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OCD-like behavior is caused by dysfunction of thalamo-amygdala circuits and upregulated TrkB/ERK-MAPK signaling as a result of SPRED2 deficiency

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Obsessive-compulsive disorder (OCD) is a common neuropsychiatric disease affecting about 2% of the general population. It is characterized by persistent intrusive thoughts and repetitive ritualized behaviors. While gene variations, malfunction of cortico-striato-thalamo-cortical (CSTC) circuits, and dysregulated synaptic transmission have been implicated in the pathogenesis of OCD, the underlying mechanisms remain largely unknown. Here we show that OCD-like behavior in mice is caused by deficiency of SPRED2, a protein expressed in various brain regions and a potent inhibitor of Ras/ERK-MAPK signaling. Excessive self-grooming, reflecting OCD-like behavior in rodents, resulted in facial skin lesions in SPRED2 knockout (KO) mice. This was alleviated by treatment with the selective serotonin reuptake inhibitor fluoxetine. In addition to the previously suggested involvement of cortico-striatal circuits, electrophysiological measurements revealed altered transmission at thalamo-amygdala synapses and morphological differences in lateral amygdala neurons of SPRED2 KO mice. Changes in synaptic function were accompanied by dysregulated expression of various pre- and postsynaptic proteins in the amygdala. This was a result of altered gene transcription and triggered upstream by upregulated tropomyosin receptor kinase B (TrkB)/ERK-MAPK signaling in the amygdala of SPRED2 KO mice. Pathway overactivation was mediated by increased activity of TrkB, Ras, and ERK as a specific result of SPRED2 deficiency and not elicited by elevated brain-derived neurotrophic factor levels. Using the MEK inhibitor selumetinib, we suppressed TrkB/ERK-MAPK pathway activity in vivo and reduced OCD-like grooming in SPRED2 KO mice. Altogether, this study identifies SPRED2 as a promising new regulator, TrkB/ERK-MAPK signaling as a novel mediating mechanism, and thalamo-amygdala synapses as critical circuitry involved in the pathogenesis of OCD.
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OPEN
ORIGINAL ARTICLE
OCD-like behavior is caused by dysfunction of
thalamo-amygdala circuits and upregulated
TrkB/ERK-MAPK signaling as a result of SPRED2 deciency
M Ullrich
1
, M Weber
2
, AM Post
3,4
, S Popp
4
, J Grein
1
, M Zechner
1
, H Guerrero González
1
, A Kreis
5
, AG Schmitt
5
, N Üçeyler
6
,
K-P Lesch
4,7
and K Schuh
1
Obsessive-compulsive disorder (OCD) is a common neuropsychiatric disease affecting about 2% of the general population. It is
characterized by persistent intrusive thoughts and repetitive ritualized behaviors. While gene variations, malfunction of cortico-
striato-thalamo-cortical (CSTC) circuits, and dysregulated synaptic transmission have been implicated in the pathogenesis of OCD,
the underlying mechanisms remain largely unknown. Here we show that OCD-like behavior in mice is caused by deciency of
SPRED2, a protein expressed in various brain regions and a potent inhibitor of Ras/ERK-MAPK signaling. Excessive self-grooming,
reecting OCD-like behavior in rodents, resulted in facial skin lesions in SPRED2 knockout (KO) mice. This was alleviated by
treatment with the selective serotonin reuptake inhibitor uoxetine. In addition to the previously suggested involvement of cortico-
striatal circuits, electrophysiological measurements revealed altered transmission at thalamo-amygdala synapses and
morphological differences in lateral amygdala neurons of SPRED2 KO mice. Changes in synaptic function were accompanied by
dysregulated expression of various pre- and postsynaptic proteins in the amygdala. This was a result of altered gene transcription
and triggered upstream by upregulated tropomyosin receptor kinase B (TrkB)/ERK-MAPK signaling in the amygdala of SPRED2 KO
mice. Pathway overactivation was mediated by increased activity of TrkB, Ras, and ERK as a specic result of SPRED2 deciency and
not elicited by elevated brain-derived neurotrophic factor levels. Using the MEK inhibitor selumetinib, we suppressed TrkB/ERK-
MAPK pathway activity in vivo and reduced OCD-like grooming in SPRED2 KO mice. Altogether, this study identies SPRED2 as a
promising new regulator, TrkB/ERK-MAPK signaling as a novel mediating mechanism, and thalamo-amygdala synapses as critical
circuitry involved in the pathogenesis of OCD.
Molecular Psychiatry advance online publication, 10 January 2017; doi:10.1038/mp.2016.232
INTRODUCTION
Obsessive-compulsive disorder (OCD) is a neuropsychiatric condi-
tion characterized by persistent intrusive thoughts (obsessions)
and repetitive ritualized actions (compulsions). Factor analytic
studies have identied four primary subtypes of OCD: contamina-
tion obsessions with cleaning compulsions, symmetry obsessions
with ordering compulsions, hoarding obsessions with collecting
compulsions, and aggressive/sexual/religious/somatic obsessions
with checking compulsions.
1
However, OCDs vary greatly in
the types of obsessions and compulsions, reecting both
heterogeneity in clinical phenotypes and the underlying
pathophysiology.
2,3
Furthermore, there are various OCD-related
disorders, for example trichotillomania and excoriation disorder,
tic disorders like Tourettes syndrome, and autism spectrum
disorders that share considerable overlapping features with OCD.
4
As with many neuropsychiatric disorders, the neurobiological
basis of OCD still remains obscure. A large body of functional
neuroimaging studies has related OCD symptoms to alterations in
the activity of cortico-striato-thalamo-cortical (CSTC) circuits.
5,6
Especially hyperactivity in orbitofrontal cortex and ventromedial
striatum seems to be crucial in the pathogenesis of OCD.
7
The
amygdala is the integrative center for emotions and emotional
behavior and its role in mediating fear and anxiety is the most
commonly referenced to date.
8,9
However, a possible impact of
the amygdala on the development of OCDs is indicated and
extensively discussed but needs additional investigation.
10
Although family and twin studies support a signicant genetic
contribution to OCD and related conditions, no particular gene
has reached the stringent level of statistical evidence to be
considered a denitive risk gene.
4
Since selective serotonin
reuptake inhibitors (SSRIs), such as uoxetine, are the rst-line
pharmacological treatment for OCDs, one of the strongest
candidates for the cause of OCD is the gene encoding the
serotonin transporter.
11
The same meta-analysis also implicated
glutamatergic and dopaminergic neurotransmitter systems as well
as brain-derived neurotrophic factor (BDNF) and tropomyosin
receptor kinase B/neurotrophic tyrosine kinase receptor type 2
(TrkB/NTRK2) as possible genetic factors.
11
The latter two are part
of a cerebral signaling pathway, which is essential for the
regulation of neuronal gene transcription, neurogenesis, and
1
Institute of Physiology I, University of Wuerzburg, Wuerzburg, Germany;
2
Institute of Physiology II, University of Frankfurt, Frankfurt am Main, Germany;
3
Department of
Psychiatry, Psychosomatic Medicine and Psychotherapy, University of Frankfurt, Frankfurt am Main, Germany;
4
Division of Molecular Psychiatry, Clinical Research Unit on
Disorders of Neurodevelopment and Cognition, Laboratory of Translational Neuroscience, Center of Mental Health, University of Wuerzburg, Wuerzburg, Germany;
5
Department
of Psychiatry, Psychosomatics and Psychotherapy, Center of Mental Health, University of Wuerzburg, Wuerzburg, Germany;
6
Department of Neurology, University of Wuerzburg,
Wuerzburg, Germany and
7
Department of Translational Neuroscience, School for Mental Health and Neuroscience (MHeNS), Maastricht University, Maastricht, The Netherlands.
Correspondence: Professor K Schuh, University of Wuerzburg, Institute of Physiology I, University of Wuerzburg, Roentgenring 9, 97070 Wuerzburg, Germany.
E-mail: kai.schuh@uni-wuerzburg.de
Received 9 June 2016; revised 20 October 2016; accepted 1 November 2016
Molecular Psychiatry (2017) 00, 115
www.nature.com/mp
neuronal differentiation. In the adult nervous system, BDNF/TrkB
signaling regulates synaptic strength, transmission, and
plasticity.
12,13
Because BDNF plays a critical role in brain
development and plasticity, it is widely implicated in psychiatric
diseases, including major depressive, bipolar, anxiety-related, and
neurodevelopmental disorders but also in neurodegenerative
diseases.
14,15
Although several studies demonstrated the involve-
ment of BNDF in OCD as well, the outcomes were inconclusive in a
way that it is still unclear whether BDNF sequence variants like the
common Val66Met substitution are protective or predictive for
OCD.
16,17
Alterations in BDNF plasma levels are indicative of
various psychiatric disorders
14
and may also be associated with
OCD.
18
Genetic variations of the NTRK2 gene encoding TrkB were
suggested to contribute to OCD in humans, however, the
pathomechanism is unknown.
19
The impact of BDNF and its
receptor TrkB on anxiety-related disorders has been investigated
in mouse models but this also revealed contradictory results.
20
The neurotrophin BDNF is the most prevalent growth factor in
the central nervous system and the preferred ligand of TrkB, a
transmembrane receptor tyrosine kinase that is phosphorylated at
several tyrosine residues.
21
After BDNF-mediated activation of
TrkB, signals are mediated by different intracellular cascades,
of which the Ras/ERK-MAPK pathway is one of the most
prominent.
12,13
Ras is activated after receptor phosphorylation
via different adapter molecules. From Ras, signals can be
propagated by phosphorylation and activation of sequential
kinases including Raf, MEK1/2, and ERK1/2. ERK activation results
in phosphorylation and activation of transcription factors and
other regulatory target proteins.
22
Important intrinsic regulators of
Ras/ERK-MAPK pathway activity are the SPRED proteins (Sprouty-
related, EVH1 domain-containing protein 2), a family of the three
homologs SPRED1, 2 and 3.
23
SPREDs very potently and
exclusively inhibit the Ras/ERK-MAPK cascade downstream of
multiple receptor tyrosine kinases and in response to a wide range
of mitogenic stimuli, for example, growth factors, cytokines, and
chemokines.
2325
The expression pattern of SPRED proteins,
especially that of SPRED2 is widespread in humans and mice
but most pronounced in the central nervous system.
26,27
SPREDs
are also functionally required in neuronal development by
regulating neurogenesis through control of ERK-dependent neural
progenitor cell proliferation and maintenance of germinal zone
integrity.
28
Because of the co-localization of SPREDs with various
endosome markers, a role in synaptic vesicle transport is also
assumed.
2729
SPRED1 deciency in humans causes Legius
syndrome, a disease of the rasopathy spectrum, which is
associated with learning disabilities, developmental delays, and
macrocephaly.
30
Similarly, SPRED1 KO mice also show disabilities
in hippocampus-dependent learning
31
and facial abnormalities.
32
The loss of functional SPRED2 in mice leads to hyperactivity of
the hypothalamic-pituitary-adrenal (HPA) axis, demonstrated by
increased release of stress hormones including corticotropin-
releasing hormone, adrenocorticotropic hormone, and
corticosterone.
33
Here we show that SPRED2 deciency caused an OCD-like
behavioral phenotype. The observation of OCD-like grooming
resulting in severe self-inicted facial lesions in SPRED2 KO mice
prompted us to investigate anxiety-related behavior and skin
sensitivity, and to treat SPRED2 KO mice with uoxetine. Since our
results supported the assumption of an OCD-like phenotype in
SPRED2 KO mice, we performed electrophysiological recordings at
cortico-striatal and thalamo-amygdala synapses in SPRED2 KOs.
We recorded distinct changes of synaptic excitability at thalamo-
amygdala synapses, which was accompanied by altered neuron
morphology in the lateral amygdala. Hence, we aimed to unravel
the cause of thalamo-amygdala malfunction and to verify its
contribution to OCD. Investigation of SPRED2 expression and of
various pre- and postsynaptic proteins in wildtype (WT) and
SPRED2 KO amygdala identied the molecular basis for
dysregulated activity in thalamo-amygdala circuits. Given the
described inhibitory effect of SPRED2 on Ras/ERK-MAPK signaling,
which is an essential mediator of BDNF/TrkB signals in brain, we
considered TrkB/ERK pathway dysregulation as a cause of synaptic
protein level alterations in the amygdala. We investigated
expression and activity of crucial signaling components like BDNF,
TrkB, Ras and ERK and demonstrated that pathway upregulation is
a specic result of SPRED2 deciency. Our hypothesis that TrkB/
ERK-MAPK pathway overactivation contributes to OCD was
conrmed by articial pathway downregulation using selumetinib,
which restored normal behavior in SPRED2 KO mice. With this
study, we discovered a link between SPRED2 deciency, TrkB/ERK
signaling, thalamo-amygdala malfunction, and psychiatric condi-
tions like OCD.
MATERIALS AND METHODS
SPRED2 KO mice
SPRED2 KO mice were generated by a gene trap approach as described
previously.
34,35
To generate mice with a disrupted, non-functional Spred2
gene, the embryonic stem cell line XB228 (International Gene Trap
Consortium, Davis, CA, USA) was used. It contained the pGT0 gene trap
vector, which functionally disrupted the Spred2 gene (Figure 1b). Mice
were housed in a daily 12/12 h light-dark cycle under controlled room
temperature (21 ± 1 °C) and humidity (55 ± 5%) conditions with tap water
and standard mouse chow ad libitum unless stated otherwise. To minimize
possible inbred effects, mice were raised on a mixed 129/Ola ×C57Bl/6
genetic background. SPRED2 KO mice were obtained by mating SPRED2
heterozygous animals. All mouse experiments were conducted using
SPRED2 KO mice and WT littermates as controls. Unless stated otherwise,
we used SPRED2 KO mice aged 612 months with apparent OCD-like
phenotype of mixed gender. Mice too severely affected by their behavior
and thereby not suitable for testing were excluded from the analyses.
Required sample sizes were calculated based on effect size and error
probabilities using G*Power 3.1.9.2.
36
Numbers of samples (n) used in each
experiment are indicated in gure legends. All experiments were approved
by the local councils for animal care (Regierung von Unterfranken: #98/14;
#03/12; #2-375) and were conducted according to the European law for
animal care and use.
Behavioral analysis
Open eld. The open eld (OF) consisted of a quadratic black opaque
PERSPEX XT box (50 × 50 × 40 cm), which was semipermeable to infrared
light (TSE Systems, Bad Homburg, Germany) and illuminated by infrared
LEDs from below.
37
The area of the OF was divided into a 36 × 36 cm
central zone (100 lx) and the surrounding periphery (50 lx). Mice were
placed in the periphery and their behavior was recorded for 5 min using
the VideoMot2 system (TSE Systems). Variables measured included time
spent, distance traveled, and visits in each zone, total time spent moving,
total distance traveled, vertical rears, number of grooming bouts, and
defecation/urination.
Elevated plus maze. An elevated plus maze (EPM) made from black
PERSPEX (TSE Systems) and semipermeable for infrared light was used and
illuminated by infrared LEDs from below.
37
The apparatus was elevated to
a height of 60 cm above oor level and comprised a central platform
(5 × 5 cm, 15 lx) extending to two opposing open arms (30 × 5 × 0.25 cm,
30 lx) and two opposing closed arms (30 × 5 × 15 cm, 5 lx). Mice were
placed in the center facing an open arm, and their behavior was recorded
for 5 min using the VideoMot2 system (TSE Systems). Behavioral analysis
included time spent, distance traveled, and visits in each zone, total
distance traveled, total time spent moving, vertical rears, number of
grooming bouts, and defecation/urination.
Light/dark box. The light/dark box (LDB) contained a central gate (5 × 5 cm)
separating a transparent, brightly illumina ted 'lit' compartment (40 × 40 × 27 cm,
300 lx) from a small enclosed 'dark' compartment (40 × 20 × 27 cm, 05lx).
38
Mice were placed into the light compartment and their behavior was
recorded for 5 min. Measured behavioral parameters included time spent in
each compartment, latency to cross from lit to dark area, number of
grooming bouts, and defecation/urination.
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
2
Molecular Psychiatry (2017), 1 15
Mechanical sensitivity
The von Frey test based on the up-and-down-method
39
was used to
examine paw withdrawal thresholds to mechanical stimulation. Mice were
placed in plexiglass cages on a wire mesh and the plantar surface of the
hindpaws was touched with a von Frey lament starting at 0.69 g. If
the mouse withdrew its hindpaw upon administration of mild pressure, the
next thinner von Frey lament was used. If the mouse showed no reaction
to this stimulation, the next thicker von Frey lament was applied. Each
hindpaw was tested three times. The 50% withdrawal threshold (that is
force of the von Frey hair to which an animal reacts in 50% of the
administrations) was recorded. Tests were performed by an investigator
blinded for mouse genotype and study objectives.
Thermal sensitivity
Paw withdrawal latencies to heat were determined applying a standard
algesiometer (Ugo Basile, Gemonio, Italy) based on the method of
Hargreaves.
40
Mice were placed on a glass surface and a radiant heat
source was positioned under one hindpaw. The time until paw withdrawal
was recorded automatically. To avoid tissue damage, a time limit for heat
application of 15 s was used. Each hindpaw was tested three times. Tests
were performed by an investigator blinded for mouse genotype and study
objectives.
Grooming behavior
Grooming included face-wiping, full-body grooming, and scratching and
rubbing of head and ears. Grooming bouts lasted at least 3 s; bouts after
pauses longer than 3 s were regarded as new bouts. Number of grooming
bouts were counted during EPM, OF and LDB tests. Mouse behavior was
also determined by video observations before and after experimental
rescue with uoxetine. Mice were placed in a standard cage, which was
covered by a closed chamber containing a webcam (Philips, Amsterdam,
Netherlands) and infrared LEDs for illumination on top. Every mouse was
recorded for 30 min and movies were analyzed for the times mice spent
with grooming, digging, rearing, locomotion and the distance traveled
using VideoMot2 (TSE Systems).
Experimental therapy
Fluoxetine treatment. SPRED2 KO mice and WT littermate controls were
treated with the SSRI uoxetine (Stada, Bad Vilbel, Germany) for 2 weeks.
Mice of each genotype were randomly selected for either the placebo or
uoxetine group. Fluoxetine was administered at a dose of
20 mg kg
1
day
1
within standard mouse diet; the placebo group was
fed with standard mouse diet. Documentation by photos (Canon EOS
1000D digital camera, Tokyo, Japan) and videos (Philips PixelPlus webcam)
was performed before and after 2 weeks of treatment.
Selumetinib treatment
SPRED2 KO mice were treated with the MEK1/2 inhibitor selumetinib
(AZD6244, Selleck chemicals, Houston, TX, USA) for one week. Selumetinib
was administered at a dose of 8 mg kg
1
day
1
within standard mouse
diet. Photo documentations were performed with a Canon EOS 1000D
digital camera before and after one week of treatment.
Electrophysiology
Mice were terminally anesthetized with isourane, decapitated, brains
were rapidly removed and transferred into ice-cold preparation solution
containing (in mM): 210 sucrose, 26 NaHCO
3
, 1.3 MgSO
4
, 1.2 KH
2
PO
4
,
Figure 1. Self-inicted facial lesions result from OCD-like grooming in SPRED2 KO mice. (a) Severe facial skin lesions developed mostly uni-
and sometimes bilaterally in SPRED2 KO mice from the age of 4 months. These lesions were a result of excessive and injurious self-grooming,
appeared on head, neck and snout regions and were characterized by ulcerations and hemorrhage. (b) SPRED2 KO mice were generated by
insertion of a gene trap vector between exons 4 and 5 of Spred2. Gene trapping resulted in a non-functional Spred2 gene and the Spred2
promoter-driven expression of the β-geo reporter gene. (c)In vivo monitoring of Spred2 expression by X-Gal staining of coronal brain sections
of SPRED2 heterozygous mice demonstrated widespread SPRED2 expression in the brain including cortex, hippocampus, amygdala and
striatum, the latter two brain regions associated with the development of OCDs. (d) Exemplary Western blot analyses revealed SPRED2
expression in WT amygdala lysates and the complete absence of SPRED2 protein in amygdala of SPRED2 KO mice. Expression of related
SPRED1 was not compensatory upregulated as conrmed by quantication of relative SPRED1 expression (n=11 for WT and KO). Data are
mean ±s.e.m. KO, knockout; n.s., not signicant; OCD, obsessive-compulsive disorder; WT, wildtype.
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
3
Molecular Psychiatry (2017), 1 15
2 MgCl
2
, 2 KCl, 2 CaCl
2
, 10 glucose, 3 myoinositol, 2 sodium-pyruvate, and
0.4 ascorbic acid, equilibrated with 95% O
2
/5% CO
2
. Coronal slices
(250 μm) were cut with a vibratome (Leica VT1000S, Wetzlar, Germany) in a
submerged chamber lled with ice-cold preparation solution. Slices were
transferred to a holding chamber lled with articial cerebrospinal uid
containing (in mM): 124 NaCl, 26 NaHCO
3
, 2 KCl, 1.2 KH
2
PO
4
, 1.3 MgSO
4
,
2 CaCl
2
, and 10 glucose, equilibrated with 95% O
2
/5% CO
2
. The holding
chamber was heated for 1 h to 34 °C to improve patch success before
slices were kept at room temperature. For recording, slices were
transferred into a superfusion recording chamber mounted on an upright
xed stage microscope (Zeiss, Oberkochen, Germany) with infrared
differential interference optics. Superfusion rate was 2-3 ml articial
cerebrospinal uid per minute. Patch-clamp recordings were made at
room temperature under visual guidance by an infrared sensitive camera
(Kappa CF6, Gleichen, Germany). Patch electrodes were pulled from
borosilicate capillaries (Science Products, Hofheim, Germany) and lled
with a solution containing (in mM): 95 K-gluconate, 20 K
3
-citrate, 10 NaCl, 1
MgCl
2
, 0.5 CaCl
2
, 1 BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-
tetraacetic acid), and 10 HEPES, pH 7.2 (KOH), 270290 mosm/l. Measure-
ments of miniature excitatory postsynaptic currents (mEPSCs) were made
with a high chloride solution containing (in mM): 140 KCl, 5 NaCl, 1 CaCl
2
,
10 EGTA, 2 MgCl
2,
2K
2
-ATP, 0.5 Na-GTP, and 10 HEPES, pH 7.2 (KOH),
270290 mosm/l. Electrodes had a resistance of 24MΩ.
Synaptic input to lateral amygdala and putamen neurons was
investigated by placing a SNEX-200 concentric tungsten electrode (Science
Products, Hofheim, Germany) in the afferent ber tract. The electrode was
connected to a stimulator (ISO-Flex, A.M.P.I., Jerusalem, Israel). As marked
in Figure 3, appropriate brain regions were identied according to the
mouse brain atlas by Paxinos and Franklin.
41
During whole cell recordings
a presynaptic stimulus was applied in voltage-clamp conguration at
70 mV for 150 μs. Amplitudes of 10 responses were always averaged
(1 s repeat interval). The stimulation threshold was the minimal stimulation
amplitude (in mA) eliciting a postsynaptic response. Recordings were
made using an EPC10 amplier (HEKA Elektronik, Lambrecht/Pfalz,
Germany). Data were ltered with a 10 kHz Bessel and 2.9 low-pass
Bessel lter with a sampling rate of 430 kHz. The Pulse and Pulset
software (HEKA Elektronik) was used for data acquisition and analysis.
mEPSC were measured in voltage clamp conguration (70 mV) for
1 min with 200 μM CdCl
2
in articial cerebrospinal uid. For data
analysis we used Mini Analysis 6.0 software (Synaptosoft, Decatur, GA,
USA). Resistance and seal quality were monitored at the beginning and
several times during recordings to assure consistent measurement
conditions.
Neuron morphology
Mouse neurons were stained using a modied Golgi-Cox impregnation
method as described previously.
42
In brief, dissected brains were
impregnated in Golgi solution for 30 days, 150 μm serial coronal slices
were prepared using a sliding microtome and mounted on glass
microscopic slides using Vitro Clud (R. Langenbrinck, Emmendingen,
Germany). Golgi-stained pyramidal neurons in the lateral amygdala were
reconstructed by an experimenter blind to the genotype using the
Neurolucida system (MBF Bioscience, Williston, VT, USA). Only neurons
located in the center of sections, displaying intense staining of dendritic
arborizations and allowing unequivocal identication of dendritic spines
were chosen for reconstructions. Per genotype 2030 neurons were used
to determine dendritic parameters such as length of dendrites, spine
numbers, and spine densities/10 μm in total or of a particular branch order.
Quantitative real-time PCR
Amygdala was punched out from mouse brains, tissues were homogenized
with a Polytron PT 3100 homogenizer (Kinematica, Luzern, Switzerland)
and RNA was extracted using TRIzol reagent according to the manufac-
turers instructions (Invitrogen, Carlsbad, CA, USA). 500 ng of total RNA
were reverse transcribed to complementary DNA (cDNA) using TaqMan
Reverse Transcription Reagents (Applied Biosystems, Waltham, MA, USA) in
a 100 μl PCR reaction additionally containing: 10 × Reaction Buffer (10 μl),
10 mM dNTPs (20 μl), 25 mM MgCl
2
(22 μl), Random Hexameres (5 μl),
RNAse Inhibitor (2 μl) and 50 U/μl Multiscribe Reverse Transcriptase
(6.25 μl). The 96-well GeneAmp PCR System 9700 cycler was used and
the following cycler conditions obtained: 10 min, 38 °C; 60 min, 48 °C;
25 min, 95 °C. Five μl of cDNA entered quantitative real-time PCR
(qRT-PCR) using TaqMan Universal Master Mix and the following target
specic predesigned mouse TaqMan Gene Expression Assays
(Applied Biosystems; Assay-IDs in brackets): PSD 95 (Mm00492193_m1),
mGluR2 (Mm01235831_m1), mGluR5 (Mm00690332_m1), and ERC1
(Mm00453569_m1). 18 s rRNA (Hs99999901_s1) was used as an endogen-
ous control. The 2
ΔΔCq
method was applied for relative quantication of
gene expression as previously described.
43
Mean value of WT samples was
set to 1 and mean value of KO samples was expressed as x-fold of WT.
Preparation of amygdala lysates
Amygdala was dissected from mouse brains on a metal plate cooled with
ice and homogenized in an assay-dependent buffer using a plastic
douncer tting into a 1.5 ml microreaction tube. Protein content of
samples was measured using the Bradford method.
Western blot
Amygdala lysates were prepared by adding 1 ml of 2% SDS in PBS
supplemented with Complete Protease Inhibitor Cocktail (Roche, Basel,
Switzerland) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche) to
50 mg of tissue. Proteins were separated by 515% SDSPAGE under
reducing conditions and electrotransferred to Protran nitrocellulose
membranes using semi-dry blotters. Blots were probed using primary
antibodies against GAPDH (#2118, 1:10,000, Cell Signaling Technology,
Danvers, MA, USA), SPRED1 (1:500,
34
), SPRED2 (1:500,
34
), PSD95 (#P246,
1:2,000, Sigma-Aldrich, St Louis, MO, USA), mGluR5 (#53090, 1:500, Abcam,
Cambridge, UK), mGluR2 (#15672, 1:1,000, Abcam), ERC1 (#50312, 1:500,
Abcam), Bassoon (# 141003, 1:500, Synaptic Systems, Goettingen,
Germany), Rab3A (#107111, 1:1,000, Synaptic Systems), Rab6 (#sc310,
1:200, Santa Cruz Biotechnology, Dallas TX, USA), α-Tubulin (#T6074,
1:5,000, Sigma-Aldrich), β-Tubulin (#T8320, 1:2,000, Sigma-Aldrich), p44/42
MAP kinase (#9102, ERK1/2, 1:2,000, Cell Signaling Technology), phospho-
p44/42 MAP kinase (P-ERK1/2, #9101, 1:1,000, Cell Signaling Technology),
TrkB (#07225, 1:1,000, Merck Millipore, Billerica, MA, USA), phospho-TrkB
Y515 (P-TrkB Y515, #ab109684, 1:150, Abcam) and phospho-TrkB Y817 (P-
TrkB Y817, #bs-3732R, 1:200, Bioss, Woburn, MA, USA) followed by goat
anti-rabbit (#111-035-144, 1:10,000, Jackson Immuno Research, West
Grove, PA, USA) or goat anti-mouse (#115-035-146, 1:5,000, Jackson
Immuno Research) horseradish peroxidase-conjugated secondary antibo-
dies, all diluted in 5% non-fat dry milk in PBS supplemented with 0.05%
Tween20. Signals were developed using CheLuminate-horseradish perox-
idase FemtoDetect reagent (Applichem, Darmstadt, Germany) and
recorded by a FluorChem SP Imager (Alpha Innotech, Biozym, Hessisch
Oldendorf, Germany).
Ras activity assay
A pan-Ras Activation Assay Kit (#STA-400, Cell Biolabs, San Diego, CA, USA)
was used to detect active Ras in mouse amygdala lysates according to the
manufacturers protocol. Tissue was lysed at a ratio of 50 mg per 1 ml assay
buffer and 1 mg total protein was used for each Ras pull-down.
Quantication of protein expression
Band intensities of Western blots were quantied using ImageJ software
(National Institutes of Health, Bethesda, MD, USA). Expression levels of
proteins were normalized to GAPDH expression, P-ERK levels were
normalized to total ERK, P-TrkB levels to total TrkB, and levels of active
Ras to total Ras signals. Mean value of WT samples was set to 1 and mean
value of KO samples was expressed as x-fold of WT.
Phospho-RTK array
Active phosphorylated receptor tyrosine kinases (RTK) in amygdala lysates
were identied using the membrane-based Proteome Proler Mouse
Phospho-RTK Array (#ARY014, R&D Systems, Minneapolis, MN, USA). For
incubation of each membrane 500 μg of protein was used, following the
instructions of the manufacturer's protocol. Signals were detected by a
FluorChem SP Imager (Alpha Innotech, Biozym).
BDNF ELISA
For determination of mature free BDNF levels in mouse amygdala lysates,
we used the BDNF Emax ImmunoAssay System (#G7610, Promega,
Madison, WI, USA) without an acid treatment procedure according to
the manufacturers instructions. All samples were analyzed in duplicate,
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
4
Molecular Psychiatry (2017), 1 15
BDNF concentrations were calculated from a standard curve using
nonlinear regression curve tting (Prism5, GraphPad, La Jolla, CA, USA)
and related to the corresponding protein content.
SPRED2 phosphorylation assay
Embryonic mouse hypothalamic cells (cell line mHypoE-44, Cedarlane,
Burlington, ON, Canada) were cultivated in high glucose DMEM (Invitro-
gen), supplemented with 10% FBS and 1% Pen/Strep at 37 °C and 5% CO
2
and grown to 90100% conuency. To starve for growth factors, cells were
rinsed twice with sterile PBS and incubated at 37 °C and 5% CO
2
in DMEM
without FBS. After 6 h, cells were stimulated with 50 ng ml
1
BDNF in
DMEM (Merck Millipore) for 5, 15, 30 or 60 min. Cells were lysed in 1 ml of
ice-cold buffer containing 150 mM NaCl, 1% Igepal CA-630, 0.5% Sodium
deoxycholate, Complete Protease Inhibitor Cocktail (Roche) and PhosSTOP
Phosphatase Inhibitor Cocktail (Roche). Lysates were centrifuged for
10 min at 12 000gand 4 °C and supernatants were collected. As an input
control 50 μl of each sample was mixed with 50 μl Laemmli buffer and
incubated for 5 min at 95 °C. For antibody incubation, 5 μg phospho-
tyrosine (PY) antibody mix containing 3 μg anti-PY 100 and 2 μg anti-PY
102 (#9411 and #9416, Cell Signaling Technology) was added to each
sample before rotating for 1 h at 4 °C. Precipitation of the antibody-protein
complexes was performed by adding 100 μl of 50% v/v Protein G
Sepharose 4 Fast Flow beads (GE Healthcare, Chalfont St Giles, UK) to
protein samples and incubating for 1 h at 4 °C under gentle rotation. After
washing of beads 2 times with 1 ml lysis buffer and once with 1 ml 50 mM
Tris buffer (pH 8.0) they were mixed with 50 μl Laemmli buffer
and incubated for 5 min at 450 r.p.m. and 95 °C. Samples were stored at
20 °C and analyzed by Western blot.
X-Gal staining
Dissected mouse brains were embedded in Tissue-Tek OCT-compound
(Sakura Finetek Europe, Leiden, Netherlands) and snap-frozen in liquid
nitrogen. 10 μm cryosections were cut using a CM1950 microtome (Leica)
and X-Gal staining was performed as previously described.
26,35
Stained
sections were photographed by a Canon EOS 1000D digital camera
connected to a Stereomicroscope 2000-C (Zeiss).
Statistics
Data obtained from the uoxetine rescue experiment were rst analyzed
via three-way mixed ANOVA with genotype (WT/KO) and treatment
(uoxetine/placebo) as between-subjects factors and time (baseline/
2 weeks) as repeated measures factor. Post hoc t-tests were performed
to further analyze signicant time × treatment × genotype interactions. In
the remaining experiments, signicant differences between genotypes
were analyzed by MannWhitney Utest for not normal distributed data or
by two-sided two-sample t-tests and Welch´s tests, respectively, depend-
ing on the homogeneity of variances. Normal distribution of data was
tested using the ShapiroWilk test in combination with graphical analysis
tools like box and histogram plots. Statistical analyses were performed
using SPSS Statistics 19 (IBM, Armonk, NY, USA) or Prism5 (GraphPad).
Results are expressed as mean ± s.e.m. A P-value of *Po0.05 was
considered statistically signicant, whereas **Po0.01 represented high
and ***Po0.001 highest signicance. P-values 0.05 were considered as
statistically not signicant (n.s.).
RESULTS
Self-inicted facial lesions of SPRED2 KO mice are indicative of
obsessive-compulsive grooming
Starting at ~ 4 months of age, SPRED2 KO mice developed
apparent skin lesions on head, neck, and snout regions. These
lesions occurred uni- and bilaterally and progressed to ulcerations
with hemorrhage over time (Figure 1a). The penetrance of this
phenotype increased with age, and 80% of the KOs were affected
at the age of 12 months. We did not detect any lesions in WT
littermates, even when they were housed in the same cage with
SPRED2 KO mice from birth. This indicated that the lesions in KO
mice were not a result of aggressive encounters between cage
mates. However, SPRED2 KO mice were often seen engaged in
self-grooming regardless of whether they were housed alone or
with littermates (Supplementary Video). Thus, we hypothesized
that the phenotype of SPRED2 KOs could be the result of excessive
and injurious self-grooming, indicating an OCD-like behavior
caused by SPRED2 deciency in brain regions relevant for the
onset of OCD.
In SPRED2 KO mice, the Spred2 gene was disrupted by insertion
of a gene trap vector between exons 4 and 5 of Spred2 (Figure 1b).
The gene trap vector comprised a β-geo reporter gene, which is
expressed under control of the endogenous Spred2 promoter.
Therefore, in vivo monitoring of Spred2 expression by X-Gal
staining is possible. Indicated by the blue color after X-Gal staining
of coronal brain sections from heterozygous mice, SPRED2 is
expressed in various regions of the brain, including cortex and
hippocampus. High promoter activity was especially detected in
amygdala and striatum, both brain regions associated with the
development of OCD-like behaviors (Figure 1c). Western blot
analysis using amygdala lysates from SPRED2 KO mice and WT
controls demonstrated SPRED2 expression in WT amygdala but
the complete loss of full-length SPRED2 protein in KO amygdala.
The deciency of functional SPRED2 was not compensated by
increased expression of homologous SPRED1, demonstrated
by unaltered SPRED1 expression after normalization to GAPDH
(Figure 1d).
Obsessive-compulsive grooming is associated with changes in
anxiety-like behavior in SPRED2 KO mice
Excessive grooming or other OCD-related conditions are often
associated with additional behavioral phenotypes in mice. To
assess anxiety-like behavior in SPRED2 KO mice, we performed OF,
EPM and LDB tests. We used male mice aged 710 months,
displaying excessive grooming and facial lesions. In the OF an
elevated number of grooming events was recorded in SPRED2 KO
mice (Figure 2a), supporting our hypothesis of an obsessive-
compulsive behavior as cause of the self-inicted skin lesions. The
total distance traveled was reduced in SPRED2 KO mice, indicating
diminished locomotor and exploratory activity as a consequence
of obsessive grooming (Figure 2a). In comparison to WTs, SPRED2
KO mice tended to spend more time in the center of the OF
(Supplementary Table 1), which was indicative of less anxiety. In
the EPM, SPRED2 KOs in fact spent a longer time span in the open
arms (Figure 2b), traveled a longer distance in, and payed more
visits to the open area as compared with control mice; the
parameters in the guarded area were accordingly decreased
(Supplementary Table 1). Normally, mice avoid exploration of the
potentially dangerous open arms, which pointed to reduced
anxiety in SPRED2 KO mice. Similar to the OF test, the total
distance traveled during the EPM test was decreased (Figure 2b),
again suggesting a basically impaired locomotion. Species-
conform behaviors, for example, rearing and digging were also
generally reduced in favor of compulsive grooming (Supple-
mentary Table 1 and Supplementary Figure 1). In the LDB test,
SPRED2 KO mice again preferred the stressful environment and
spent more time in the brightly lit chamber. The latency to cross to
the save and dark compartment was prolonged, again indicating a
less anxious phenotype in SPRED2 KOs (Figure 2c). Like in the OF
test, the number of grooming bouts was higher in SPRED2 KO
mice compared with WTs (Supplementary Table 1). We also
examined the anxiety behavior in a group of younger male mice
aged 24 months, which did not show the grooming phenotype
or skin lesions. However, we could not detect any behavioral
changes of these younger SPRED2 KO mice in the above described
tests compared with WT controls (Supplementary Table 2).
Therefore, we conclude that reduced anxiety of SPRED2 KO mice
correlates with the occurrence of obsessive-compulsive grooming.
SPRED2 KO mice did not stop grooming even when they
already had apparent lesions. Therefore, we investigated the
sensitivity to thermal and mechanical stimuli in SPRED2 KOs and
WT controls. We used 710 months old mice without apparent
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
5
Molecular Psychiatry (2017), 1 15
lesions and only showing rst signs of OCD-like behavior. SPRED2
KOs displayed similar heat and mechanical paw withdrawal
latencies and thresholds compared with WT littermates, suggest-
ing intact skin sensitivity (Figure 2d). Since nociceptive testing is
normally conducted in younger mice and not well suited for mice
permanently engaged in self-grooming, assays were also
performed with mice aged 2-3 months. Again, we observed no
differences in nociception between young WT and SPRED2 KOs
(Supplementary Table 2).
Fluoxetine treatment reduces obsessive-compulsive grooming in
SPRED2 KO mice
We next evaluated whether drugs used to treat OCD in humans
would be effective in reducing the abnormal grooming in SPRED2
KO mice. Because SSRIs are a rst-line treatment for OCD, we
treated SPRED2 KO mice with apparent OCD-like phenotype and
WT controls aged 710 months either with uoxetine or placebo
for 2 weeks. We monitored treatment effects on behavior and the
occurrence of skin lesions by photo and video documentations.
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
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Molecular Psychiatry (2017), 1 15
Photo monitoring revealed no occurrence of lesions in placebo-
treated WT mice. In contrast, in placebo-treated SPRED2 KO mice
facial lesions resulting from overgrooming remained or ulcerations
and hemorrhages even worsened (Figure 2e). In the uoxetine
group, no treatment effects were visible in WT mice, while in
SPRED2 KOs the occurrence and severity of self-inicted lesions
were diminished (Figure 2f).
These ndings were conrmed by video recordings of single
mice at baseline and after 2 weeks of placebo vs uoxetine
treatment. Here, ANOVA revealed a signicant time × treatment ×
genotype interaction (P= 0.008) and a highly signicant main
effect of genotype (Po0.001) for the time mice were engaged
in grooming. Overall, SPRED2 KO mice did not only display a
higher number of grooming events (Figure 2a) but also a
longer grooming time compared with WT mice. Neither placebo
nor uoxetine treatment affected the duration of grooming
events in WT mice (Figure 2g and h). In placebo-treated
KO mice, however, grooming time seemed slightly increased after
2 weeks compared with baseline (Figure 2g), which is in line
with aggravation of facial lesions over time. Interestingly,
uoxetine-treated SPRED2 KO mice spent less time grooming
after 2 weeks of treatment compared with baseline (Figure 2h).
This conrms that grooming can be interpreted as an
OCD-like behavior in SPRED2 KOs, which can be treated with
uoxetine.
Digging is a species-typical behavior in mice but responsive to
SSRI treatment.
44
Therefore, it can also be regarded as OCD-like
and is commonly used as control parameter for uoxetine effects.
Digging was reduced by uoxetine treatment in addition to
excessive grooming, conrming the efciency of uoxetine
treatment and the OCD-like nature of the self-grooming behavior
in SPRED2 KOs (Supplementary Figure 1).
Changed synaptic transmission at cortico-striatal and thalamo-
amygdala synapses in SPRED2 KO mice
The pathogenesis of OCD is associated with dysregulation in CSTC
circuits. SPRED2 is highly expressed in cortex, striatum, and
thalamus but also in other parts of the central nervous system. To
detect possible defects in striatal neurotransmission caused by
SPRED2 deciency, we performed whole cell patch clamp
measurements on acute brain slices of SPRED2 KO mice with
apparent OCD phenotype and WT controls aged 68 months. The
stimulation electrode was placed in bers of the corpus callosum
and the recording electrode in single neurons of the putamen,
which is part of the striatum (Figure 3a). We stimulated putamen-
innervating corpus callosum bers with two consecutive pulses of
1 mA and with an interstimulus interval of 50 ms. This presynaptic
ber stimulation elicited two elevated excitatory postsynaptic
currents (EPSCs) in the putamen of SPRED2 KO mice, indicating
an increased synaptic transmission at cortico-striatal synapses
compared with WT controls (Figure 3b and d). According to that,
the stimulation threshold tended to be reduced in putamen
neurons of SPRED2 KOs (Figure 3c). Since both EPSC1 and EPSC2
were elevated to a similar extent in SPRED2 KO mice (Figure 3d),
the paired pulse ratio in KOs was comparable to WTs (Figure 3e),
indicating no relevant changes in the presynaptic vesicle release
probability.
The effects seen in cortico-striatal neurotransmission, however,
were even more pronounced in thalamo-amygdala circuits.
Accordingly, we performed another set of whole cell patch clamp
measurements by placing the stimulation electrode in thalamic
bers and the recording electrode in single neurons of the lateral
amygdala (Figure 3f). Stimulation of thalamic afferents with two
consecutive pulses of 1 mA and with an interstimulus interval of
50 ms elicited an elevated EPSC1 in lateral amygdala neurons of
SPRED2 KO mice (Figure 3g and i). This indicates an increased
transmission also at thalamo-amygdala synapses and is in line
with the reduced stimulation threshold in lateral amygdala
neurons of SPRED2 KOs (Figure 3h). The provoked EPSC2 was
comparable in WTs and SPRED2 KOs (Figure 3g and i). This
leads to a reduced paired pulse ratio in SPRED2 KO mice and
reects changes in the presynaptic vesicle release probability
at these amygdaloid synapses (Figure 3j). Therefore, we
additionally measured response parameters of spontaneously
released vesicles by recording mEPSCs (Figure 3k) in lateral
amygdala neurons. The frequency of mEPSCs recorded within
1 min was reduced in SPRED2 KO mice, which again indicates
presynaptic alterations in vesicle release probability (Figure 3l).
mEPSC magnitudes were also decreased in SPRED2 KOs, which
might either be caused by changes in postsynaptic sensitivity or in
vesicle transmitter load (Figure 3m).
Altered morphology in lateral amygdala neurons is accompanied
by dysregulated transcription and expression of synaptic genes
Changes in synaptic input are correlated with morphological
alterations in the respective neurons. Hence, we reconstructed
pyramidal neurons from the lateral amygdala of SPRED2 KO
mice showing apparent OCD-like behavior and of WT controls
aged 912 months. We detected a higher total spine number on
dendritic branches of branch orders 14 in SPRED2 KOs
(Figure 4a). Spine density per 10 μm of dendritic length was also
elevated across these branch orders compared with WTs
(Figure 4b).
Since SPREDs are suppressors of Ras/ERK-MAPK signaling and
thus critical regulators of cell proliferation and gene expression,
we further examined whether changed synaptic excitability and
neuron morphology was associated with altered expression of
synaptic proteins in the amygdala. Western blot analyses of pre-
and postsynaptic proteins demonstrated different expression
levels in 1012 months old SPRED2 KO mice compared with WT
Figure 2. Obsessive-compulsive grooming is accompanied by altered anxiety-like behavior and can be alleviated by uoxetine in SPRED2 KOs.
(a) In the open eld, the number of grooming bouts was elevated in SPRED2 KO mice (n=9), whereas the total distance traveled by SPRED
KOs was reduced compared with WT controls (n=7). (b) In the elevated plus maze, SPRED2 KOs (n=9) spent more time in the open arms and
the distance traveled was again reduced in comparison to WT littermates (n=7). (c) In the light/dark box, SPRED2 KO mice (n=9) spent more
time in the brightly lit chamber than the WT controls (n=7) and took longer to cross from the lit to the dark compartment. (d) Compared with
WTs (n=5), SPRED2 KO mice (n=7) showed neither differences in withdrawal thresholds of the hindpaw upon mechanical stimulation with
von Frey laments nor in hindpaw withdrawal latencies upon thermal stimulation with radiant heat. (e) Photo documentations of mice within
the placebo group revealed an unaltered or even worsened state of facial lesions in SPRED2 KO mice (n=7) but no occurence of wounds in
WT controls (n=6) after 2 weeks. (f) Photo documentations of mice treated with uoxetine for 2 weeks demonstrated a clear recovery of
occurrence and severity of self-inicted lesions due to reduced hemorrhages and ulcerations in SPRED2 KOs (n=11) but no visible effects of
treatment in W T mice (n=7). (g-h) Videotaping of single mice before and after 2 weeks of uoxetine treatment revealed an increased duration
of grooming events in SPRED2 KO mice (n=11) as compared with WT (n=7) at baseline. (g) Duration of grooming events was not affected by
placebo treatment in WT mice but seemed slightly increased in SPRED2 KOs (P=0.073) after 2 weeks. (h) Fluoxetine treatment had no effect
on grooming time in WT mice but decreased it in SPRED2 KOs after 2 weeks of treatment. Data are mean ±s.e.m; *Po0.05, **Po0.01,
***Po0.001. KO, knockout; n.s., not signicant; WT, wildtype.
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
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Molecular Psychiatry (2017), 1 15
controls (Figure 4c). The expression of PSD95, an important anchor
of various synaptic proteins at the postsynapse, was upregulated
in SPRED2 KOs compared with WT littermates. Protein levels of
metabotropic glutamate receptor 5 (mGluR5), which is primarily
located at the periphery of postsynaptic densities, were also
elevated in SPRED2 KO mice as well as levels of mGluR2, which is
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
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Molecular Psychiatry (2017), 1 15
primarily distributed at presynaptic axon terminals but may also
be expressed at postsynaptic sites. ERC1 (ELKS/RAB6-interacting/
CAST family member 1), a structural and functional determinant of
the presynaptic active zone, was also upregulated, while
presynaptic bassoon, a direct interaction partner of ERC1 in the
active zone, was downregulated. The expression of the small
GTPases Rab3A and Rab6, which are involved in the
regulation of synaptic vesicle transport along microtubules and
exocytosis at the presynapse, was not altered. Furthermore,
levels of α- and β-tubulin were unchanged (Figure 4c).
Quantication of protein amounts by normalization to
GAPDH conrmed the dysregulated expression of various
pre- and postsynaptic proteins in the amygdala of SPRED2 KOs
(Figure 4d).
Differences in protein expression are mainly caused either by
altered gene transcription or by protein turnover rate. To address
a possible dysregulation at transcriptional level, we performed
quantitative RT-PCRs using RNA from the amygdala and gene-
specic primers for PSD95, mGluR2, mGluR5 and ERC1. We
detected that the dysregulated protein expression observed in
Western blots was accompanied by altered expression levels of
the selected genes (Figure 4e). Because mRNA and protein levels
were changed in the same direction, transcriptional dysregulation
is most likely causative for alterations in synaptic protein
expression.
SPRED2 deciency leads to increased TrkB/ERK-MAPK signaling
and induces OCD-like grooming in SPRED2 KO mice
Given the dysregulated synaptic gene expression in the amygdala,
we focused on the upstream regulatory mechanism that might
contribute to the molecular and physiological changes at
amygdaloid synapses. An essential regulator of neuronal gene
transcription, proliferation and differentiation but also of synaptic
transmission and potentiation is the BDNF/TrkB signaling path-
way. Upon binding of the neurotrophin BDNF to its preferred
receptor tyrosine kinase TrkB, downstream signals are mediated
by the Ras/ERK-MAPK cascade. Because SPRED2 is a critical
inhibitor of Ras/ERK-MAPK signaling, we investigated if SPRED2
deciency impacts BDNF/TrkB/ERK-MAPK signaling in SPRED2 KO
mice. We analyzed the expression and phosphorylation of ERK1/2
in amygdala of 1012 months old SPRED2 KO mice and WT
littermates by Western blot. The expression of unphosphorylated
ERK was not altered, however, P-ERK was increased in SPRED2 KO
mice. The 2.5-fold elevated P-ERK/ERK ratio clearly reected
pathway overactivation due to the loss of SPRED2-mediated
inhibition (Figure 5a). To unravel whether the increase in Ras/ERK-
MAPK signaling is also involved in the development of the
OCD-like behavior in SPRED2 KOs, we specically blocked the
Ras/ERK-MAPK in vivo using the MEK1/2 inhibitor selumetinib. In
this prospective study, we treated SPRED2 KO mice aged 8 months
with selumetinib for one week. Photo documentation revealed
reduced hemorrhage and ulceration of self-inicted wounds in
SPRED2 KOs after one week of treatment (Figure 5b). This
indicated that SPRED2, as an endogenous suppressor of
Ras/ERK-MAPK signaling, is required to ensure normal behavior.
Investigation of factors further upstream in this pathway revealed
that also Ras was activated 1.9-fold in the amygdala of SPRED2 KO
mice compared with WTs (Figure 5c). To test whether pathway
activation is elicited by elevated BDNF expression, we
estimated BDNF levels in the amygdala of 612 months old
SPRED2 KO mice and WT controls and found no differences
between genotypes (Figure 5d). SPRED2 can be phosphorylated at
various conrmed tyrosine residues and might therefore be a
direct target of TrkB. To address this, we immunoprecipitated
tyrosine-phosphorylated proteins from BDNF-stimulated
murine hypothalamic cells and analyzed SPRED2 and TrkB by
Western blot. The input controls demonstrated constant
expression of both TrkB and SPRED2 in mHypoE44 cells after
different times of BDNF stimulation. In the IP samples, BDNF
stimulation resulted in increasing TrkB phosphorylation over time.
After 60 min of BDNF stimulation, BDNF-mediated TrkB activation
provoked phosphorylation of SPRED2 (Figure 5e). Consequently,
SPRED2 is a target of TrkB itself or of a kinase downstream of TrkB.
Given the unaltered BDNF levels in the amygdala and the
interaction between SPRED2 and TrkB, we further investigated
whether the augmented activity of Ras and ERK in SPRED2 KO
mice might be a result of specically increased TrkB
activation. We used a phospho-RTK array to identify active
phosphorylated RTKs in amygdala of 10 months old SPRED2 KO
and WT mice. Independently of the genotype, only PDGF-Rαwas
markedly phosphorylated among the 39 different murine RTKs
included in the array. In the amygdala lysates of SPRED2
KOs, we detected highest phosphorylation levels in EGFR, ErbB2
and TrkB (Figure 5f). This demonstrated activation of the TrkB
receptor in response to loss of SPRED2; however, a parallel
phosphorylation of EGFR and ErbB2 might contribute to
induction of downstream pathways. To support our hypothesis
that TrkB is responsible for activation of the Ras/ERK-MAPK
pathway, we examined possible alterations in TrkB
phosphorylation and expression quantitatively. TrkB expression
levels were 1.4-fold higher in SPRED2 KO amygdala after
normalization to GAPDH in comparison to WT controls
(Figure 5g). In addition to TrkB receptor overexpression,
phosphorylation of Y515, the tyrosine residue indicative for
Ras/ERK-MAPK pathway activation in mouse TrkB, was 1.3-fold
elevated in amygdala of SPRED2 KO mice. In contrast, Y817
phosphorylation level, which is relevant for phospholipase C
activation, was not altered, indicating that the downstream
actions of activated TrkB are specically mediated by the
Ras/ERK MAPK pathway (Figure 5g). Although a contribution of
Figure 3. Altered synaptic excitability in amygdala and striatum of SPRED2 KO mice. (a) Schematic brain slice showing representative positions
of afferent ber stimulation (asterisk) in the corpus callosum and of whole cell current measurements in the putamen. The location of each
measured cell is marked by a circle. (b) Exemplary excitatory postsynaptic currents (EPSC1, EPSC2) for SPRED2 KO and WT recorded after two
consecutive afferent ber stimulations (asterisks). (c) Stimulation threshold in putamen of SPRED2 KOs tended to be reduced after stimulation
of afferent corpus callosum bers. (d) Both EPSC1 and EPSC2 were elevated comparably in putamen of SPRED2 KO mice after two consecutive
afferent ber stimulations (paired pulses). (e) Paired pulse ratio of WTs and KOs was similar in putamen. n=5 for WT and SPRED2 KO in each
experimental setup of cortico-striatal neurotransmission measurement. (f) Schematic brain slice showing representative positions of thalamic
afferent ber stimulation (asterisk) and of whole cell current measurements in the lateral amygdala. (g) Exemplary excitatory postsynaptic
currents (EPSC1, EPSC2) for SPRED2 KO and WT recorded after two consecutive afferent ber stimulations (asterisks). (h) Stimulation threshold
in lateral amygdala of SPRED2 KOs was decreased after stimulation of afferent thalamic bers. (i) In comparison to WTs, consecutive afferent
ber stimulations (paired pulses) revealed an increased EPSC1 but a similar EPSC2 in lateral amygdala of SPRED2 KO mice. (j) Accordingly, the
paired pulse ratio was reduced in lateral amygdala of SPRED2 KOs. (k) Exemplary miniature excitatory postsynaptic currents (mEPSCs)
measured in lateral amygdala of WT and SPRED2 KO cells. (l) mEPSC frequency was decreased in SPRED2 KO mice. (m) mEPSC magnitude was
reduced in SPRED2 KOs. n=8 for WT and n=9 for SPRED2 KO in each experimental setup of thalamo-amygdala neurotransmission
measurement. Data are mean ±s.e.m; *Po0.05. KO, knockout; n.s., not signicant; WT, wildtype.
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
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Molecular Psychiatry (2017), 1 15
activated EGFR and ErbB2 cannot be excluded, TrkB seems to be a
crucial modulator of upregulated ERK-MAPK pathway in SPRED2
KO mice as demonstrated by TrkB overexpression, activation, and
association with phosphorylation of SPRED2. Missing inhibition of
BDNF/TrkB/ERK-MAPK signaling resulted in OCD-like behavior in
SPRED2 KO mice whereas SPRED2-mediated pathway down-
regulation seems to be necessary for coordinated neuronal
protein expression, synaptic function and behavior in vivo.
Figure 4. Differences in lateral amygdala neuron morphology are associated with dysregulated gene transcription and protein expression. (a)
Morphological analysis of reconstructed neurons from lateral amygdala of WT (n=26) and SPRED2 KO mice (n=22) revealed a higher total
spine number on dendritic branches of branch orders 14. (b) Spine density per 10 μm of dendritic length was also higher within these branch
orders. (c) Exemplary Western blot analyses showed changed expression levels of different pre- and postsynaptic proteins in the amygdala of
SPRED2 KO mice in comparison to WT controls. Protein levels of PSD95, mGluR5, mGluR2, and ERC1 were increased, those of bassoon were
decreased. Protein expression of Rab3A, Rab6, α-tubulin, β-tubulin and GAPDH was unaltered; GAPDH was used as loading control. (d)
Quantied signals of investigated synaptic proteins after normalization to GAPDH conrmed changed expression levels in the amygdala of
SPRED2 KOs (n=11) compared with WTs (n=11). (e) Quantitative RT-PCRs with mRNA isolated from amygdala of SPRED2 KO mice (n=11) and
WT controls (n=11) using gene-specic primers conrmed the increased expression of PSD95, mGluR5, mGluR2, and ERC1 on mRNA level.
Data are mean ±s.e.m; *Po0.05, **Po0.01, ***Po0.001. KO, knockout; n.s., not signicant; WT, wildtype.
OCD-like behavior in SPRED2 decient mice
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DISCUSSION
Identication of SPRED2 as a new factor for the pathogenesis of
OCD
Our study demonstrates that deciency of SPRED2 causes
excessive and pathological self-grooming in mice. One rst clear
indication for the presence of an OCD in SPRED2 KOs is that they
do not stop grooming even if they already have severe facial
lesions with ulcerations and hemorrhage. Reactions to mechanical
and heat stimuli were not altered in SPRED2 KO mice, suggesting
normal skin sensitivity but compulsive actions up to the point of
being self-injurious. Increased anxiety is a common feature
associated with certain forms of OCD and also occurs in SAPAP3
or SLITRK5 KO mice, two comparable models of OCD-like
grooming.
45,46
Unexpectedly, SPRED2 KO mice did not show
increased anxiety-like behavior but seemed less anxious. However,
in contrast to the other mouse models, behavioral tests revealed a
generally decreased locomotor activity in SPRED2 KOs in addition
to the reduced anxiety, which may also be interpreted as lack of
drive, lethargy, or signs of depression-like behavior. In line with
this, other species-conform behaviors, for example, rearing, were
also reduced, indicating that SPRED2 KO mice are massively
captured by compulsive actions, which inuence experimental
readouts. Furthermore, SPRED2 KO mice might also display
learning defects similar to mice decient for the related SPRED1
(ref. 31) and SPRED1-decient humans.
30
Altogether, these
disabilities possibly impact appropriate reactions to anxiogenic
stimuli or their interpretation. OCD in humans is to a great extent
associated with anxiety, specically avoidance. However, common
comorbid conditions also include other psychiatric disorders.
Estimated comorbidity with generalized anxiety disorder ranges
from below 20% to nearly 76% (overview in
47,48
). To a similar
extend, OCD is associated with major depressive disorder (4065%
according to
47,48
). This may also be the case in SPRED2 decient
mice and explain the reduced locomotor activity and the observed
lack of drive. Moreover, OCD and anxiety disorders vary greatly in
features like neurocircuitry, neurochemistry, symptoms and
cognitive-emotional processing,
49
and therefore in DSM-V (5th
edition of the Diagnostic and Statistical Manual of Mental
Disorders), OCD is no longer categorized as an anxiety disorder,
thus emphasizing the differences between both entities.
50
A second important feature of OCD is its susceptibility to stress,
that is, symptoms increase at times of stress and stressful events
may precede the onset of OCD.
51,52
Accordingly, increased stress
hormone levels such as corticotropin-releasing hormone, adreno-
corticotropic hormone, and corticosterone are often detected in
OCD patients.
53,54
This is in line with our previous ndings of a
hyperactive HPA axis in SPRED2 KO mice, leading to elevated
release of these stress hormones.
33
Third, SPRED2 KOs were responsive to uoxetine, a treatment
shown to be effective in the reduction of OCD-like grooming and
digging.
7,4446
Altogether, these data identify SPRED2 as a
candidate factor involved in the pathogenesis of OCD-like
disorders and SPRED2 KO mice as a suitable new model to
investigate OCD-like behaviors.
Excitability changes at cortico-striatal and thalamo-amygdala
synapses contribute to OCD pathogenesis in SPRED2 KO mice
Both neuroimaging studies in humans
5,6
and studies with mutant
mice implicated dysregulation within CSTC circuits in the
pathogenesis of OCD. Mice decient for SAPAP3 or SLITRK5, like
SPRED2 highly expressed in the striatum, displayed alterations in
the activity of cortico-striatal neurons in combination with OCD-
like grooming.
45,46
In SPRED2 KO mice, we detected increased
transmission at cortico-striatal synapses. In line with our results,
repeated experimental cortico-striatal stimulation in mice also
provoked excessive grooming,
7
indicating that especially hyper-
activity of cortico-striatal synapses might be causative for OCD-like
behaviors and that SPRED2 is crucially involved in regulation of
cortico-striatal circuit activity.
Interestingly, we also detected elevated synaptic excitability at
thalamo-amygdala synapses of SPRED2 KO mice. Although
aberrant function of CSTC circuits is at present the most widely
accepted neurobiological explanation for OCD, the underlying
pathology is not necessarily limited to orbitofronto-striatal
regions. Recent evidence suggests that limbic structures such as
hippocampus, anterior cingulate, and amygdala contribute to the
pathology of OCD.
10,55,56
In fact, functional magnetic resonance
imaging studies in humans correlate OCD disorders with elevated
amygdala activity,
5557
which is in line with the observed
hyperactivity in the lateral amygdala of SPRED2 KO mice.
Especially the basolateral amygdala sends prominent projections
to the ventral striatum,
8,9,56
underlining the functional connectiv-
ity of both brain regions. Since the amygdala is central to
processing emotion and to multiple aspects of cognition that are
impaired in OCD, aberrant communication between amygdala
and striatum could mediate compulsive behavior. Additionally,
dysregulation of amygdala activity may contribute to compulsivity
by imparting excessive affective inuence on behavioral selection.
Here, we provide a mouse model showing similar changes of
synaptic excitability in both functionally related brain regions
associated with OCD, which is unique amongst previously
published mouse models.
45,46,58
These data suggest that the
altered activity of CSTC circuits observed in OCD may indeed be
triggered upstream by changes in amygdala activity and that
SPRED2 is a crucial modulator of synaptic transmission from
thalamus to lateral amygdala.
Changed synaptic input into lateral amygdala results from a
combination of altered neuron morphology and dysregulated
expression of pre- and postsynaptic proteins
Detailed analysis of electrophysiological data revealed both
pre- and postsynaptic changes in SPRED2 KO mice, which
contributed to the observed increased synaptic transmission in
amygdala and striatum. In the lateral amygdala, diminished
paired pulse ratio and decreased frequency of spontaneously
released vesicles indicate a reduced presynaptic vesicle release
probability. In line with these ndings we detected altered
expression of ERC1 and bassoon, both scaffolding proteins
interacting at the active zone of presynapses. They are not only
critical for the integrity of active zone structures,
59
but also for the
regulation of presynaptic neurotransmitter release.
60,61
Especially
ERC1, a homolog of Drosophila´s bruchpilot, is required to
maintain Ca
2+
-channel density and synaptic transmission after
evoked stimuli,
60
which is in line with the ERC1 overexpression
and increased excitability of amygdala synapses in SPRED KOs.
Consistent with the changes in synaptic transmission, we also
found dysregulated expression of glutamate receptors in the
amygdala. Because SPRED2 is rather associated with intracellular
signaling than with regulation of ion channels, we focused on
metabotropic glutamate receptors and revealed a higher
expression of mGluR5 and mGluR2. While mGluR2 can be located
at both pre- and postsynaptic sites and generally decreases
neuronal excitability, mGluR5 acts exclusively at postsynaptic sites
by increasing neuronal excitability.
62
Dysregulation of mGluRs is
generally associated with different psychiatric and anxiety-related
disorders, and particularly mGluR5 antagonists and mGluR2
agonists are promising antipsychotic compounds.
63
Hence, the
observed higher expression of mGluR5 in SPRED2 KOs might
contribute to the OCD-like behavior and increased synaptic
transmission, whereas the upregulated mGluR2 expression
could already be a counterregulatory mechanism and associated
with the reduced anxiety. PSD95 is the major scaffolding protein
at the postsynapse of mature glutamate synapses and clusters
with ionotropic but also with metabotropic glutamate receptors.
64
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
11
Molecular Psychiatry (2017), 1 15
This coupling explains the higher expression of both mGluRs and
PSD95 at amygdala postsynapses. Alterations in the expression of
PSD95 or of interacting proteins are again associated with a
variety of mood disorders in humans,
65
in mice especially with
OCD-like behaviors, as also observed in the SAPAP3 KO mouse
model.
45
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
12
Molecular Psychiatry (2017), 1 15
Taken together, the mostly presynaptic effects detected by
electrophysiological recordings in the amygdala are correlated
with the dysregulated expression of proteins that have been
shown to regulate presynaptic active zone structure and
neurotransmitter release like bassoon, ERC1 and mGluR2. The
dysregulation of proteins expressed at the postsynapse, for
example, PSD95 and mGluR5, is consistent with the detected
higher branch order-dependent spine number and spine density
in lateral amygdala neurons of SPRED2 KO mice. In fact, PSD95,
mGluR5 and also TrkB/ERK-MAPK signaling are associated with
regulation of spine morphology, primarily investigated in
hippocampus
6669
but also in amygdala.
70
Dendritic spines are the main target for excitatory inputs.
Excitatory drive from thalamus to lateral amygdala was increased
in SPRED2 KOs as conrmed by higher provoked EPSCs and
reduced stimulation threshold. Therefore, higher spine number
and density in SPRED2 KOs is supposed to be the structural
manifestation of enhanced excitability at thalamo-amygdala
synapses. This in turn might contribute to general amygdala
hyperactivity, which is a feature of OCD but also of other anxiety
disorders.
5557
Although SPRED2 KO mice did not show increased
anxiety in the conducted standard tests, other features of anxiety-
related behavior are highly apparent, including increased stress
hormone levels due to HPA axis hyperactivity,
33
and reduced
exploratory locomotion. Accordingly, the amygdala has a high
density of CRH and glucocorticoid receptors and stress exposure
can induce both amygdala activation and increases in spine
density.
9,42,71
Taken together, the altered synaptic excitability in
the amygdala contributes to the OCD-like phenotype in SPRED2
KO mice and is a result of both pre- and postsynaptic effects.
Changes in thalamo-amygdala synaptic transmission are caused
by dysregulated protein expression as a consequence of altered
gene transcription, altogether a result of SPRED2 deciency and
the associated upregulation of TrkB/ERK signaling.
Increased TrkB/BDNF-ERK pathway activity is associated with
OCD-like behavior in SPRED2 KO mice
In combination with the OCD-like behavior, we detected
hyperactivation of TrKB/ERK-MAPK signaling at critical levels of
the cascade. In amygdala of SPRED2 KO mice, TrkB was not only
overexpressed, but also phosphorylation of TrkB was increased, as
demonstrated by a phospho-RTK-array and by Western blots.
More precisely, phosphorylation at Y515 was elevated in amygdala
lysates of SPRED2 KO mice, phosphorylation at Y817, however,
was unchanged. Whereas P-TrkB Y817 is responsible for induction
of downstream phospholipase C-mediated cascades, P-TrkB Y515
triggers Ras/ERK-MAPK pathways,
13
indicating that they are
specically activated by active TrKB in SPRED2 KOs. Consequently,
both Ras activity and ERK phosphorylation were augmented in
SPRED2 KO amygdala. BDNF levels in amygdala were not changed,
demonstrating that overactivation of Ras/ERK-MAPK signaling in
fact results from loss of SPRED2-mediated pathway inhibition.
Furthermore, we detected that SPRED2 is phosphorylated after
BDNF-stimulated TrkB activation. TrkB is activated by autophos-
phorylation and in turn activates various downstream targets by
phosphorylation. SPRED2 can be phosphorylated at different
tyrosine residues in response to stimulation by various growth
factors.
23
SPRED phosphorylation is functionally required for
regulation of the inhibitory effect
72,73
or as prevention of
proteasomal degradation.
72
We assume that TrkB-dependent
tyrosine phosphorylation of SPRED2 might be preventive for
protein degradation and might enable the suppression of Ras/ERK
signaling stimulated by BDNF-induced TrkB activation. This
mechanism could regulate SPRED2 steady-state levels, maintain
physiological TrkB/BDNF-ERK pathway activity in vivo, and ensure
normal behavior.
BDNF/TrkB pathways are crucially involved in nearly all stages of
neural circuit development and associated with multiple neuro-
psychiatrc diseases.
1215
Untill now, however, only rst descriptive
studies associate the BDNF/TrkB system with OCD
16,17
or related
disorders in humans and mice, indicating that genetic mutations
and altered protein levels might play a role.
1820
ERK, a critical
regulator of proliferation, is present in presumably all cells and
tissues. In brain, ERK contributes to the induction of transcription
of plasticity-related genes, mediates synaptic transmitter release
and is therefore implicated in learning and memory.
7476
However,
not much is known about the role of ERK in neuropsychiatric
disorders, especially in OCD-like disorders. In SPRED2 KO mice, we
determined the mechanism how SPRED2 deciency leads to an
increase of active TrkB receptor and signaling, which is mediated
downstream by increased Ras activity and ERK-phosporylation and
induces OCD-like behavior.
The contribution of dysregulated TrkB/ERK-MAPK signaling to
OCD-development was conrmed by administration of selumeti-
nib, a MEK1/2 inhibitor and specic blocker of Ras/ERK-MAPK
activity. Selumetinib is experimentally used for the treatment of
cancer in mouse models and clinical trials have also been
conducted in humans.
77,78
Although we used selumetinib not as
a putative medication but as an experimental rescue, it robustly
alleviated self-inicted lesions resulting from excessive grooming
in SPRED2 KO mice. Our prospective in vivo study showed that the
TrkB-activated ERK-MAPK pathway is specically involved in the
development of OCD and conrmed that physiological SPRED2-
regulated pathway activity is required to maintain normal
behavior in vivo.
Here we provide evidence that upregulation of the Ras/ERK-
MAPK pathway is not only involved in cancer pathogenesis and
developmental disorders like rasopathies, but also in the
development of OCD-related disorders. We ascertained thalamo-
amygdala circuits as affected brain region in addition to the
known cortico-striatal circuitry. In the amygdala, the upstream
Figure 5. Increased TrkB/ERK-MAPK signaling caused by SPRED2 deciency leads to OCD-like grooming in SPRED2 KO mice. (a)Westernblot
analyses of ERK expression and phosphorylationinamygdalaofSPRED2KOmice(n=8) and WT littermates (n=8) revealed no differences in levels
of unphosphorylated ERK but increased levels of phosphorylated ERK in SPRED2 KOs. Upregulation of ERK-MAPK signaling was veried by the 2.5-
fold elevated P-ERK/ERK ratio. (b)In vivo inhibition of Ras/ERK-MAPK signaling in SPRED2 KO mice (n=3) by administration of the MEK1/2 inhibitor
selumetinib for one week alleviated hemorrhage and ulceration of facial lesions. (c) Ras activation was 1.9-fold higher in SPRED2 KO amygdala (n=8)
compared with WT (n=8). (d) BDNF levels in relation to total protein content were unchanged in amygdala lysates of WT (n=18) and SPRED2 KO
mice (n=24). (e) Western blot analyses (IB) of SPRED2 and TrkB after BDNF stimulation of murine hypothalamic cells revealed constant TrkB and
SPRED2 expression over time (Input). After immunoprecipitation of tyrosine-phosphorylated (PY) proteins from BDNF-stimulated mHypoE44 cells,
Western blot analyses detected increasing phosphorylation of TrkB over time and, mediated by active TrkB, phosphorylation of SPRED2 60 min after
BDNF stimulation (IP). (f) Parallel determination of phosphorylation of 39 different mouse receptor tyrosine kinases revealed a higher
phosphorylation of TrkB specically in amygdala of SPRED2 KO mice (n=2forWTandKO).(g) Western blot analyses of TrkB in amygdala indicated
an increased expression in SPRED2 KOs reected by the 1.4-fold TrkB/GAPDH ratio as compared with WTs. Phosphorylation of TrkB at Y515 was
1.3-fold elevated but unchanged at Y817. n=8 for WT and KO. Data are mean ±s.e.m; **Po0.01, ***Po0.001. BDNF, brain-derived neurotrophic
factor; KO, knockout; n.s., not signicant; OCD, obsessive-compulsive disorder; TrkB, tropomyosin receptro kinase B; WT, wildtype.
OCD-like behavior in SPRED2 decient mice
M Ullrich et al
13
Molecular Psychiatry (2017), 1 15
trigger of OCD-like behavior is hyperactivity of BDNF/TrkB
signaling, a result of the loss of SPRED2-mediated pathway
inhibition. BDNF/TrkB/ERK-MAPK pathway dysregulation leads to
changes in pre- and postsynaptic mRNA and protein expression
and to alterations of thalamo-amygdala synaptic transmission.
Both the SSRI uoxetine and the Ras/ERK pathway inhibitor
selumetinib reduced OCD-like grooming in SPRED2 KO mice
(Supplementary Figure 2). Thus, our study identies SPRED2 as a
considerable factor in the pathogenesis of OCD, as a critical
regulator of synaptic transmission in different brain regions and as
a new regulator of BDNF/TrkB pathways. SPRED2 is highly
conserved, the most ubiquitously expressed SPRED family
member, and its expression is especially widespread in brain.
Therefore, SPRED2 is a very promising target for further and more
specic studies of brain function and associated neuropsychiatric,
-developmental and -degenerative diseases both in mice and
humans.
CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGMENTS
We thank Lydia Biko for expert technical help with mechanical and thermal sensitivity
assays. We are also very grateful to Marion Winning for her excellent technical
expertize in Golgi-Cox staining. We acknowledge Marco Abesser for technical
assistance in photography and videotaping and Nadine Ehmann and Martin Pauli for
generously providing reagents. The position of Melanie Ullrich was funded by the
DFG (SCHU1600/3-1).
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OCD-like behavior in SPRED2 decient mice
M Ullrich et al
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... In previous studies, SPRED2-knockout (KO) mice showed clear signs of neurohumoral disturbances [21] and OCD-like (obsessive compulsive disorder) behavior [22]. Since language problems (dysphonia, dysarthria) are related to developmental diseases, neurodegeneration, and advancing age [23], we wanted to examine the call usage to demonstrate developmental delay and neurodegeneration through a different approach. ...
... SPRED2-KO mice were generated by a gene trap approach as described previously [25]. In brief, the SPRED2 gene was disrupted by insertion of a gene trap vector, resulting in a disrupted, non-functional Spred2 gene and the expression of a truncated, dysfunctional SPRED2 protein [22]. For our recordings, we used either mice with a homozygous KO in both alleles or bi-allelic SPRED2 wild-type (WT) mice as controls. ...
... In all experiments, we used pairs of SPRED2 mutant mice and pairs of WT mice as controls, which were not littermates or siblings and were naïve to male/female social encounters. Mutant and WT littermate mice were identified as described before [22]. The young groups of mice were between 3 and 6 months old, the old groups of animals were one year and older. ...
Article
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Vocalization is an important part of social communication, not only for humans but also for mice. Here, we show in a mouse model that functional deficiency of Sprouty-related EVH1 domain-containing 2 (SPRED2), a protein ubiquitously expressed in the brain, causes differences in social ultrasound vocalizations (USVs), using an uncomplicated and reliable experimental setting of a short meeting of two individuals. SPRED2 mutant mice show an OCD-like behaviour, accompanied by an increased release of stress hormones from the hypothalamic–pituitary–adrenal axis, both factors probably influencing USV usage. To determine genotype-related differences in USV usage, we analyzed call rate, subtype profile, and acoustic parameters (i.e., duration, bandwidth, and mean peak frequency) in young and old SPRED2-KO mice. We recorded USVs of interacting male and female mice, and analyzed the calls with the deep-learning DeepSqueak software, which was trained to recognize and categorize the emitted USVs. Our findings provide the first classification of SPRED2-KO vs. wild-type mouse USVs using neural networks and reveal significant differences in their development and use of calls. Our results show, first, that simple experimental settings in combination with deep learning are successful at identifying genotype-dependent USV usage and, second, that SPRED2 deficiency negatively affects the vocalization usage and social communication of mice.
... neural circuits j dopaminergic modulation j SNc j excessive self-grooming j OCD Obsessive-compulsive disorder (OCD) is characterized by unwanted distressing thoughts (obsessions) and repetitive acts (compulsions), with severe disruption of daily activities (1,2). Recent evidence suggests that various structures, such as the cortex (3)(4)(5), striatum (6)(7)(8)(9), hypothalamus (10), hippocampus (11,12), amygdala (13)(14)(15), and even spinal cord (16), contribute to the pathology of OCD. Imbalanced cortical-striatal activities, such as the dynamics in cortico-striato-thalamo-cortical (CSTC) circuit are recognized as the core neurobiological substrate for OCD (17)(18)(19). ...
Article
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The neurobiological understanding of obsessive-compulsive disorder (OCD) includes dysregulated frontostriatal circuitry and altered monoamine transmission. Repetitive stereotyped behavior (e.g., grooming), a featured symptom in OCD, has been proposed to be associated with perturbed dopamine (DA) signaling. However, the precise brain circuits participating in DA's control over this behavioral phenotype remain elusive. Here, we identified that DA neurons in substantia nigra pars compacta (SNc) orchestrate ventromedial striatum (VMS) microcircuits as well as lateral orbitofrontal cortex (lOFC) during self-grooming behavior. SNc-VMS and SNc-lOFC dopaminergic projections modulate grooming behaviors and striatal microcircuit function differentially. Specifically, the activity of the SNc-VMS pathway promotes grooming via D1 receptors, whereas the activity of the SNc-lOFC pathway suppresses grooming via D2 receptors. SNc DA neuron activity thus controls the OCD-like behaviors via both striatal and cortical projections as dual gating. These results support both pharmacological and brain-stimulation treatments for OCD.
... A negative age-related CpG is located near tshz3 (Teashirt Zinc Finger Homeobox 3), rare protein-altering variants of which are highly enriched in a cohort of supercentenarians [70]. It is noteworthy that negative age-related CpG associated gene orthologs tend to be involved in synaptic transmission, including tshz3, dgcr8, spred2, gria3, gria4, kcnip4, and rims1 [71][72][73][74][75][76]. Indeed, the term 'synaptic transmission' was retrieved in GREAT functional enrichment analysis among the genes that lose methylation with age (Fig. 4). ...
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DNA methylation-based biomarkers of ageing (epigenetic clocks) have been developed for many mammals, but not yet for amphibian species. We generated DNA methylation data from African clawed frogs (Xenopus laevis) and Western clawed frogs (Xenopus tropicalis), from adult tissues, whole embryos, and tadpoles. We used an array platform designed for CpGs that are highly conserved in mammals to build multiple DNA methylation-based estimators of age for Xenopus. We found that dual species clock could be developed that apply to both humans and frogs (human-clawed frog clocks), and whose high accuracy supports that epigenetic ageing processes are evolutionary conserved outside mammals. Crossing vast evolutionary distances, we characterize age-related CpGs in Xenopus and mammalian species. Highly conserved positively age-related CpGs are located in neural-developmental genes such as uncx, tfap2d as well as nr4a2 implicated in age-associated disease. As in human clocks, positively age-related CpGs are associated with Polycomb repressive complex 2 (PRC2) target sites. Negative age-related CpGs are associated with genes involved in synaptic transmission. We conclude that signatures of epigenetic ageing are evolutionary conserved between frogs and mammals and that the associated genes relate to neural-developmental processes, altogether opening opportunities to employ Xenopus as a model organism to study development and ageing.
... For instance, SPRED2 deficiency activates ERK/MAPK pathway to enhance innate immune responses and protect mice from polymicrobial sepsis [40]. The deficiency of SPRED2 also induces obsessive-compulsive disorder-like behavior in mice through the overactivation of Ras/ERK-MAPK signaling pathway [41]. Importantly, MAPK pathway exhibits a key regulatory function in DR. ...
Article
Full-text available
Diabetic retinopathy (DR) is a common complication of diabetes, and the leading cause of blindness in adults. Sprouty-related proteins with EVH1 domain (SPRED2) play an important role in diabetes and are closely related to the lens and eye morphogenesis. This study attempted to investigate the role and related mechanism of SPRED2 in DR. DR rat model was established by administration streptozocin. Human retinal endothelial cells (HRECs) were treated with high glucose (HG) to mimic DR. The results showed that SPRED2 expression was decreased in the retinal tissues of DR rats and HG-treated HRECs. MTT assay and flow cytometry data showed that SPRED2 overexpression reduced cell viability of HG-treated HRECs. SPRED2 overexpression enhanced Caspase-3 activity and promoted apoptosis of HG-treated HRECs. Furthermore, the expressions of endothelial cell markers CD31 and E-cad were down-regulated, whereas the expressions of mesenchymal cell markers FSP1, SM22, and α-SMA were up-regulated in the HG-treated HRECs. SPRED2 overexpression reversed HG-induced endothelial-mesenchymal transition in HRECs. The expressions of tight junction components claudin 3, occludin, and ZO-1 were increased in HG-treated HRECs following SPRED2 up-regulation. In addition, SPRED2 overexpression downregulated the expression of p-ERK1/2, p-p38, and p-JNK in the HG-treated HRECs. In conclusion, this study demonstrated that SPRED2 overexpression repressed endothelial-mesenchymal transition and endothelial injury in HG-treated HRECs by suppressing MAPK signaling pathway. These findings suggested that SPRED2 may be a novel potential therapeutic target implicated in DR progression.
... Increased activity and changes in functional connectivity of the amygdala are observed in OCD patients when viewing symptom-provoking stimuli [54,55]. Also, increased molecular signalling from the amygdala has been shown to cause OCD-like behaviour in an animal model and this ceases once the hyperactivity is normalized [56]. DBS for the treatment of OCD appears to be effective, in part, by regulating amygdala hyperactivity [46,47,57]. ...
Article
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Background: Obsessive-compulsive disorder (OCD) is a debilitating disease with a lifetime prevalence of 2-3%. Neuromodulatory treatments have been successfully used in severe cases. Deep brain stimulation (DBS) targeting the inferior thalamic peduncle (ITP) has been shown to successfully alleviate symptoms in OCD patients; however, the brain circuits implicated remain unclear. Here, we investigate the efficacious neural substrates following ITP-DBS for OCD. Methods: High-quality normative structural and functional connectomics and voxel-wise probabilistic mapping techniques were applied to assess the neural substrates of OCD symptom alleviation in a cohort of 5 ITP-DBS patients. Results: The region of most efficacious stimulation was located in the regions of the ITP and bed nucleus of the stria terminalis. Both functional and structural connectomics analyses demonstrated that successful symptom alleviation involved a brain network encompassing the bilateral amygdala and prefrontal regions. Limitations: The main limitation is the small size of the ITP-DBS cohort. While the findings are highly consistent and significant, these should be validated in larger studies. Conclusions: These results identify a tripartite brain network - composed of the bilateral amygdala and prefrontal regions 24 and 46 - whose engagement is associated with greater symptom improvement. They also provide information for optimizing targeting and identifying network components critically involved in ITP-DBS treatment response. Amygdala engagement in particular seems to be a key component for clinical benefits and could constitute a biomarker for treatment optimization.
... Importantly, postnatal MEK inhibition with PD-0325901 does not affect the spatial learning and memory phenotypes but does rescue the altered social behavior phenotypes (Borrie et al., 2021a,b). Knockout of the related Spred2 results in an obsessive-compulsive behavioral phenotype (Ullrich et al., 2018), as well as impaired growth, cardiac hypertrophy, splenomegaly and craniofacial malformation (Ullrich et al., 2019). Interestingly, a recent report described a case series of patients with an autosomal-recessive NS-like phenotype resulting from loss of SPRED2 function (Motta et al., 2021), further validating SPRED2 as a RASopathy gene. ...
Article
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The RASopathies are a group of disorders caused by a germline mutation in one of the genes encoding a component of the RAS/MAPK pathway. These disorders, including neurofibromatosis type 1, Noonan syndrome, cardiofaciocutaneous syndrome, Costello syndrome and Legius syndrome, among others, have overlapping clinical features due to RAS/MAPK dysfunction. Although several of the RASopathies are very rare, collectively, these disorders are relatively common. In this Review, we discuss the pathogenesis of the RASopathy-associated genetic variants and the knowledge gained about RAS/MAPK signaling that resulted from studying RASopathies. We also describe the cell and animal models of the RASopathies and explore emerging RASopathy genes. Preclinical and clinical experiences with targeted agents as therapeutics for RASopathies are also discussed. Finally, we review how the recently developed drugs targeting RAS/MAPK-driven malignancies, such as inhibitors of RAS activation, direct RAS inhibitors and RAS/MAPK pathway inhibitors, might be leveraged for patients with RASopathies.
... Specifically, the TrkB/ERK-MAPK pathway overactivation contributes to restoring normal behavior in an animal model of OCD. Moreover, up-regulation of the Ras/ERK-MAPK pathway was involved in the development of OCD-related disorders [53]. ERK and RAS cascade reactions signal to MAPKs, activating signal transduction, transcription factors and regulating gene expression. ...
Article
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Background Obsessive-compulsive disorder (OCD) is characterized by intrusive thoughts and repetitive actions, that presents the involvement of the cortico-striatal areas. The contribution of environmental risk factors to OCD development suggests that epigenetic mechanisms may contribute to its pathophysiology. DNA methylation changes and gene expression were evaluated in post-mortem brain tissues of the cortical (anterior cingulate gyrus and orbitofrontal cortex) and ventral striatum (nucleus accumbens, caudate nucleus and putamen) areas from eight OCD patients and eight matched controls. Results There were no differentially methylated CpG (cytosine-phosphate-guanine) sites (DMSs) in any brain area, nevertheless gene modules generated from CpG sites and protein-protein-interaction (PPI) showed enriched gene modules for all brain areas between OCD cases and controls. All brain areas but nucleus accumbens presented a predominantly hypomethylation pattern for the differentially methylated regions (DMRs). Although there were common transcriptional factors that targeted these DMRs, their targeted differentially expressed genes were different among all brain areas. The protein-protein interaction network based on methylation and gene expression data reported that all brain areas were enriched for G-protein signaling pathway, immune response, apoptosis and synapse biological processes but each brain area also presented enrichment of specific signaling pathways. Finally, OCD patients and controls did not present significant DNA methylation age differences. Conclusions DNA methylation changes in brain areas involved with OCD, especially those involved with genes related to synaptic plasticity and the immune system could mediate the action of genetic and environmental factors associated with OCD.
Article
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Context Fibraurea recisa Pierre. (Menispermaceae) (FR) is a traditional Chinese medicine known as “Huangteng.” The total alkaloids of FR (AFR) are the main active ingredients. However, the pharmacological effects of AFR in the treatment of depression have not been reported. Objectives This study investigates the antidepressant effects of AFR by network pharmacology and verification experiments. Materials and methods Compound-Target-Pathway (C-P-T) network of FR and depression was constructed through network pharmacology. In vitro, HT-22 cells were treated with corticosterone (CORT) solution (0.35 mg/mL), then AFR (0.05 mg/mL) solution and inhibitor AZD6244 (14 μM/mL) or BAY11-7082 (10 μM/mL) were added, respectively. The cell viability was detected by CCK-8. In vivo, C57BL/6 mice were divided into 5 groups, namely the normal group, the CUMS group, the AFR (400 mg/kg) group, and the 2 groups that were simultaneously administered the inhibitory group AZD6244 (8 mg/kg) and BAY11-7082 (5 mg/kg). Western blotting was used to assess the expression level of the proteins. Results AFR could protect HT-22 cells from CORT-induced damage and increase the cell viability from 49.12 ± 3.4% to 87.26 ± 1.5%. Moreover, AFR significantly increased the levels of BDNF (1.3, 1.4-fold), p-ERK (1.4, 1.2-fold) and p-CERB (1.6, 1.3-fold), and decreased the levels of NLRP3 (11.3%, 31.6%), ASC (19.2%, 34.2%) and caspase-1 (18.0%, 27.6%) in HT-22 cells and the hippocampus, respectively. Discussion and conclusions AFR can improve depressive-like behaviours and can develop drugs for depression treatment. Further studies are needed to validate its potential in clinical medicine.
Article
Background Brain-derived neurotrophic factor (BDNF) is a growth factor that plays many critical functions in the central nervous system (CNS) and may be involved in the development of a range of psychopathologies, including depression, dementia, and neurodegenerative disorders. Methods In the present study, we performed the first systematic review with a meta-analysis to quantitatively compare the peripheral blood BDNF levels between patients with obsessive-compulsive disorder (OCD) and healthy controls (HCs). A systematic search was conducted using PubMed and Web of Science databases to identify the relevant articles. Results Nine studies encompassing 474 adults with OCD and 436 HCs were included in this meta-analysis. A random-effects meta-analysis showed that patients with OCD had significantly decreased peripheral blood levels of Brain-derived neurotrophic factor (BDNF) when compared with the HCs (Hedges' g = −0.722, 95% confidence interval [CI] = −1.152 to −0.292, P = 0.001). Subgroup analyses revealed decreased BDNF levels in plasma of patients (Hedges' g = −1.137, 95% CI = −1.463 to −0.810, P = 0.000) and drug-free patients (Hedges’ g = −1.269, 95% CI = −1.974 to −0.564, P = 0.000) as compared to patients on active drug therapy and HCs. Meta-regression analyses showed that age, sex, sample size, Y-BOS total score, and publication year had no moderating effects on the outcome. Conclusion Although the relationship between our findings and the pathophysiology of OCD and the role BDNF plays in the development of the disease remains to be determined, the outcomes suggest that BDNF may serve as a potential biomarker of OCD.
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Pharmacological activation of the serotonin (5-HT) 1B and 5-HT1A receptors has been shown to induce OCD-like perseverative circling and locomotor stereotypy in rodents. Although, several studies have examined how activation of these receptors facilitates these motor-associated OCD-like behaviors, it is not known how acute 5-HT1B and 5-HT1A activation impacts behavioral inflexibility, a common trait related to OCD. The current study examined how acute 5-HT1B/1 A receptor agonist RU24969 treatment at 0.01, 0.1, and 1.0 mg/kg impacted behavioral flexibility in both female and male C57BL/6 J mice. Behavioral flexibility was tested using a spatial reversal learning task, with probabilistic reward contingencies. In addition, locomotor activity and anxiety-like behaviors were also measured. RU24969 at 0.1 and 1.0 mg/kg impaired behavioral flexibility in both female and male C57BL/6 J mice. RU24969 treatment at 1.0 mg/kg reduced locomotor activity in male mice, although RU24969 treatment did not significantly reduce locomotor activity in female mice. In the open field, 1.0 mg/kg elevated anxiety-like behavior in male mice only. Overall, these results demonstrate that acute 5-HT1B and 5-HT1A receptor activation leads to impairments in behavioral flexibility, a common trait associated with OCD.
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Obsessive-compulsive disorder encompasses a broad range of symptoms that represent multiple psychological domains, including perception, cognition, emotion, social relatedness, and diverse motor behaviors. The purpose of these analyses was to evaluate the correlational relationships of the symptoms of obsessive-compulsive disorder. This study examined the 13 a priori categories used to group types of obsessions and compulsions in the Yale-Brown Obsessive Compulsive Scale symptom checklist in two independent groups of patients with obsessive-compulsive disorder (N = 208 and N = 98). A principal-components factor analysis with varimax rotation was performed, followed by a series of other exploratory analyses. The two data sets yielded nearly identical results. Four factors--obsessions and checking, symmetry and ordering, cleanliness and washing, and boarding--emerged in each data set, in total accounting for more than 60% of the variance. Obsessive-compulsive disorder is a multidimensional and etiologically heterogeneous condition. The four symptom dimensions identified in this study are largely congruent with those identified in earlier reports. These factors may be of value in future genetic, neurobiological, and treatment response studies.
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Significant interest in the mechanistic underpinnings of obsessive-compulsive disorder (OCD) has fueled research on the neural origins of compulsive behaviors. Converging clinical and preclinical evidence suggests that abnormal repetitive behaviors are driven by dysfunction in cortico-striatal-thalamic-cortical (CSTC) circuits. These findings suggest that compulsive behaviors arise, in part, from aberrant communication between lateral orbitofrontal cortex (OFC) and dorsal striatum. An important body of work focused on the role of this network in OCD has been instrumental to progress in the field. Disease models focused primarily on these regions, however, fail to capture an important aspect of the disorder: affective dysregulation. High levels of anxiety are extremely prevalent in OCD, as is comorbidity with major depressive disorder. Furthermore, deficits in processing rewards and abnormalities in processing emotional stimuli are suggestive of aberrant encoding of affective information. Accordingly, OCD can be partially characterized as a disease in which behavioral selection is corrupted by exaggerated or dysregulated emotional states. This suggests that the networks producing OCD symptoms likely expand beyond traditional lateral OFC and dorsal striatum circuit models, and highlights the need to cast a wider net in our investigation of the circuits involved in generating and sustaining OCD symptoms. Here, we address the emerging role of medial OFC, amygdala, and ventral tegmental area projections to the ventral striatum (VS) in OCD pathophysiology. The VS receives strong innervation from these affect and reward processing regions, and is therefore poised to integrate information crucial to the generation of compulsive behaviors. Though it complements functions of dorsal striatum and lateral OFC, this corticolimbic-VS network is less commonly explored as a potential source of the pathology underlying OCD. In this review, we discuss this network's potential role as a locus of OCD pathology and effective treatment.
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Patients with triple-negative breast cancer (TNBC) have a poor prognosis because TNBC often metastasizes, leading to death. Among patients with TNBC, those with extracellular signal-regulated kinase 2 (ERK2)-overexpressing tumors were at higher risk of death than those with low-ERK2-expressing tumors (hazard ratio, 2.76; 95% confidence interval, 1.19-6.41). The MAPK pathway has been shown to be a marker of breast cancer metastasis, but has not been explored as a potential therapeutic target for preventing TNBC metastasis. Interestingly, when we treated TNBC cells with the allosteric MEK inhibitor selumetinib, cell viability was not reduced in two-dimensional culture. However, in three-dimensional culture, selumetinib changed the mesenchymal phenotype of TNBC cells to an epithelial phenotype. Cells that undergo epithelial-mesenchymal transition (EMT) are thought to contribute to the metastatic process. EMT leads to generation of mesenchymal-like breast cancer cells with stem celllike characteristics and a CD44+CD24-/low expression pattern. We tested the hypothesis that targeted inhibition of the MAPK pathway by selumetinib inhibits acquisition of the breast cancer stem cell phenotype and prevents lung metastasis of TNBC. TNBC cells treated with selumetinib showed inhibition of anchorageindependent growth, an indicator of in vivo tumorigenicity (P < 0.005), and decreases in the CD44+CD24-/low fraction, ALDH1 activity, and mammosphere-forming efficiency. Mice treated with selumetinib formed significantly fewer lung metastases than control mice injected with vehicle (P < 0.05). Our data demonstrate that MEK inhibitors can inhibit breast cancer stem cells and may have clinical potential for the prevention of metastasis in certain cases in which tumors are MAPK dependent.
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We applied and validated a quantitative allodynia assessment technique, using a recently developed rat surgical neuropathy model wherein nocifensive behaviors are evoked by light touch to the paw. Employing von Frey hairs from 0.41 to 15.1 g, we first characterized the percent response at each stimulus intensity. A smooth log-linear relationship was observed, with a median 50% threshold at 1.97 g (95% confidence limits, 1.12-3.57 g). Subsequently, we applied a paradigm using stimulus oscillation around the response threshold, which allowed more rapid, efficient measurements. Median 50% threshold by this up-down method was 2.4 g (1.81-2.76). Correlation coefficient between the two methods was 0.91. In neuropathic rats, good intra- and inter-observer reproducibility was found for the up-down paradigm; some variability was seen in normal rats, attributable to extensive testing. Thresholds in a sizable group of neuropathic rats showed insignificant variability over 20 days. After 50 days, 61% still met strict neuropathy criteria, using survival analysis. Threshold measurement using the up-down paradigm, in combination with the neuropathic pain model, represents a powerful tool for analyzing the effects of manipulations of the neuropathic pain state.
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• In light of prior data that the central administration of vasopressin in animals is associated with abnormal persistence of behaviors acquired under aversive conditioning, we studied the secretion of arginine vasopressin into the cerebrospinal fluid and plasma in patients with obsessivecompulsive disorder and controls. Patients with obsessivecompulsive disorder had significantly elevated basal levels of arginine vasopressin in the cerebrospinal fluid and significantly increased secretion of arginine vasopressin into the plasma in response to hypertonic saline administration. Moreover, seven of 12 patients with obsessivecompulsive disorder showed a loss of the normal linear relationship between plasma arginine vasopressin level and osmolality. In addition, cerebrospinal fluid corticotropin releasing hormone, which has synergistic effects with arginine vasopressin centrally and at the pituitary gland, was also significantly elevated in patients with obsessivecompulsive disorder compared with controls.
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The amygdala has long been associated with emotion and motivation, playing an essential part in processing both fearful and rewarding environmental stimuli. How can a single structure be crucial for such different functions? With recent technological advances that allow for causal investigations of specific neural circuit elements, we can now begin to map the complex anatomical connections of the amygdala onto behavioural function. Understanding how the amygdala contributes to a wide array of behaviours requires the study of distinct amygdala circuits.
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Twin and family studies support a significant genetic contribution to obsessive-compulsive disorder (OCD) and related disorders, such as chronic tic disorders, trichotillomania, skin-picking disorder, body dysmorphic disorder, and hoarding disorder. Recently, population-based studies and novel laboratory-based methods have confirmed substantial heritability in OCD. Genome-wide association studies and candidate gene association studies have provided information on specific gene variations that may be involved in the pathobiology of OCD, though a substantial portion of the genetic risk architecture remains unknown.