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Fragile X Syndrome

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Background

• Caused by a CGG expansion in the FMR1 gene

• Males severely affected, females are mosaic

• Fragile X syndrome can be a cause of autism or related disorders, although not all children with fragile X syndrome have these conditions

• Symptoms include• Developmental delays: crawling, walking• Hand clapping or hand biting• Hyperactive or impulsive behavior• Mental retardation• Speech and language delay • Tendency to avoid eye contact• Physical signs: Flat feet, flexible joints and low muscle tone, large body size, large forehead or

ears with a prominent jaw, long face, large testicles, soft skin

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Bakker et al., 2004; Han et al;. 2015; Huber et al. 2002; Krueger et al., 2011; Koga et al., 2015

FMR1 KO mice

• These mice have a knockout allele of the fragile X mental retardation syndrome 1 gene (Fmr1) on the X chromosome and exhibit many phenotypic characteristics of the Fragile X Syndrome in humans including hyperactivity, repetitive behavior and seizures.

• Absence of the Fragile X Mental Retardation protein (FMRP) in the mice causes activation of RAC1 protein resulting in abnormalities in dendritic spines in various regions of the brain. and altered synaptic function.

• The absence of FMRP also alters synaptic plasticity which results in an impairment of long-term potentiation in the cortex and hippocampus, as well as an augmentation of long-term depression in the hippocampus and cerebellum.

• Male FMR1 KO mice on FVB/n background bred at PsychoGenics are used in all studies

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Behavioral Tests in Adult Mice

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Male KO mice show similar BW compared to WT mice

Age (weeks)

8 9 10 11 12

Bo

dy W

eig

ht

(g)

15

20

25

30

35

40

FMR1_WTFMR1_KO

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Male mice tested at 9 weeks of age

FMR1 KO mice are hyperactive compared to WT mice

Time (min)

5 10 15 20 25 30 35 40 45 50 55 60

Dis

tan

ce

Tra

ve

led

(c

m)

0

250

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1500FMR1_WTFMR1_KO

Dis

tan

ce T

ravele

d in

Cen

ter

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)

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To

tal

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Dis

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n C

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500FMR1_WTFMR1_KO

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Male mice tested at 9 weeks of age

Male FMR1 KO mice show increased rearing activity

Reari

ng

Fre

qu

en

cy (

co

un

t)0

250

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2000FMR1_WTFMR1_KO

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Time (min)

5 10 15 20 25 30 35 40 45 50 55 60

Re

ari

ng

Fre

qu

en

cy (

co

un

t)

0

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250FMR1_WTFMR1_KO

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KO mice show deficits in Contextual and Cued Fear Conditioning %

Fre

ezin

g

0

20

40

60

80

100FMR1_WTFMR1_KO

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Time (min)

1 2 3 4 5

% F

reezin

g

0

20

40

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100FMR1_WT FMR1_HMZ KO Context

Pre-Cue Cue Post-Cue

% F

ree

zin

g

0

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70FMR1_WTFMR1_KO

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Context

Male mice tested at 11 weeks of age

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FMR1 KO mice show reduced startle response

Startle Intensity (dB)

70 75 80 85 90 95 100 105 110 115 120

Mean

Sta

rtle

Res

po

nse

0

200

400

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FMR1_WTFMR1_KO

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Male mice tested at 10 weeks of age

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Audiogenic Seizures in 3 week old mice

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Audiogenic seizures

• KO mice are tested at 3 weeks of age

• Mice are are individually placed in a Plexiglas chamber and allowed to explore for 15 sec. They are then exposed to a 125 dB tone for 2 minutes, followed by 1 minute of no sound, and then a repeat 2 minute tone. The mice are scored based on their response, latency, and seizure intensity:

0: no response 1: wild running and jumping2: clonic seizures3: clonic/tonic seizures4: tonic seizures5: respiratory arrest

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Late

nc

y t

o s

eiz

e (

sec

)

0

50

100

150

200

250

300

350

Salineseizure: 66.6 seizure: 256 seizure: 300 seizure: 270

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SalineMPEP 3 mg/kg MPEP 10 mg/kg MPEP 30 mg/kg MTEP 30 mg/kg

Effects of MPEP and MTEP on audiogenic seizures

Late

ncy t

o r

esp

irato

ry a

rrest

(sec)

0

50

100

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arrest arrest: 99.875 arrest: 101.3 arrest: 275.4 arrest: 300 arrest: 300

* * *SalineMPEP 3 mg/kg MPEP 10 mg/kg MPEP 30 mg/kg MTEP 30 mg/kg

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Effects of MPEP and MTEP on survival

Vehicle

MTEP (30 mg/kg)

MPEP 1 mg/kg 3 mg/kg 30 mg/kg

died

survived

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Effects of diazepam on audiogenic seizures

Vehicle Diazepam 0.03 mg/kgDiazepam 0.1 mg/kgDiazepam 0.3 mg/kgDiazepam 1 mg/kg

Late

ncy t

o s

eiz

e (

sec)

0

50

100

150

200

250

300

350

seizure: 151

seizure: 168.75

seizure: 246.375

seizure: 300

seizure: 300

* * *

Late

ncy t

o r

esp

irato

ry a

rrest

(sec)

0

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350

Col 2: 162.6667 Col 2 Col 2: 162.6667 Col 2: 180.625 Col 2: 277.5 Col 2: 300 Col 2: 300

* * *

died

survived

Diazepam 0 0.03 0.1 0.3 1 mg/kg

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Effects of dicyclomine on audiogenic seizures

Late

ncy t

o r

esp

irato

ry a

rrest

(sec)

0

50

100

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*

* *

Dicyclomine 0 3 10 30 mg/kg

Late

nc

y t

o s

eiz

e (

se

c)

0

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*

**

Dicyclomine 0 3 10 30 mg/kg

Dicyclomine 0 3 10 30 mg/kg

died

survived

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Electrophysiology

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Fmr1 mice exhibit enhanced hippocampal mGluR-dependent long-term potentiation (LTD), which is reversed by mGluR antagonist 8-OH-DPAT

Time course of the changes in responses recorded inCA1 area of hippocampus induced with an applicationof an mGluR agonist (S)-DHPG (100μM). DHPG wasapplied from 0 to 5 min and the subsequent mGluRantagonist 8-OH-DPAT (100nM) was applied from 10to 15 minutes

fEP

SP

Slo

pe (

% B

aselin

e)

0

20

40

60

80

100

120FMR1_WT FMR1_KOFMR1_KO - 8OH DPAT(100uM)

*

#

TIme (min)

-10 0 10 20 30 40 50 60

fEP

SP

Slo

pe

(%

Ba

se

lin

e)

20

40

60

80

100

FMR1_WT FMR1_ KO FMR_KO - 8OH DPAT

Summary of the data for the last 5 min ofexperiment. *p

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A significant reduction in tonic inhibitory currents, a critical factor modulating neuronal excitability, was observed in dentate granule cells of 2 month-old male FMR1-KO mice in agreement with prior observations (Zhang, N. et al (2017). Exp. Neurol., PMID: 28822839).

Tonic inhibitory currents are significantly reduced in dentate granule cells of FMR1-KO mice.Whole-cell patch clamp recordings were made in dentate granule cells of hippocampal slices from 2-monthold male wild-type (WT) (n=22 cells, 6 mice) and FMR1-KO mice (n=24 cells, 5 mice), Vhold=-70 mV.Left Following a stable baseline (-THIP) and subsequent enhancement of tonic GABA currents by the dsubunit-selective agonist THIP (gaboxadol, 1 mM; +THIP), tonic currents were unmasked by blockingGABAA receptors with 100 mM picrotoxin. *p