatry. 2010
Tsetsenis T, Ma XH, Lo Iacono L, Beck SG, Gross C. Suppression of conditioning to ambiguous cues
by pharmacogenetic inhibition of the dentate gyrus. Nat Neurosci. 2007; 10:896–902. [PubMed:
17558402]
Wellman CL, Izquierdo A, Garrett JE, Martin KP, Carroll J, Millstein R, Lesch KP, Murphy DL,
Holmes A. Impaired stress-coping and fear extinction and abnormal corticolimbic morphology in
serotonin transporter knock-out mice. J Neurosci. 2007; 27:684–691. [PubMed: 17234600]
Zhang J, Huang XY, Ye ML, Luo CX, Wu HY, Hu Y, Zhou QG, Wu DL, Zhu LJ, Zhu DY. Neuronal
nitric oxide synthase alteration accounts for the role of 5-HT1A receptor in modulating anxiety-
related behaviors. J Neurosci. 2010; 30:2433–2441. [PubMed: 20164327]
Richardson-Jones et al. Page 16
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Figure 1.
(A) Adult mice homozygous for the regulatable Htr1atetO allele and possessing one copy of
a tTS transgene express 5-HT1A receptors in normal patterns in the brain when maintained
on doxycycline, assessed by 125I-labelled MPPI autoradiography and shown in coronal
sections at the level of the hippocampus and raphe nucleus. A schematic of normal 5-HT1A
receptor binding in the sagittal plane is shown for reference. (B) Adult mice homozygous for
the regulatable Htr1atetO allele and possessing one copy of the α-CamKII-tTS transgene
express 5-HT1A raphe autoreceptors normally, but show almost no binding of forebrain
receptors when maintained in the absence of doxycycline. (C) Adult mice homozygous for
the regulatable Htr1atetO allele and possessing one copy of the Pet1-tTS transgene express 5-
HT1A forebrain heteroreceptors normally, but show almost no binding of raphe
autoreceptors when maintained in the absence of doxycycline. (D) Mice lacking 5-HT1A
autoreceptors (Auto-KO) and mice with autoreceptors in tact (Control) were generated by
maintaining Htr1atetO/tetO Pet1-tTS+ mice in the absence or presence of doxycycline
throughout life. (E) Mice lacking 5-HT1A heteroreceptors (Hetero-KO) and mice with
heteroreceptors intact (Control) were generated by maintaining Htr1atetO/tetOα CamKII-tTS+
mice in the absence or presence of doxycycline throughout life. Mice lacking
heteroreceptors beginning in adulthood (Adult-Hetero-KO) were maintained on doxycycline
until 7 weeks of age.
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Figure 2.
(A) Quantitative 125I-labelled MPPI autoradiography for 5-HT1A receptors revealed a
significant decrease in the dorsal raphe (i) of Auto-KO mice compared to their matched
Controls. Conversely, no difference was detected in forebrain regions (ii), such as the
ventral dentate gyrus of the hippocampus (HPC), the amygdala (AMG), and the entorhinal
cortex (EC). (B) (i)No difference was detected in binding in the dorsal raphe of Hetero-KO
or Adult-Hetero-KO mice compared to their matched controls. Conversely, binding was
significantly decreased in dorsal CA1 of the hippocampus (HPC) in both groups compared
to control (ii) (N=3–7/group). All values are mean ± SEM. ***p<0.0001. Matched
autoradiograms showing detailed expression patterns of 5-HT1A receptors binding across the
rostrocaudal extent of the brain in coronal section comparing (C) Auto-KO mice and their
Controls; and (D) Hetero-KO and Adult-Hetero-KO mice and their Controls. Low levels of
heteroreceptors remain in parts of the septum, olivary nucleus, and entorhinal cortex. (E)
Receptor binding in the postnatal developmental period in Hetero-KO mice and their
Controls. Matched sections are shown at the level of the dorsal hippocampus and the dorsal
raphe nucleus. Forebrain receptor suppression is complete by P14.
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Figure 3.
(A) Schematic depicting level of sections taken for slice recordings in the dorsal raphe of
Auto-KO mice. (B) Representative current traces from whole cell recordings in the dorsal
raphe of Auto-KO mice and their Controls in response to 5-CT. (C) Mean outward current
amplitude in response to 100 nm 5-CT was decreased in Auto-KO mice. Values are mean
±SEM. (N = 12–24/group). (D) Schematic depicting level of sections taken for slice
recordings in CA1 of the hippocampus in Hetero-KO mice. (E) Representative current traces
from whole cell recordings in CA1 hippocampal pyramidal cells of Hetero-KO mice and
their Controls in response to the 5-HT1A/7 agonist 5-CT. (F) Mean outward current
amplitude in response to 100 nm 5-CT was decreased in Hetero-KO mice. Values are mean
±SEM. (N = 19–22/group). **p =0.01, ***p<0.0001
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Figure 4.
(A) Hypothermic response to the 5-HT1A/7 agonist 8-OH DPAT is normal in Hetero-KO
mice (i) and their Controls (ii). (B) Hypothermic response to the 5-HT1A agonist 8-OH
DPAT is normal in Adult-Hetero-KO mice (i) and their Controls (ii). Hypothermic response
to the 5-HT1A agonist 8-OH DPAT is abolished in Auto-KO mice (i) and normal in their
Controls (ii), (n=3–6 mice/group/dose). Values are mean ±SEM; ***p<0.001.
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Figure 5.
(A) Extracellular serotonin levels measured by in vivo microdialysis in the vHPC (A) and
PFC (B) following injection of saline (t0) and 18mg/kg fluoxetine (t105). Values are mean ±
SEM for each time point. Total extracellular serotonin, measured by area under the curve
analysis compared to baseline, increases in the vHPC (C) and PFC (D) of both Auto-KO and
Control mice in response to acute fluoxetine treatment. Auto-KO mice display a larger
percentage increase than Controls in response to fluoxetine in the vHPC but not PFC. Values
are ± SEM (n=7–9 mice/group). *p<0.05, ***p<0.001.
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Figure 6.
(A) No group differences were detected between Hetero-KO mice and their Controls in the
total exploration (i) or percentage exploration of the center (ii) of the open field, (n=32). (B)
No group differences were detected between Hetero-KO mice and their Controls in total
exploration (i), percentage exploration of the light, (ii), or entries into the light (iii) in the
light/dark choice test, (n=41). (C) No group differences were detected between Adult-
Hetero-KO mice and their Controls in the total exploration (i) or percentage exploration of
the center (ii) of the open field, (n=47). (D) No group differences were detected between
Adult-Hetero-KO mice and their Controls in total exploration (i), percentage exploration of
the light, (ii), or entries into the light (iii) in the light/dark choice test, (n=30). (E) Auto-KO
mice displayed decreased total exploration (i) and decreased percentage exploration of the
center (ii) of the open field, compared to their Controls, (n=50). (F) Auto-KO mice
displayed less total exploration (i) and fewer entries into the light compartment (iii), but no
difference in percentage exploration of the light (ii) compared to their Controls in the light/
dark choice test, (n=37). All values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.
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Figure 7.
Hetero-KO mice displayed decreased mobility compared to the their Controls (A) across a
two day forced swim test, (N=48). No group differences were detected between Adult-
Hetero-KO (B) or Auto-KO mice (C) and their Controls across a two day forced swim test,
(N=34 and 44, respectively). All values are mean ± SEM. *p<0.05, **p<0.01.
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Figure 8.
Schematic depicting predicted behavioral roles of endogenous 5-HT1A auto- and
heteroreceptors in the juvenile and adult. Raphe autoreceptors are coded blue and forebrain
heteroreceptors in regions including the hippocampus (Hpc), amygdala (Amg) and cortex
(Ctx) are coded yellow. Endogenous autoreceptors, which limit levels of serotonin released
by raphe neurons, affect anxiety related circuitry in development or circuitry mediating
forced swim test behavior in the adult. Endogenous heteroreceptors, which exert inhibitory
control over certain forebrain neurons, affect forced swim test behavior but not anxiety in
the juvenile.
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Richardson-Jones et al. Page 25
Table 1
Mean basal serotonin levels (fmol/20μl dialysate) ± SEM in ventral hippocampus (HPC) and prefrontal cortex
(PFC).
5-HT (FMOL/20UL)
HPC PFC
CONTROL 2.8 ± 0.3 1.9 ± 0.1
AUTO-KO 3.7 ± 0.5 3.7 ± 0.7*
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