• Anxiety – behavioural state arising in
anticipation of potential threat
- Protective role by preparing an organism for potential
danger
- Becomes pathological when exaggerated or
uncontrollable
• Anxiety disorders - abundant and costly
- 20% of the general population suffer from at least one form of anxiety disorder at some point of their life
- Financial cost of more than 65 billion dollars in Canada alone
Neural substrates underlying anxiety
1. A neural substrate defined by gene
expression pattern in the developing embryo
2. A neural substrate defined by connectivity in the adult brain
Neural substrates underlying anxiety
1. A neural substrate defined by gene
expression pattern in the developing embryo- Genetically distinct subset of serotonergic neurons
2. A neural substrate defined by connectivity in the adult brain
- Hippocampus to amygdala connection
BRAINSTEM
Serotonin (5HT)-producing Serotonin (5HT)-producing neuronsneurons
9 clusters (nuclei) B1-B9
BRAINSTEM
Serotonin (5HT)-producing Serotonin (5HT)-producing neuronsneurons
9 clusters (nuclei) B1-B9
• Autonomic body physiology modulation of breathing,
body temperature blood pressure heart rate
• learning and memory• sensorimotor gating• modulation of mood states
BRAINSTEM
Serotonin (5HT)-producing Serotonin (5HT)-producing neuronsneurons
9 clusters (nuclei) B1-B9
Serotonergic neurons are implicated in a range of clinical disorders:
• Sudden infant death syndrome
• Fetal alcohol syndrome• Autism
• Anxiety• Depression• Addiction and Compulsive behaviors
• Autonomic body physiology modulation of breathing,
body temperature
blood pressure heart rate
• learning and memory• sensorimotor gating• modulation of mood states
Serotonergic neurons have different functional properties
Message: there are many subtypes of serotonergic neurons
• different Anatomical locations
• different Projection patterns
• different Cell morphologies
• different Physiological properties
Which serotonin neurons underlie which behaviors and physiological processes?
?• breathing• heart rate• blood pressure• learning and memory• sensorimotor gating• anxiety• aggression
• breathing• heart rate• blood pressure• learning and memory• sensorimotor gating• anxiety• aggression
Defining serotonergic neurons by gene expression pattern during early development
?
Ser
oto
nerg
ic
pro
geni
tors
dorsal view
r1
r4
r7
embryonichindbrain
Ser
oto
nerg
ic
pro
geni
tors
Serotonergic neuron progenitors are located in embryonic hindbrain
Ser
oto
nerg
ic
pro
geni
tors
dorsal view
embryonichindbrain
r1
r2
r3
r4
r5
r6
r7
Embryonic hindbrain is composed of morphologically distinct structures called rhombomeres
r1
r2
r3
r4
r5
r6
r7
embryonichindbrain
Ho
xa-4
Ho
xb-4
Ho
xd-4
Ho
xd-3
Ho
xb3
Ho
xa-3
Ho
xb-2
Ho
xa-2
Ho
xb-1
Ho
xa-1
Elk-L
3
Elf- 2
Elk-L
Eb
kS
ek-4
Se
k-3
Se
k-2S
ek-1
Kro
x-20
Kre
isler
Adapted from Lumsden and Krumlauf, 1996
rhom
bom
ere
s
Ser
oton
ergi
c p
roge
nito
rs
Rhombomeres are molecularly distinct structures expressing different sets of genes
dorsal view
embryonichindbrain
Ser
oton
ergi
c p
roge
nito
rs
Rhombomeres are molecularly distinct structures expressing different sets of genes
Message:serotonergic neuron progenitors in different rhombomeres have different gene expression pattern
dorsal view
Ser
oton
ergi
c p
roge
nito
rs
embryonichindbrain
Question:
How the embryonic gene expression patterns are related to serotonergic neuron function?
loxP loxP
OFFBAP
Dual recombinase-mediated gene activation
FRT FRT
effector
loxP loxP
OFFBAPFRT FRT
effector
Creexcision
BAP effector
FRT FRT
OFF
Dual recombinase-mediated gene activation
loxP loxP
OFFBAPFRT FRT
effector
Flpexcision
BAP effector
loxP loxP
OFF
Dual recombinase-mediated gene activation
loxP loxP
OFFBAPFRT FRT
effector
BAP effector ON
FlpCre
Dual recombinase-mediated gene activation
loxP loxP
OFFBAPFRT FRT
effector
BAP effector ON
GeneB::FlpGeneA::Cre
creFlp
Dual recombinase-mediated gene activation
loxP loxP
OFFBAPFRT FRT
effector
BAP effector ON
GeneB::FlpGeneA::Cre
EffectorON
Dual recombinase-mediated gene activation
r1
r2
r3
r4
r5
r6
r7
ONPet1-FlpeSerotonergic-specific Flpe
Rhombomere-specific Cre lines
GFP OFF
ONF P
FRT
GFP-TOX GFP
FRTrosa26
rosa26
CAG promoter
CAG promoter
Flpe
FF
Cre
P P
loxP loxP
r1
r2
r3
r4
r5
r6
r7
ONPet1-FlpeSerotonergic-specific Flpe
Rhombomere-specific Cre lines
GFP OFF
ONF P
FRT
GFP-TOX GFP
FRTrosa26
rosa26
CAG promoter
CAG promoter
Flpe
FF
Cre
P P
loxP loxP
Jensen et al, Nature Neuroscience 2008
Genetic fate map of serotonergic neurons
contributionsfrom a singlerhombomere
Jensen et al, Nature Neuroscience 2008
Genetic fate map of serotonergic neurons
contributionsfrom a singlerhombomere
contributions from multiplerhombomeres
contributions from multiplerhombomeres
Genetic fate map of serotonergic neurons
Jensen et al, Nature Neuroscience 2008
contributionsfrom a singlerhombomere
Message #1: Genetic Fate map = Classical, Anatomical Map
contributions from multiplerhombomeres
contributions from multiplerhombomeres
contributionsfrom a singlerhombomere
Message #1:
Message #2: provide framework to study the function of genetically defined serotonergic neuron subtypes
Genetic Fate map = Classical, Anatomical Map
contributions from multiplerhombomeres
contributions from multiplerhombomeres
Question:
How the embryonic gene expression patterns are related to serotonergic neuron function?
GOAL - Silence individual serotonergic neuron subtypes to determine their function
loxP loxP
BAPFRT FRT
GFP-TOX
GOAL - Silence individual serotonergic neuron subtypes to determine their function
loxP loxP
BAPFRT FRT
GFP-TOX
Tetanus toxin light chain : blocks neurotransmitter releaseTetanus toxin light chain : blocks neurotransmitter release
FIRST - silence all serotonergic neurons to reveal the full extent of attainable phenotypes
loxP loxP
BAPFRT FRT
GFP-TOX
anxiety-related behaviors
contextual learning sensorimotor gating
Assayed a set of 3 behaviors known to be modulated by serotonin (5-HT):
• contextual fear conditioning
• open field • prepulse inhibition of acoustic startle
• elevated zero maze
• light-dark exploration test
CollaboratorMelloni Cook
Tennessee Mouse Genome ConsortiumNeuromutagenesis Project
24hrs
repeat x3 at 2.5 min intervalsassay fear-induced freezing
Contextual Fear Conditioning Paradigm
Can they learn to associate an aversive stimulus to a particular cage context?
24hrs
repeat x3 at 2.5 min intervalsassay fear-induced freezing
p<.044
enhanced fear induced freezing behavior
Contextual Fear Conditioning Paradigm
24hrs
repeat x3 at 2.5 min intervalsassay fear-induced freezing
p<.044
No difference
triple Control
Act
ivit
y in t
he a
ltere
d c
onte
xt No difference
“silenced” mice show enhanced contextual learning and memory
enhanced fear induced freezing behavior
Elevated zero maze(advancement over Plus maze)
Open field test
Light-dark exploration test
3 tests of anxiety-related behaviors
Mice with higher level of anxiety-related behavior spend more time here.
innate fear innate fear of open areasof open areas
vs.vs.desire to exploredesire to explorenovel environmentsnovel environments
Light-dark explorationtest
Elevated zero maze Open field test
n=34 n=32
*p< 0.03
A 2-factor (genotype x sex) multivariate analysis of variance (MANOVA) was used. F(1,62)=4.81, p<.032
*
anxiety-related behaviors
contextual learning sensorimotor gating
“silenced” mice
(ePet1::Flpe, hßact::Cre, RC::PFtox)
sensorimotor gating: a neurological gating process that prevents distracting sensory inputs from generating unnecessary motor outputs
regulation is disrupted in Schizophrenia and Huntington disease
operationally measured by inhibition of the acoustic startle response after a leading (prepulse) stimulus
prepulse
TIME
Sti
mulu
s in
tensi
ty
85 dB
Sti
mulu
s in
tensi
ty pulse
TIME
Startle response
120 dB
% inhibition: indication of sensorimotor gating
Prepulse Inhibition (PPI) test
Mea
n pe
rcen
tage
In
hibi
tion
ofS
tart
le R
esp
onse
“Silenced” mice exhibited less of a startle in presence of a prepulse
control
“silenced” - Pet1Flpe, hßact-cre, RC::PFtox
P<.023
“Silenced” mice show enhanced sensorimotor gating.
background white noise
P<.007
anxiety-related behaviors
contextual learning sensorimotor gating
“silenced” all serotonergic neurons
anxiety-related behaviors
contextual learning sensorimotor gating
“silenced” all serotonergic neurons
Do serotonergic neurons with different embryonic origins serve different functions?
r1-derived5-HT
neurons
TOX inAll
5-HT neurons
What if we “silence” just the r1-derived 5-HT neurons?
? ? ?
anxiety-related behaviors
contextual learning sensorimotor gating
silence
all5-HT neurons
r1-derived
anxiety-related behaviors
contextual learning sensorimotor gating
no difference
silence
all5-HT neurons
r1-derived no difference
anxiety-related behaviors
contextual learning sensorimotor gating
silence
all5-HT neurons
r1-derived
anxiety-related behaviors
contextual learning
sensorimotor gating
no difference no difference
Anxiety-related behaviors
R1-derived 5-HT neurons may modulate anxiety-related behaviors
silence
all5-HT neurons
r1-derived
anxiety-related behaviors
contextual learning
sensorimotor gating
no difference no difference
Modulated more by r2- and/or r3-derived 5-HT neurons?
Anxiety-related behaviors
Contextual learning
Sensorimotor gating
silence
all5-HT neurons
r1-derived
anxiety-related behaviors
no difference no difference
Modulated more by r2- and/or r3-derived 5-HT neurons?
r2-derived
r3-derived
r5-derived
in p
rog
ress
contextual learning
sensorimotor gating
anxiety-related behaviors
contextual learning
sensorimotor gating
XXserotonergic neuron subtype
? ?
GOAL - Assigning specific serotonergic neuron subtypes to specific behaviors
Neural substrates underlying anxiety
1. A neural substrate defined by gene
expression pattern in the developing embryo- Genetically distinct subset of serotonergic neurons
2. A neural substrate defined by connectivity in the adult brain
- Hippocampus to amygdala connection
Amygdala and Anxiety
• c-Fos induction upon exposure to anxiogenic stimuli (Hale et al., 2006; Knapska et al., 2007)
• Stimulating the BLA increases anxiety : GABA antagonist bicucullin infusion into the BLA (Sajdyk et al., 1999, 2002; Spiga et al., 2006)
• Suppressing the BLA decrease anxiety : glutamate blocker infusion into the BLA (Sajdyk et al., 1997)
Hippocampus and Anxiety
• Sustained anxiety activates the vHPC : Brain imaging with human and rhesus monkeys (Oler et al., 2010; Hasler et al., 2007)
• Stimulating the vHPC increases anxiety : GABA antagonist Bicuculline or picrotoxin infusion (Rezvanfard et al. 2009; Bast T et al., 2001)
• Suppressing the vHPC reduces anxiety : GABA agonist infusion or mechanical lesion (Kjelstrup et al., 2002; Bannerman et al., 2003; Mchugh et al.,
2004; Trent et al., 2010; Mceown et al. 2010)
Amygdala and HPC are reciprocally connected
Basolateral Amygdala Ventral hippocampus
Amygdala and HPC likely coordinate with each other to control anxiety behaviours
Q. How does the neural communication between the two structures contributes to anxiety?
Optogenetic manipulation
Behavioural consequence of activating the vHPC to BLA inputs
AAV-hSyn-ChR2-YFP
473nm blue laser
Which subregions of the vHPC project to the BLA?
Cholera toxin B chain (CTB)
Ventral CA1, subiculum, lateral entorhinal cortex project to the basolateral amygdala
intermediate
ventral
dorsal
AAV-hSyn-ChR2-YFP
mPFC
Hypothalamus
BLA
AAV-hSyn-ChR2-YFP
mPFC
Hypothalamus
BLA
200um optic fiber delivering 473nm laser
Optogenetic stimulation of vHPC axon terminals at the basolateral amygdala
N= 12
Mice were tested in 4 consecutive sessions of 5 min EPM (OFF/ON/OFF/ON)
Acute stimulation of vHPC terminals at the BLA induced anxiety behaviors in the EPM
Acute stimulation of vHPC terminals at the BLA induced anxiety behaviors in the EPM
Optogenetic stimulation of vHPC axon terminals at the basolateral amygdala
Summary
A neural substrate defined by gene
expression pattern in the developing embryo- Genetically distinct subtype of 5-HT neurons derived by
rhombomere 1
A neural substrate defined by connectivity in the adult brain
- vHPC to BLA connection- Acute stimulation of the vHPC to BLA inputs is sufficient
to induce anxiety behaviours in mice
Genetic approaches to link neurons to behaviors
Canadian Foundation Fighting Blindness
National Institute of Heath
Part 1.
Natural Sciences and Engineering Research Council of Canada
University of Toronto
•Shadi Bakir•Robin Nguyen•Wendy Xin
Harvard University
•Susan Dymecki•Wade Reghre•Patricia Jensen•Jia Jia Mai
University of Memphis
•Melloni Cook
Part 2.
Cre
loxP loxP
DNA target
excision
Site specific DNA excision by recombinase
Flp
FRT FRT
DNA target
excision
Site specific DNA excision by recombinase
Recombinase-mediated gene activation
loxP loxP
OFFBAP effector
Transcriptional STOP
sequences
: BroadlyActive
Promoter
Recombinase-mediated gene activation
loxP loxP
OFFBAP effector
excision
ONBAP effector
Cre
• Activity modulator (silence/activate neuron activity)
• Ablator (kill neurons)
• Fluorescent reporter (label neurons)
embryonichindbrain
Ser
oton
ergi
c p
roge
nito
rs
Where do serotonergic neurons originating from different rhombomeres settle in the adult brain?
?
dorsal view
Hippocampus and Anxiety
• Sustained anxiety activates the vHPC : Brain imaging with human and rhesus monkeys (Oler et al., 2010; Hasler et al., 2007)
• Stimulating the vHPC increases anxiety : GABA antagonist Bicuculline or picrotoxin infusion (Rezvanfard et al. 2009; Bast T et al., 2001)
• Suppressing the vHPC reduces anxiety : GABA agonist infusion or mechanical lesion (Kjelstrup et al., 2002; Bannerman et al., 2003; Mchugh et al., 2004; Trent et al., 2010;
Mceown et al. 2010)
Spatial learning
Anxiety