Anxiety – behavioural state arising in anticipation of potential threat

• 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



expression pattern in the developing embryo- Genetically distinct subset of serotonergic neurons


- Hippocampus to amygdala connection

BRAINSTEM

Serotonin (5HT)-producing Serotonin (5HT)-producing neuronsneurons

9 clusters (nuclei) B1-B9

BRAINSTEM



• Autonomic body physiology modulation of breathing,

body temperature blood pressure heart rate

• learning and memory• sensorimotor gating• modulation of mood states

BRAINSTEM



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


loxP loxP

OFFBAPFRT FRT

effector

Flpexcision

BAP effector

loxP loxP

OFF


loxP loxP

OFFBAPFRT FRT

effector

BAP effector ON

FlpCre


loxP loxP

OFFBAPFRT FRT

effector

BAP effector ON

GeneB::FlpGeneA::Cre

creFlp


loxP loxP

OFFBAPFRT FRT

effector

BAP effector ON

GeneB::FlpGeneA::Cre

EffectorON


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




contributions from multiplerhombomeres





Message #1: Genetic Fate map = Classical, Anatomical Map




Message #1:

Message #2: provide framework to study the function of genetically defined serotonergic neuron subtypes

Genetic Fate map = Classical, Anatomical Map



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


p<.044

enhanced fear induced freezing behavior

Contextual Fear Conditioning Paradigm

24hrs


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

*



“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



“silenced” all serotonergic neurons



“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?

? ? ?



silence

all5-HT neurons

r1-derived



no difference

silence

all5-HT neurons

r1-derived no difference



silence

all5-HT neurons

r1-derived


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


contextual learning

sensorimotor gating


Modulated more by r2- and/or r3-derived 5-HT neurons?

Anxiety-related behaviors

Contextual learning

Sensorimotor gating

silence

all5-HT neurons

r1-derived



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


contextual learning

sensorimotor gating

XXserotonergic neuron subtype

? ?

GOAL - Assigning specific serotonergic neuron subtypes to specific behaviors



expression pattern in the developing embryo- Genetically distinct subset of serotonergic neurons


- 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

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

Anxiety – behavioural state arising in anticipation of potential threat

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